Master’s Project

December 25, 2011

In the lab of Prof. A.J.M. Driessen (Molecular Microbiology) I am currently working on the first of my two master’s projects. This project has an approximate duration of six months and is centered on some molecular aspects of bacterial protein secretion across the cytoplasmic membrane. In order to give you the chance of learning something about what I do,  I summarized my work-plan and some of the theory behind it.  If you have questions, do not hesitate to contact me!


Cells are no closed and just internally highly complex entities that are sealed from the outside world. In fact in one way or the other every cell directly or indirectly communicates with its environment through processes which are mediated by channels or receptors. For multicellular eukaryotic organisms it is evident and well-known that cells secrete substances which serve as substrates for a wide array of physiological functions. These substances are ranging from simple ions for neuronal signaling purposes or pH homeostasis to more complex peptide hormones and other proteins. A general issue which was overlooked by science for decades is the fact that secretion and communication also appears in and among prokaryotic cells. Since these organisms most of the time consist of single cells, systemically relevant excretion of compounds which are synthesized within the cell is occurring in a different manner and on a different scale. Bacteria, representing one of the two prokaryotic domains, indeed possess highly complex systems which enable interaction with the environment in the form of other cells or an adherence substrate.

A key role for a bacterial cells ability to survive and to secrete molecules into the environment plays the bacterial secretory (Sec) system. Genetic, biochemical, biophysical, and structural approaches during the last two decades have shaped a detailed understanding of the functional elements and dynamics of this system. The so-called translocase system encompasses an array of proteins which are functionally centered around the translocon channel which mediates the export of proteins across the bacterial cytoplasmic membrane and the insertion of membrane proteins into it. The focus of this work lies on the study the SecA motor proteins that occur in bacteria that next to the general Sec system also possess a so-called accessory Sec system which is responsible for the export of a subset of proteins. A significant number of pathogenic bacteria make use of this subsystem to export protein virulence factors. SecA is a motor protein and ATPase which is thought to be mainly responsible for the movement of proteins across the membrane via the SecYEG translocon channel. How the two different SecA motor proteins that occur in the general and accessory Sec systems interact and what their exact functional relationship is, is currently not understood. By making use of genetic, biochemical, and fluorescence microscopic techniques the focus of the present study is to elucidate a possible interaction between Staphylococcus aureus’ general Sec systems SecA1 and its accessory Sec systems counterpart SecA2. Parts of the bacterial Sec system are conserved in all three kingdoms of life and among others also function in the eukaryotic endoplasmic reticulum in order to secrete proteins into the cytoplasm. Embedding the performed research into its context is essential and therefore brief introductions to bacterial cell structure and the two Sec systems follow with a focus on SecA structure and function.

It does, however, not lie within the scope of this work to formulate a wholesome review on the translocase system. A number of reviews have already been published on this topic, which offer an in-depth overview and cover this subject more explicitly (1, 2, 3).

The Sec translocase system

Since the SecA proteins of S. aureus, which stand central in my research project, are part of the functional complex Sec translocase system, here a brief overview is given about this system. Fig. 1 summarizes the most important aspects of this kind of bacterial protein translocation across the cytoplasmic membrane.

Fig. 1: Schematic overview of the bacterial protein translocation system termed Sec translocase. Proteins which are synthesized within the ribosome are exported from the cytoplasm over the cytoplasmic membrane into the periplasm by the proton motive force (PMF) and the ATPase SecA via the heterotrimeric membrane channel SecYEC. Two major ways of protein translocation exist: posttranslational secretion and cotranslational insertion into the cytoplasmic membrane. Secretory proteins are either directly targeted to SecA by means of their N-terminal signal sequence or are bound by the chaperone SecBfirst and translocate later via SecA and SecYEG. Membrane proteins are translocated cotranslational. Their C-terminal signal sequence is bound by the signal recognition particle (SRP) and targeted to the SRP receptor (FtsY). Consequently SecAtranslocates these proteins via a lateral gate in the SecYEG channel into the membrane. SecDF(yajC) is an accessory factor which seems to improve preprotein translocation. YidC associates with the translocon during protein insertion into the membrane (figure derived from (1)).

Some bacteria posses two different versions of SecA

In bacteria SecA universally functions as the ATPase which delivers the chemical energy to physically translocate proteins across the cytoplasmic membrane in a post-translational manner. However, some bacteria posses two homologues of this protein termed SecA1 and SecA2. SecA1 functions as the essential housekeeping protein for translocation, while SecA2 seems to be especially important for a subset of proteins. Studies on bacteria which are expressing  SecA2 have shown, that this subset of proteins often includes virulence factors, as for example adhesion molecules (4). In addition to SecA1/A2 some of these bacteria also contain a homologue monomer of the heterotrimeric translocon channel SecYEG (Fig. 1) termed SecY2.  Summarizing, bacteria containing either only SecA1/A2 or SecA1/A2 + SecY2 are said to express an accessory Sec system next to the general Sec system which is depicted in Fig. 1.

The question

The intriguing research questions of my project therefore is: Do SecA1 and SecA2 dimerize with each other or which other combinations are required to be functional? This is an important question, since SecA1/A2 might function as a link between the general and accessory Sec systems. As the accessory Sec system is important for virulence in some bacteria it is essential to learn more about its protein molecular basis. Fig. 2 summarizes the hypothetical dimers which SecA1/A2 might form in bacteria with or without an additional SecY2 homologue. Currently I am investigating the SecA proteins of S. aureus which is not expressing an additional SecY2. Some of the combinations depicted below (Fig. 2, (D) through (F)) therefore are not predicted to apply to the current state of my project.

Fig. 2: Schematic overview of the hypothetical combinations of two SecA motor proteins in S. aureus with the two SecYEG homologues when assuming a functional SecAdimer. Dimers (A) through (C) might function with the canonical SecY1EG translocation channel, while dimers (D) through (F) might also interact with the accessory SecY2EG translocation channel which is present in S. aureus. In addition it is also theoretically possible that dimer (A) binds to SecY2EG and dimer (D) to SecY1EG (created by myself, based on earlier work of Irfan Prabudiansyah).

How to get there

Here I will present a very brief work-plan, just to give you an idea about which techniques and experimental approaches my project entails in order to investigate the properties of SecA1/A2 and their functional relation to each other in S. aureus. The aim is to use molecular genetics to overexpress both proteins, which is followed by using knowledge of biochemistry to purify and fluorescently label the proteins. The final step is to use confocal microscopy (cross-correlation) and FRET microscopy to examine the potential interaction between both proteins.


The molecular cloning of of both secA1 and secA2 gene sequences from S. aureus into plasmid vectors involves some bioinformatics work and the designing of the needed primers. In addition DNA sequencing is performed to confirm correct sequence insertion. The Isopropyl β-D-1-thiogalactopyranoside (IPTG) inducible plasmids are isolated and heat-shock transformed into E. coli cells. These cells containing either secA1 or secA2 are consequently grown in large amounts at 25°C to 30°C to reduce inclusion body formation after overexpression induction by the addition of IPTG into the growth medium. After cell harvesting and cell membrane disruption by french pressing or sonication, the overexpressed protein-containing cytoplasmic fraction is isolated by ultracentrifugation. Results are confirmed by SDS-PAGE and Western blots.


Overexpressed SecA1/A2 is purified by HPLC mediated cation exchange and size-exclusion chromatography. After desalting on a size-exclusion column both proteins are labeled with two fluorophores (Cy5 & fluoresceine) via a maleimide group to their external cysteines (50% of purified SecA1 is labeled with first fluorophore, other half with second, same procedure for SecA2). Labeling efficiency is checked by photometry at different wavelengths specific to excitations spectra of fluorophores. Removal of excess fluorohore molecules is essential for avoiding too strong background noise during microscope steps. This is achieved by HPLC cation exchange at protein specific salt concentration. Results are checked on SDS-PAGE under fluorescence detection conditions.


Analysis of dimer formation is first achieved by an array of different concentrations of SecA1 and SecA2 combined to each other. Secondly, the potential dimers are analyzed with the help of a confocal microscope observing a volume of approximately 90 femtoliters. Fluorescence cross-correlation spectroscopy is applied to this volume of the solution in order to determine the diffusion coefficient of the fluorescently labeled particles (5). Potential dimer formation can then be described by altered dual color absorption/emission spectra of the proteins (FRET), as well as their unchanged or slower diffusion time through the observed volume in the case of dimer formation.

Final remarks

If you also consider the approach of my work as interesting as I do, you can always contact me back via mail. In the future I would like to continue my work and extend my knowledge into the direction of biophysics. Describing the properties of proteins by interdisciplinary means has definitively caught my eye.

(1) Du Plessis, Nouwen, Driessen, The Sec translocase, Biochimica et BiophysicaActa (2010).

(2) Driessen & Nouwen, Protein Translocation Accross the Bacterial Cytoplsmic Membrane. Annual Reviews of Biochemistry (2008).

(3) Papanikou, Karamanou, Economou, Bacterial protein secretion through the translocase nanomachine, Nature Reviews Microbiology (2007).

(4) Rigel & Braunstein, A new twist on an old pathway – accessory Sec systems, Molecular Microbiology (2008).

(5) Schwille, Mayer-Almes, Rigler, Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution, Biophysical Journal (1997)


The biology of light

December 16, 2011

If you want to know something about the connection of biomolecules and light, then this might be of interest to you. I also thought it is pretty interesting, so I wrote this piece on proteins, linker-molecules, emission, and fluorophores. First of all because I wanted to understand the principles of fluorescence better since it is important for my project (FRET and confocal microscopy), but secondly also because I like the interplay between biology and physics. For my work on the hypothetical interaction of Staphylococcus areus SecA1 and SecA2 proteins I use two molecules that have fluorophore properties. Fig.1 depicts these molecules called fluorescein-5-maleimide (A) and Cy5-maleimide (B). Obviously both names are not the IUPAC nomenclature names for chemical compounds, however in everyday lab-life, these two names seemed more practical.


Fig. 1: Chemical structure of two fluorophores that can be used to label proteins via sulfur containing thiol groups on external cysteines. Left fluorescein-malamide is depicted and on the right side Cyanine (Cy5) maleimide. The maleimide group is the lowest identical portion of both molecules.  It functions as a linker between the thiol group and the actual fluorophores.

To be fluorescent actually describes a property that some molecules have. This includes the absorption of light at a specific wavelength (relatively high energy) and the subsequent emission of light with a longer wavelength and lower energy per photon. The relation between the energy of a photon and its wavelength is important when talking about fluorophores (Fig. 2). The frequency of a photon (≈ internal energy) is inversely proportional to its wavelength, thus photons with a short wavelength contain more energy than photons with longer wavelength.

 Fig. 2: The relation of a photon energy with its wavelength. Long wavelength means lower energy, because the frequency of the photon decreases (not shown in this formula) (1).

If one concentrates on the chemical structures depicted in Fig. 1 it becomes apparent that they both contain a large number of double bond containing cyclic carbon rings. And this is also the case for most fluorophores in nature. These structures are one secret of fluorescent properties.

Light low wavelength transfers energy on electrons in double bond which becomes excited and “jumps up” a band, thereby stabilizing the energy which has just been absorbed. This excited state of the electron does not last forever and it will fall back to its original and more stable state sooner or later. However, thermodynamics tells us that energy does not just disappear so the energy which is lost (because the electron has fallen back) must be somewhere. Indeed the energy is somewhere. The fluorescence effect that we see is the electromagnetic radiation in the form of photons that is emitted from the falling electron. Important to notice is, however, that not all energy is emitted as visible electromagnetic radiation. The physical turnover processes also create other emissions that cannot simply be seen by eye and in addition energy is “lost” through collisions of our fluorophores with other molecules. Since this is the case, the emitted energy is somewhat lower than the absorbed energy and therefore also the wavelength of the emitted photons becomes longer, as Fig. 2 tells us. As light color depends on wavelength, the emitted color that can be observed is different from the previously absorbed one (Fig. 3).

 Fig. 3: Emission spectra of green fluorescent protein (GFP). The wavelength of the emitted light is longer than the wavelength of the absorbed light. Therefore also the color of the light appears different (2).

Fluorescent properties of molecules enable biologists to mark proteins or other biomolecules in order to make them easily detectable by eye or with the help of photomultipliers. Two or more fluorescent markers can even be combined and might transfer energy to each other when their carrier molecules make contact. Changes emission spectra can consequently be observed. This is the basis of FRET microscopy and allows me to study the potential dimerization of proteins that are thought to function in the same protein translocation pathway.



Lamarck’s revenge

December 7, 2011

Lamarck himself of course did not know about molecular genetics, but in biology it is becoming more and more clear that inheritance of acquired traits might also be possible. However, most of these traits are transmitted via epigenetics (methylation, acetylation, etc.) which is essentially chromatin and thus DNA based. A mechanism which enables the “inheritance” of acquired traits and in which DNA is not the central element, has now been published in the Cell journal (Transgenerational Inheritance of an Acquired Small RNA-Based Antiviral Response in C. elegans).   The group of  Oliver Hobert from the Department of Biochemistry at Columbia University in New York has reported that upon viral infection of their model organism Caenorhabditis elegans, the worm activates its defense mechanisms in such a way that it is also transferred to later generations which did not have contact with the virus at all (1). In this case the immunological response of C. elegansis is based on the so-called RNA interference (RNAi) mechanism. RNAi is generally known to silence genes in the cadre of genetic regulation and works by the binding of specially synthesized small RNA molecules to regular mRNA which is subsequently up- or downregulated thereby influencing protein production. For quite some years it has also been known that RNAi is used in viral defense by utilizing the double stranded RNA (dsRNA) of the virus as a template for the production small RNAs (viRNA) that subsequently lead to the breakdown of the viral genes and an effective protection. Also in the present study this behavior of the RNAi system is important.

By saying “gene-silencing effects evoked by exogenously added dsRNA can be observed not only in the treated animals, but […] also in the progeny of the treated worms” the authors summarize the findings of other scientists, who had found in previous studies that upon RNAi system stimulation by foreign dsRNA (for example from a virus) the viRNA parts of this defense mechanisms could also be traced back in generations that never encountered the dsRNA (i. e. virus). Therefore some kind of “inheritance” must have taken place. In this paper Hobert and coworkers tried to identify whether this “inheritance”  is rather traditional and therefore DNA or chromatin mediated (for example epigenetic) or whether it involves some kind of novel mechanism that transgenerationally “directly” transmits the RNA .

In order to test this the scientists introduced dsRNA containing the FR1gfp transgene into the C. elegans worms. This dsRNA was responsive to a heat-shock signal due to a promoter which was able to sense abrupt increase of temperature. By means of raising the temperature the virus could thus be induced. Under conditions where the worm RNAi machinery is functional the replication and distribution of the foreign viral RNA is of course inhibited. The worms keep their regular phenotype. When, however, the RNAi machinery is mutated the worm cannot defend itself against the replication of the foreign RNA. In this case also the FR1 gene which is fused to a green fluorescent protein (gfp) gene is expressed. The worms appear greenish after excitation with light of the fitting wavelength. Mutations where induced in two genes called rde-1 and rde-4. These two genes are normally responsible for the expression of dsRNA binding proteins that induce the RNAi response. Fig. 1 (left) depicts the general experimental setup in which immunodeficient mutant worms are crossed with immunocompetent worms (parental generation) what results in heterozygotic (for this allele) worms in the F1 generation (kids). When these worms are crossed with each other a Mendl-like inheritance pattern results in the F2 generation (grandchildren). The essential question is: Are the homozygotic mutants of the F2 generation resistant to the virus dsRNA even though they have never encountered the virus before and do not possess the machinery to combat the dsRNA? In case they are not resistant the researchers would observe a green fluorescence signal due to the replicating RNA (see also above).

Fig. 1: The genetic principle behind this study (left) and some results showing the generational transmittance of viRNA into mutant worms what consequently renders them resistant to the virus (no green fluorescent signal) (right). The transmission effect eventually “wears off” after 4-5 generations.

After performing the above described heat-shock induction of the F2 generation worms, the hypothesis derived results could indeed be observed (Fig. 1 (right)). Even viRNA transmission up to the 5th generation was present. The authors excluded the possibility that the resistance of the homozygous mutants is based exclusively on maternal deposition by the heterozygous parents since it has been demonstrated before that none of the gene activity is transmitted between generations (2). In addition the necessary viRNA is transmitted over several generations also if no viral stimulus is added. However the virus resistance “wears off” after 4-5 generations.

That viRNA is really transmitted among generations is proven by a physical RNA detection assay in which the 20–30 nt viRNAs are quantified in accordance to their alignment on the viral RNA and qualified based on their strandedness.

All together these results indicate that classical genetics based on DNA and even relatively new elements of it such as epigenetics are not the whole picture of biological adaption and evolution. However, important molecular components of this viRNA transmittance system remain unidentified. Why does the protective effect “wear off”? How are the viRNAs transmitted from generation to generation if not by maternal deposition? What is the code if no viral dsRNA template is present and nothing is deposited?

In their conclusion the authors stress the point that the above described mechanism can have a strong evolutionary significance for organisms since it renders it possible to rapidly transmit the mechanisms of an adaptive response to the following generations. Short-term environmental stress can thus be effectively counter acted.

Even though many important questions, especially concerning the molecular basis, remain unanswered, this study shows that biology is far from being elucidated completely. In the search for answers it can thus sometimes be helpful to look into a direction that seems the least probable. Lamarck might agree.

This blog-article is especially based on the two articles named below:

(1) O. Rechavi, G. Minevich, O. Hobert, Transgenerational Inheritance of an Acquired Small RNA-Based Antiviral Response in C. elegans. Cell147 (2011). (online publication)

(2) D. Blanchard, P. Parameswaran, J. Lopez-Molina, J. Gent, J.F. Saynuk and A. Fire, On the nature of in vivo requirements for rde-4 in RNAi and developmental pathways in C. elegans. RNA Biol.,  8  (2011).

For an amino acid, which are one of the building blocks of life, pyrrolysine was discovered quite recently. Just in 2002 it was discovered by mass spectrometric analysis and chrystallographic approaches. Until present pyrrolysine has been found in some enzymes of the methanogenic pathway called MtmB, MtbB, and MttB of archea belonging to the order of Methanosarcinales and two bacterial species. This rare amino acid is just regularly encoded by the natural genetic code. UAG, which is normally an amber-type stop codon on mRNA and stops translation, is the nucleotide code for pyrrolysine. Only a special nucleotide composition which surrounds the UAG triplets allows the interaction of the pyrrolysine carrying tRNA with its codon. In most other organisms this “exception from the rule” is not possible since the chemical forces of UAG do not allow tRNA binding of any kind. UAG thus functions as stop codon in the majority of all organisms. As its name already suggests it is similar to lysine and features a pyrroline ring at the lysine side chain (Fig. 1).

Fig 1: Chemical structure of pyrrolysine. A pyrrol ring is added to the lysine (right part of molecule) side chain (stretching from NH2 to NH).

Two genes are required in order to make out of the UAG stop codon a “normal” amino acid encoding codon. These genes are called pylT and pylS. Respectivly they encode a fitting, but unusual tRNA (of course with the antisense codon CUA) and an aminoacyl-tRNA synthetase which loads the pylT encoded tRNA with pyrrolysine. As a third requirement and as mentioned before also the nucleotide environment around UAG is important. Next to the two required genes also a special, so-called pyrrolysine insertion sequence (PYLIS sequence), which lies downstream of  UAG seems to be essential. Within the mRNA this sequence forms a stemloop and confers structural stability during tRNA binding.

After these genetic and molecular biology questions were (at least partly) answered, one question remained: How is pyrrolysine itself synthesized by the cell? Which pathway is employed and which enzymes take part in the catalyzed reactions?

A few weeks ago researchers from the Technical University of Munich shed some light on the biochemistry of pyrrolysine biosynthesis. In the following I want to explain briefly what they found out. However I want to keep it quite simple and will not mention which molecular clusters within the enzymes active site initiate and facilitate the electron transfer in order to catalyze pyrrolysine formation. Interested readers are referred to the original publication (1).

The researchers started out by overexpression of one of the genes which lies in the gene cluster which was already known to be responsible for pyrrolysine synthesis. Also a reaction pathway for the biosynthesis of pyrrolysine had been proposed by other researchers (2). Fig. 2 presents this pathway.

Fig. 2: Proposed route of pyrrolysine biosynthesis. In addition the names of the enzymes are mentioned which catalyze the reaction steps (adapted from (1), originally proposed by (2)).

Starting out from this foundation the TUM researchers purified the first step catalyzing PylB protein and determined its structure by X-ray crystallography. Fig. 3 depicts the result of this approach. A S-adenosylmethionine (SAM) cofactor, an iron-sulfur cluster, and the reaction product of the enzyme are found in the central cavity of the structure which at the same time seems to form the active site of the enzyme. To find a reaction product within the enzyme is actually a surprise since it tells a lot about the mechanisms by which the enzyme works. In this case the molecule number 2 from Fig. 2 was detected. The researchers concluded that this was really the catalysis product of the enzyme since impurities could be excluded. The compound is no metabolite of E. coli metabolism and was also not part of any of the buffers that was used during purification and structure determination. In addition the reaction had to be bound very tightly in order to “survive” the three-step purification and crystallization procedure. This fact also supported the catalysis product theory. Since the quality of the model’s active site region was quite good, it was possible to virtually insert a 3D molecular model of PylB’s substrate (lysine, Fig. 1 without the ring structure) into the cavity which helped to develop an actual model of how PylB catalyzes the first step of pyrrolysine biosynthesis. Knowledge of organic chemistry led the researchers to the possibility that abstraction of a hydrogen ion from one special position of the substrate leads to the formation of a radical (atom with one free electron, very reactive). This process does not occur spontaneously, but requires the action of PylB’s active site. Otherwise the energy barrier to abstract the hydrogen would be too high. Upon radical formation an isomerization occurs and the essential relocation of the methyl group (CH2) can occur which gives the reaction product its final structure. This sequential reaction probably only occurs because the substrate is located in close proximity to the SAM element within the active site which catalyzes the first step of the reaction.

Fig. 3: Stereoview of the crystal structure of PyB, the enzyme which catalyzes the first pyrrolysine biosynthesis step. The same enzyme is depicted on both sides, but stereoview is applied to enhance the 3D illusion without wearing special glasses. Important to note are the chemical groups which are present in the hypothetical active center of the enzyme. They can be seen in the top part of the enzyme. The SAM subunit is located between an iron-sulfur cluster and the reaction product which the enzyme delivers (compound 2 in Fig. 2). SAM together with the iron-sulfur cluster seem to initiate the catalyzing reaction (starting with regular lysine) by hydrogen abstraction and subsequent radical formation (adapted from (1)).

In addition the researchers also describe the potential dynamics of PylB which would enable it to release the reaction product. However, the proposed movement of a certain amino acid helix structure is only hypothetical. Still it is remarkably how this study, which combines molecular and structural biology with organic chemistry, elucidated a major mechanism behind the existence of the 22nd amino acid. The researchers also stress the importance of their work in the cadre of protein synthesis with non-natural amino acid substitutions for which the unusual incorporation method of pyrrolysine into proteins (described in the beginning) might be a suitable model.

(1) Quitterer, F., List, A., Eisenreich, W., Bacher, A. and Groll, M. (2011), Crystal Structure of Methylornithine Synthase (PylB): Insights into the Pyrrolysine Biosynthesis. Angewandte Chemie International Edition. doi: 10.1002/anie.201106765

(2) Gaston, M.A., Zhang, L., Green-Church, K.B. and Krzycki, J.A. (2011), The complete biosynthesis of the genetically encoded amino acid pyrrolysine from lysine. Nature 471, 647-650. doi:10.1038/nature09918

Together with Myriam Löffler and Katja Becker I wrote this piece on mitochondrial diseases based on carrier deficiencies during the bachelors course “Molecular Mechanisms of Disease” at Radboud University Nijmegen. This course, together with a bioinformatics and two biochemistry courses lead to my wish to study more about the world of protein functioning at the molecular level. Maybe this work is of interest to some people out there.

Adenosine triphosphate is the most essential and universal high-energy containing molecule of each cell and is synthesized within the mitochondrial cell compartment. In order to maintain the complex metabolic mitochondrial equilibrium, exchange of several compounds with the surrounding cytosol is necessary and facilitated by members of the mitochondrial carrier family. Since the carrier structure contains a number of highly conserved amino acid motifs that are important for carrier specificity, point mutations can already have strong negative effects on carrier function. The spectrum of metabolic diseases therefore contains a number of mitochondrial carrier associated syndromes, some of which are present here.

1. Introduction

The mammalian mitochondrion is a metabolically highly divers compartment of the eukaryotic cell. It is required for among others gluconeogenesis, amino and fatty acids metabolism (urea cycle and β-oxidation), synthesis of proteins encoded by mitochondrial DNA (mtDNA), synthesis of its own mtDNA by making use of nucleotides, and thermogenesis through for example uncoupling. In addition and most importantly the process of oxidative phosphorylation (OXPHOS) is accomplished by different structures within the mitochondria. In order to be able to synthesize the body’s most important energy rich molecule ATP, an intense substrate and product exchange of compounds that are directly and indirectly linked to OXPHOS is necessary. Thus, several members of the mitochondrial carrier family are present to link cytosolic and mitochondrial metabolism. The substrates of the transporters include nucleotides, amino acids, co-factors, carboxylic acids and inorganic anions 1.

2. Mitochondrial carriers in health

2.1 Importance of carriers and OXPHOS

In total the human genome (nDNA and mt DNA) encodes for 48 mitochondrial carriers. It is likely that there are about 39 different functions associated to these carriers since some carriers portray an almost identical amino acid sequence similarity which is attributed to a comparable function in vivo 1. Consequently the mitochondrial carrier family can be split up into two principal groups based upon their functional properties. The first one includes carriers that have a function in the metabolic generation of energy within the cell. Most importantly those carriers are directly associated to the transport of substrates and product which are essential for OHXPHOS reactions. Secondly, the carrier family includes a group of transporters that are needed for intermediary metabolism within the mitochondria. Among this group fall carriers that are related to for example amino acid metabolism and the replication of mtDNA.

2.2 Carriers can be directly or indirectly associated with OXPHOS

In order for the OXPHOS system to be functional several substrates are required which all have to enter the mitochondrial compartment with the help of carriers (for a review of the OXPHOS system see 2 and 3). In the following a number of important compounds will be described which are transported by specific membrane molecules. Pyruvate for example which is the aliphatic end product of glycolysation is transported by a, in humans presently not identified, monocarboxylase transporter 1, 4. Pyruvate is later converted into acetyl-CoA by a reaction requiring high levels of coenzyme A (CoA). This is achieved by a recently identified carrier in human mitochondria (SLC25A42) which exchanges the externally synthesized CoA with adenosine 3′,5′-diphosphate 5. Converting pyruvate in addition also requires a number of cofactors, which also enter the mitochondrion via carriers. Except for the thiamine pyrophosphate carrier (TPC) which was recently identified in Drosophila melanogaster 6 there is presently no proof that other cofactor transporters as for lipoamide, NAD+ and FAD structurally belong to the human mitochondrial carrier family since they have only been described for some prokaryotes, Saccharomyces cerevisiae and Arabidopsis thaliana 1. In addition intermediates of the citric acid cycle which convert acetyl-CoA into reducing equivalents and GTP must enter and leave the mitochondrion. A great number of identified carriers fulfil this task. Most importantly the exchange of ATP from the in- to the outside for ADP which is transported from the out- to the inside of the mitochondrion has to take place with the help of mitochondrial carriers. This process in combination with phosphate carriers ensures the functioning of ATPase within complex V of the respiratory chain and the supply of readily generated ATP for the cell 1. Further a variety of mitochondrial uncoupling proteins (UCPs) which uncouple elements of the electron transport chain can be used for thermogenic purposes through the release of the H+ proton gradient which generates heat in cells of especially the skeletal muscles 7.

2.3 Carriers associated with the metabolism of non-OXPHOS related compounds are important for housekeeping

Several carriers are needed to maintain the more general functional properties of mitochondria in order to produce ATP molecules. Apart from the carriers that have a direct influence on the metabolic cycles and the respiratory chain there are carriers needed for the metabolism of amino acids and replication of mtDNA.

Nitrogen rich compounds which originate from the degeneration of aminoacids in the form of citrulline after a number of conversions leave the mitochondrion through carriers. The urea cycle for example depends on carriers such as the citrulline/ornithine exchanger. In addition also structurally complete amino acids are transported into or out of the mitochondrion. This is achieved by a number of single amino acid carriers or transport molecules that exchange one amino acid for another one 1.

For the intra mitochondrial metabolism of mtDNA deoxynucleotides are needed within this cell compartment. The deoxynucleotide carrier (DNC) encoded by the gene SLC25A19 seems to fulfil this function 8. However and despite the structural properties of DNC there is increasing evidence that DNC might not be the appropriate deoxynucleotide carrier of the mitochondrion based on metabolomic and enzyme kinetical studies 9. Other specific carriers are utilized to transport methyl containing compounds for the methylation of DNA, RNA and proteins 1.

2.4 General structure of the mitochondrial carrier family

Multiple sequence alignment of several carriers was applied to identify residues that play a role in substrate transport and binding points since it was expected that residues that play a role in transport are more symmetrically conserved than the asymmetric residues that provide substrate binding capabilities 10.

The atomic model of the Bos taurus ADP/ATP carrier provided first insights into the exact structural and functional properties of members of the mitochondrial carrier family. Presently this whole group of carrier proteins is classified based upon its structure. When comparing their amino acid sequences several aspects are noticeable. First of all the sequence consists out of three homologous tandem repeats of about 100 amino acids that form a pseudosymmetric structure (Fig. 1A) and a 3D barrel structure within the mitochondrial membrane. Each repeat consists out two α-helices, termed H1-H6, which are linked by a short α-helix on the mitochondrial matrix site of the carrier. Within this region also the conserved signature motif PX[DE]XX[RK] can be found. The odd numbered helices (H1, H3, and H5) are linked by the proline residues kinking them in a certain angle based on the fused backbone properties of the prolines. Charged residues within the signature motif form salt bridges and lock the odd numbered transmembrane helices together closing the carrier structure on the matrix site 10.

For the transport of substrates across the membrane the carriers exist in two distinct conformational states. During the cytoplasmic state (c-state) the carrier accepts its substrate from the cytoplasmic site and during the matrix state the carrier binds to a substrate from the mitochondrial matrix side. Binding of substrates occurs within a mid-membrane internal aqueous cavity (Fig. 1B) consistent with the two opening states at a specific and functionally conserved binding site in all mitochondrial carriers. This conservation can especially be observed at three carrier-substrate contact points named contact point I-III on the even numbered helices (H2, H4, and H6). Especially contact point II offers some interesting insights in carrier function because the conservation of amino acids at this point correlates precisely with the subfamily of the carrier. Different signature motifs of for example amino acid, keto acid or adenine nucleotide carriers at contact point II are thus able to bind and distinguish the specific substrate that belongs to the subfamily (Fig. 1B) 11.

The precise structural changes a carrier undergoes during substrate binding and release are presently unknown but modelling with the yeast ADP/ATP carrier proposed a probable mechanism. According to this mechanism carriers act as monomers and binding of the substrate disrupts the fragile but normally stable salt bridge network at the edges of the carrier enabling it to change its conformational state into one of the two possibilities 10. Other mitochondrial carriers are expected to work in a similar way.

When looking at these fragile binding and transport mechanisms it is not astonishing that already single amino acid mutations can distort carrier function heavily leading to malfunctions and disease.

 Fig. 1: (A) displays the tripartite structure of the mitochondrial carrier family consisting out of three tandem repeats (dotted red lines) each containing two conserved helices (black). Red bars mark the conserved signature motif at the matrix site which probably fulfils functions in transport and structure 1. (B) is a lateral view model of the mid membrane aqueous cavity substrate binding site, displaying the contact points I-III, of the carrier BtAAC1. H6 is removed. The purple arrow denotes the position of the substrate, the yellow arrow the position of the salt bridges 11.

3. Mitochondrial carriers in disease

In case of defects in mitochondrial carriers, involved metabolic routes get disturbed. Possible impacts of this are the accumulation of products and substrates and failure of the system due to malfunctioning of metabolic routes. Fig. 2 shows fifteen mitochondrial carriers and their roles in transport of important metabolic substrates and products. This chapter describes a few of them and which, if damaged, cause diseases. A number of mitochondrial carrier associated diseases are presently known (Tab. 1) and some of them will be discussed here.

Fig. 2: Overview of the most important carriers in the mitochondrial membrane and their interactions. The ATP/ADP carrier (red circle) function is essential for the OXPHOS system, especially for the complex V ATPsynthase (light blue structure), since it delivers substrate (ADP) and catalyzes the transport of product out of the mitochondrion (ATP). The PiC carrier (blue circle) is directly linked to the synthethis of ATP through Pi supply for the ATPsynthase. The carnitine/ arylcarnitine carrier (green circle) imports acylCoA derived from the breakdown of fatty acids into the mitochondrial lumen. This acylCoA is further used as a substrate in the β-oxidation. The glutamate carrier (black circle) transports glutamate into the mitochondrial lumen. This transport depends on an H+ gradient, provided by the OXPHOS system 12.

Tab. 1: Diseases associated with mitochondrial carriers 12.

3.1 Diseases due to defects of mitochondrial carriers directly linked to oxidative phosphorylation

Oxidative phosphorylation of ADP to ATP acquires uptake of inorganic phosphate (Pi) and ADP into the mitochondria. Therefore, two carriers in the mitochondrial membrane are needed to catalyze the uptake: the ADP/ATP translocase and the PiC carrier.


The ATP/ADP carrier (Fig. 2) catalyzes as an antiporter the transport of adenosine triphosphate (ATP) out of the mitochondria, and conversely transports adenosine diphosphate (ADP) out of cytsol into the mitochondria. ADP is needed for the synthesis of new ATP molecules inside the mitochondria.

One ATP is exchanged for one ADP molecule simultaneously:

ATP4-(matrix) + ADP3-(cytosol) → ATP4-(cytosol)+ ADP3-(matrix)

Therefore ATP preferentially moves along the membrane potential (concentration gradient) across the inner membrane, towards the cytosol.

  • AAC1 deficiency

AAC1 is the heart-/muscle-specific ADP/ATP carrier. It is encoded by the SLC25A4 gene.

In case of an AAC1 deficiency, a homozygous mutation is located in this gene. Alanine at position 123 is replaced by an aspartic acid (A123D), resulting in a complete loss of the protein’s ability to transport ADP and ATP. A123D is the first recessive mutation found in the AAC1 gene 13. When looking at crystal structure of the bovine AAC1 the structural and functional importance of A123 becomes apparent. A123 is localized between H5 and H6 in the consencus sequence GXXXG and reaches into the central substrate binding cavity which lies two helix turns above the fragile salt bridge network that controls the carriers transport conformation (cytoplasmic or matrix state, see above). It is thus imaginable that A123 substitutions can cause carrier deficiencies 14.

The symptoms which are observed in patients with AAC1 deficiency constitute exercise intolerance, lactic acidosis, hypertrophic cardiomyopathy and a mild myopathy. Significantly lower activities of the respiratory chain complexes I, III and IV were measured when compared to physiologically normal values. These complexes are encoded by mtDNA. Due to the changes of adenine nucleotides in addition mtDNA is negatively affected and therefore also the mtDNA dependent respiratory chain complexes.

AAC1 deficiency may be compensated by glycolysis and transport by other AAC isoforms or other adenine nucleotide mitochondrial carriers, for instance the ATP-Mg/Pi carrier.

  • Sengers’ syndrome

The genetic defect that causes this form of AAC1 deficiency is still unknown. Presently no mutation has been found in SLC25A4 and linkage analysis excluded that the AAC1 locus is involved. Sengers’ syndrome has been described in two unrelated patients with symptoms like congenital cataracts, hypertrophic cardiomyopathy, mitochondrial myopathy and lactic acidosis. There was reduced amount of AAC1 protein and also impaired adenine nucleotide transport found in the muscle tissues of these patients. A molecular relation to AAC1 deficiency thus is possible. It is feasible that steps within DNA metabolism processes such as defective transcription, translation or targeting of AAC1 could basis of this disease 15.

  • Autosomal dominant progressive external ophthalmoplegia (adPEO)

adPEO is a heterogeneous disorder with a dominant inheritance pattern. The disease is caused by defects in certain nuclear genes affecting the mtDNA. Thus, it is resulting in multiple deletions of mtDNA in post-mitotic tissues, in particular in brain, muscle and heart. In turn, these deletions induce the deficiency of respiratory chain proteins and therefore defective energy production 12.

adPEO usually appears at the age of 20–40 years. The symptoms found in patients with this disease are for example weakness of the external eye muscles, ptosis, mild descending myopathy, also possible symptoms imply bilateral cataract, sensorineural hypocusia,  tremor, ataxia, sensorimotor peripheral neuropathy, generalized muscle weakness, exercise intolerance, depression, parkinsonism and endocrine dysfunction.

Research showed that not only primary mtDNA deletions are responsible for PEO.  By linkage analysis it has been shown, that also mutations of three nuclear genes, leading to secondary multiple deletions of mtDNA, can cause this disorder:

a) SLC25A4, encoding the heart-/muscle-specific mitochondrial AAC1

b) Twinkle, encoding a mtDNA helicase

c) POLG1,encoding the catalytic subunit of mtDNA-specific polymerase γ.

AAC1 deficiency is described in the above section. The genes Twinkle and POLG1 are directly involved in the repair and replication of mtDNA. This type of nuclear-dependent PEO can be inherited through heterozygous mutations in the POLG1 gene, but more common as an autosomal dominant disorder 16, 17. All three mutations thus lie within nuclear DNA, but then in genes which affect the metabolism of mtDNA. These intragenome interactions lay the basis for adPEO. Treatment of PEO medicates the symptoms, such as eye props and ptosis surgery.

PiC carrier

The PiC carrier (Fig. 2) catalyzes as a symporter the transport of phosphate ions out of the cytosol into the mitochondria by H+ co-transport. Phosphate (Pi) is essential for the oxidative phosphorylation of ADP into ATP.

  • PiC deficiency

The PiC carrier is encoded by the SLC25A3 gene. Two isoforms of this carrier, which are alternative transcripts, have been identified. PiC-A is expressed only in heart and muscle, and PiC-B is expressed in all tissues 12.

A homozygous mutation in PiC-A is the cause of PiC deficiency. Glycine is substituted by a

glutamic acid residue, resulting in a damage of the protein function. It is remarkable that this mutation is localized within exon 3A, which is one of the alternatively spliced variants (PiC-A). Gly72Glu is located within the first transmembrane helix (H1, see above ). Substituting a small non-charged amino acid (Gly) with a charged and thus polar and relatively large amino acid (Glu) is expected to have severe effects on folding and thus functional and structural properties of the protein. Multiple sequence alignment of SLC25A3 from several eukaryotic organisms showed a high degree of glycine conservation at position 72 indicating an important and specific role of this residue 18. In addition this glycine lies within the conserved H1 region of the carrier family (see above) which is important for the opening and closing state of the carrier. These results indicate that Gly72Glu might be the reason for PiC deficiency.

The defect in the PiC-A-mediated uptake of Pi into the mitochondria inhibits the ATP synthesis by oxidative phosphorylation and thereby leads to a severe shortage of energy supply within the affected cells. However the ATP synthesis in patients was only defective in muscle tissue since PiC-B is not affected as it lies on another transcript version. Analysis of the heart mitochondria showed reduced levels of ADP-stimulated respiration of pyruvate, but normal activity of the respiratory chain enzymes, pyruvate dehydrogenase and oligomycinsensitive ATP-ase. This suggests that there is no defect in the SLC25A4 gene as in AAC1 deficiency. Patients with this disorder suffer from muscular hypotonia, progressive hypertrophic cardiomyopathy, elevated plasma lactate levels, and lactic acidosis. Newborns die within 4 to 9 months because of heart failure due to the missing PiC-A carrier. The function of the PiC-A carrier cannot be compensated by another protein 12.

3.2 Diseases due to defects of mitochondrial carriers involved in intermediary and housekeeping metabolism

Next to the carriers directly related to the oxidative phosphorylation, there are a lot of carriers playing an important role in processes that are not directly linked to ATP synthesis. These range from carriers that provide intermediary compounds for the oxidative phosphorylation to carriers that transport amino acids and nucleotides for the replication and translation of mitochondrial DNA (mtDNA) 1 (Fig. 2).

For example the carnitine/arylcarnitine carrier (CAC) for example is related to generating energy, but not ATP directly as opposed to PiC, because it imports acylCoA from lipid degeneration in the cytoplasm to the mitochondrial lumen so that it can be used in β-oxidation. This process also generates energy as NADH en FADH2 which are used in the oxidative phosphorylation by the complexes I-V to transport electrons (Fig 2). This carrier is thus indirectly linked to the ATP generation 19.

On the other hand, the glutamate carrier (GC) manages the symport of protons and glutamate that can be used in the synthesis of proteins from mtDNA (Fig. 2). The Py carrier (PyC) among others imports pyrimidines for the replication of mtDNA. Defects in these processes will first impair the mitochondrion itself and as a result they will disturb the ATP generation 12.

Pathological mutations in one of these carriers involved in intermediary mitochondrial metabolism are expected to cause severe diseases. There are five defects in the carriers known that play an important role in the development of severe symptoms, mainly related to the function of the heart, liver, brain and muscles. The CAC deficiency influences the β-oxidation and so the energy supply of the oxidative phosphorylation while the HHH syndrome is associated with a defect in the ornithine/citrulline carrier (ORC) impairing the urea cycle. The AGC2 deficiency, Amish microcephaly and neonatal myclonic epilepsy are related to the transport of nucleotids and amino acids (table 3.1) 12.

Carnitine/ Arylcarnitine Carrier

The carnitine/arylcarnitine carrier exchanges arylcarnitine from the cytoplasm with carnitine from the mitochondrial matrix. In the cytoplasm carnitine reacts with acylCoA derived from lipid degeneration. The product, acycarnitine, enters the mitochondrion supported by the CAC. In the mitochondrial matrix a reaction of acylcarnitine with CoA results in carnitine and acylCoA. Carnitine is exported by CAC while acylCoA takes part in the ß-oxidation (Fig. 2) 19.

  • CAC deficiency

CAC is encoded by the gene SLC25A20 which is located on chromosome 3p21.31 and contains 9 exons. It is expressed in high levels in skeletal muscles, liver and heart. This might explain the symptoms that are caused by mutations. So far, mainly missense mutations such as D32N, R133W or P230R have been identified to cause CAC deficiency 20. All three are mutations within different signature motifs that appear in all mitochondrial carriers. Other mutations related to the disease are R178Q and G81R which concern residues that are expected to be part of the substrate binding site (Fig. 3) 21. Furthermore, frameshift mutations in exon 1 and 3 in combination with missense mutations are found to cause the appearance of an early stopcodon affecting the amino acid at position 127 22.

 Fig. 3: Protein structure of CAC. The different colours represent different domains and the binding site is predicted to be around the violet area. The mutations found in CAC deficiency are also shown 12.

The symptoms of a CAC deficiency especially affect the organs showing a high expression of CAC. So, cardiac arrhythmia, muscle weakness and abnormal liver function appear due to an accumulation of acylcarnitines and fatty acids that cannot be oxidized. Normally, fatty acid oxidation provides energy in form of NADH and FADH2 in periods of fasting and physical exercise leading to severe complications in fasting patients. Furthermore, hypoketosis occurs because of the reduced fatty acid oxidation and acidosis and hyperammonemia might develop from an increase in amino acid oxidation to compensate the diminished β-oxidation 21. CAC deficiency is inherited autosomal recessivly and denotes two phenotypes. It can emerge in the neonatal period which is most common and the more severe form. Patients suffer a high mortality rate within the first hours or days after birth and present no CAC activity. However, patients displaying a later onset during childhood posses the milder variation and a rest activity of CAC remains. They can be treated by administrating medium-chain triglycerides. These can enter the mitochondrial matrix without the aid of CAC and carnitine. In the mitochondrion the triglycerides can take part in the β-oxidation and energy can be generated despite of the CAC deficiency 12. To make treatment efficient an early recognition of the disease is required. CAC deficiency can be identified by mass spectrometry which is used in neonatal screening programs. As a reaction on the diagnosis of CAC deficiency patients get a special diet consisting of frequent meals with low lipid content and rich in carbohydrates. So they will not become hypoglycemic as a result of the frequent meals. Low lipid levels in the food are required to prevent the accumulation of fatty acids and acycarnitine that lead to cardiac arrhythmia, muscle weakness and abnormal liver function 12.

Glutamate Carrier (GC)

The glutamate carrier symports glutamate with protons into the mitochondrial matrix. Within the mitochondrion a proton motive force (PMF) is used to generate ATP, and GC can profit from this H+ gradient, too (Fig. 2). Two known isoforms of GC exist. GC2 is expressed in the mitochondria of almost all tissues and is responsible for the fulfillment of basic glutamate requirements. Only in tissues with an extended demand GC1 is expressed. In the neonatal myoclonic epilepsy a deficiency in GC1 leads to severe symptoms 12.

  • Neonatal myoclonic epilepsy

The mutations found to cause neonatal myoclonic epilepsy are P206L and G236W in the SLC25A22 gene mapped on chromosome 11p15.5. Multiple sequence aligments showed that these mutational sites are located within highly conserved regions in helices (H1, H3, and H5) near the cytosolic membrane side 23. The effect might be that the carrier protein cannot fold any more or that there is no substrate binding possible due to a disturbed salt-bridge network (see above).

The symptoms are hypotony, progressive microcephaly, abnormal visual nerve conduction, encephalopathy and spasticity. These mainly neurological effects can arise due to the fact that glutamate is an important neurotransmitter and that effective transmission requires a particular intracellular glutamate concentration 23. However, the detailed mechanisms of the development of these symptoms remain elusive. Treatment could target the concentration of glutamate within cells. An increase of the concentration gradient would facilitate the transport of more glutamate.

4. Conclusion

Due to the complexity of the metabolic pathways that are present within the mitochondrial cell compartment deficiencies of mitochondrial carriers with different functions cause severe metabolic diseases. These defects are mostly based on mutations within the nDNA, but can also originate from mtDNA. The mitochondrial carrier family is organized and built up in a highly specific and conserved manner and even a small number of point mutations and therefore amino acid substitutions within these regional motifs can have great negative implications on carrier structure and thus function.

The phenotypic effects of the described malfunctions can especially be observed in organs with high energy demand such as liver, heart, skeletal muscle and brain. Due to the systemic importance of these organs severe diseases are the consequence, frequently with high mortality in early childhood.

One carrier can affect several metabolic routes directly or indirectly and therefore compensation of the described deficiencies is seldom possible due to the high specificity of the carriers. In some cases treatment might be promising and can involve bypassing the critical transport route through the supply of alternative compounds.

Since metabolic diseases are accompanied by severe health effects and cure is currently not possible, more insights in the molecular functioning of mitochondrial metabolism in general and especially carrier properties are essential.


1.    Kunji, E.R.S. The role and structure of mitochondrial carriers. FEBS Letters 564, 239-244 (2004).

2.    Smeitink, J., van den Heuvel, L. & DiMauro, S. The genetics and pathology of oxidative phosphorylation. Nat Rev Genet 2, 342-352 (2001).

3.    Boekema, E.J. & Braun, H. Supramolecular Structure of the Mitochondrial Oxidative Phosphorylation System. Journal of Biological Chemistry 282, 1 -4 (2007).

4.    Wang, X. et al. Pyruvate protects mitochondria from oxidative stress in human neuroblastoma SK-N-SH cells. Brain Research 1132, 1-9 (2007).

5.    Fiermonte, G., Paradies, E., Todisco, S., Marobbio, C.M.T. & Palmieri, F. A Novel Member of Solute Carrier Family 25 (SLC25A42) Is a Transporter of Coenzyme A and Adenosine 3′,5′-Diphosphate in Human Mitochondria. Journal of Biological Chemistry 284, 18152 -18159 (2009).

6.    Iacopetta, D. et al. The biochemical properties of the mitochondrial thiamine pyrophosphate carrier from Drosophila melanogaster. FEBS Journal 277, 1172-1181 (2010).

7.    Rousset, S. et al. The Biology of Mitochondrial Uncoupling Proteins. Diabetes 53, 130S-135 (2004).

8.    Dolce, V., Fiermonte, G., Runswick, M.J., Palmieri, F. & Walker, J.E. The human mitochondrial deoxynucleotide carrier and its role in the toxicity of nucleoside antivirals. Proceedings of the National Academy of Sciences of the United States of America 98, 2284-2288 (2001).

9.    Kang, J. & Samuels, D.C. The evidence that the DNC (SLC25A19) is not the mitochondrial deoxyribonucleotide carrier. Mitochondrion 8, 103-108 (2008).

10.  Robinson, A.J., Overy, C. & Kunji, E.R.S. The mechanism of transport by mitochondrial carriers based on analysis of symmetry. Proceedings of the National Academy of Sciences 105, 17766-17771 (2008).

11.  Robinson, A.J. & Kunji, E.R.S. Mitochondrial carriers in the cytoplasmic state have a common substrate binding site. Proceedings of the National Academy of Sciences of the United States of America 103, 2617-2622 (2006).

12.  Palmieri, F. Diseases caused by defects of mitochondrial carriers: A review. Biochimica et Biophysica Acta (BBA) – Bioenergetics 1777, 564-578 (2008).

13.  Palmieri, L. et al. Complete loss-of-function of the heart/muscle-specific adenine nucleotide translocator is associated with mitochondrial myopathy and cardiomyopathy. Hum. Mol. Genet. 14, 3079-3088 (2005).

14.  Curran, A.R. & Engelman, D.M. Sequence motifs, polar interactions and conformational changes in helical membrane proteins. Current Opinion in Structural Biology 13, 412-417 (2003).

15.  Jordens, E.Z. et al. Adenine nucleotide translocator 1 deficiency associated with Sengers syndrome. Annals of Neurology 52, 95-99 (2002).

16.  Van Goethem, G., Dermaut, B., Lofgren, A., Martin, J. & Van Broeckhoven, C. Mutation of POLG is associated with progressive external ophthalmoplegia characterized by mtDNA deletions. Nat Genet 28, 211-212 (2001).

17.  Spelbrink, J.N. et al. Human mitochondrial DNA deletions associated with mutations in the gene encoding Twinkle, a phage T7 gene 4-like protein localized in mitochondria. Nat Genet 28, 223-231 (2001).

18.  Mayr, J.A. et al. Mitochondrial Phosphate-Carrier Deficiency: A Novel Disorder of Oxidative Phosphorylation. The American Journal of Human Genetics 80, 478-484 (2007).

19.  Huizing, M. et al. Cloning of the Human Carnitine-Acylcarnitine Carrier cDNA and Identification of the Molecular Defect in a Patient. The American Journal of Human Genetics 61, 1239-1245 (1997).

20.  SOLUTE CARRIER FAMILY 25 (CARNITINE/ACYLCARNITINE TRANSLOCASE), MEMBER 20; SLC25A20 – OMIM Result. Online Mendelian Inheritance in Man (OMIM)  (2005).at <;

21.  Iacobazzi, V. et al. Molecular and functional analysis of SLC25A20 mutations causing carnitine-acylcarnitine translocase deficiency. Human Mutation 24, 312-320 (2004).

22.  Hsu, B.Y.L. et al. Aberrant mRNA Splicing Associated with Coding Region Mutations in Children with Carnitine-Acylcarnitine Translocase Deficiency. Molecular Genetics and Metabolism 74, 248-255 (2001).

23.  Molinari, F. et al. Impaired Mitochondrial Glutamate Transport in Autosomal Recessive Neonatal Myoclonic Epilepsy. The American Journal of Human Genetics 76, 334-339 (2005).


Uncovering a cover

November 5, 2011

Fig. 1: The EMBO Journal cover, showing a comparison of four genomes, which makes use of a publicly available bioinformatic visual analytic tool called Circos.

This is the cover of the European Molecular Biology Organization Journal (short: EMBO Journal) (Volume 28, Number 9
06 May 2009) (Fig. 1) is a nice example how scientific results can be made visible in a beautiful way. The freely available Circos tool was used to compare the also freely available genomes of human, chimpanzee, mouse, and zebrafish. Circos is an aesthetic appealing a visually relatively easy way of relating data sets to one another. In the times of -omics it can be handy for disciplines such as biochemistry, molecular biology, and genetics to present their data in a recognizable and interpretable manner.

An example of how Circos figures are used in scientific and popular literature in general is pictured below (Fig. 2). It gives a first idea about the impact that the visually appealing presentation of your data can have on general public readers and fellow scientists. PR of your science is getting more and more important I would say.

Fig. 2: Examples of Circos usage in scientific and popular literature as well as advertisement (originally from the Circos homepage).

In the following I would like to explain how these magnificent circular figures have to be interpreted based on the orignal publication of Martin I Krzywinski et al. in Genome Research, Vol 19, 1639-1645, 2009 where the author describes the general properties of the program and gives more examples of the programs capabilities. In order to explain the cover which is pictured above in Fig. 1, below in Fig. 3 you see the complete and more “clean” version of it.

Fig. 3: Complete visual genomic comparison, which is also displayed as a journal cover in Fig. 1. For a bigger picture size click the figure (extracted from a Circos visual guide which can be found here).

Fig. 4: Legend for Fig. 3. Click to enlarge (extracted from a Circos visual guide which can be found here).

When concentrating on the EMBO figure above, one thing is immediately apparent to the eye: The enormous amount of DNA similarity linkages between human (top right), zebrafish (top left), mouse (bottom left), and chimpanzee (bottom right) genomes. However, it is important to firstly note that this figure does NOT picture ALL possible similarity linkages between the genomes, but instead only compares the human genome to the other three. Every human chromosome has a unique color-code which is visible at the outermost edge in the “human section” of Fig. 3 next to the chromosome number (1-22, and X). This color-code also makes the similarity lines, which span across the figure, more recognizable. Every locus on every human chromosome can thus be linked to one (or more) loci on each of the three other genomes.

The most other details (inner rings) are not as easy comparable to each other, since they depict individually SELECTED information PER genome. As explained in Fig. 4 this means that the yellow and blue zone is differing per genome. The intention seemed to be to display the capabilities of the program rather than making the genomes perfectly comparable for a number of items like for example number of exons on that chromosome or types/names of genes. Instead each genome covers some unique information which can be traced back in the legend (Fig. 4).

It is thus obvious from this short summary of just one publicly available example what the advantages and drawbacks of circular gene comparison are. First of all it is appealing to the eye and with the help of a legend easily and quickly interpreted. Dealing with gene annotations actually becomes fun. However, it is also clear that this display technique rises and falls with its resolution. Of course it is impressive to compare four genomes almost literally in one blink of an eye, but the information content which can be extracted after studying the picture remains quite low. Although this is the case, in his paper Martin Krzywinski presents also ways of using Circos in a higher resolution manner. Instead of comparing four whole genomes to each other it is also possible to concentrate on one single “cancer” gene, which appears in four cancer types. Conserved mutations and recombinations become immediately visible. Circos thus, on a small-scale, is ideal to for example assist in tackling huge challenges like the interpretation of the cancer genome which is a new approach to subdivide cancer into genetic types instead of morphological ones. In the future this might allow more efficient therapies for similar typed cancers.

Always have been interested in what it would be like to live inside a hot and poisonous volcano? Then this is something for you:

In the following I want to describe a paper which was published by, among others, members of the Department of Microbiology at Radboud University Nijmegen where I did my BSc internship. The team of researchers was headed by Marjan Smeulders from Nijmegen and Thomas Barends working at the Max Planck Institute for Medical Research in Heidelberg. I am glad that their work on a archeal CS2 hydrolase ended up in a fine piece of work in Nature just last week. This enzyme is essential part of the energy metabolism of a certain species of archeon which was originally extracted from a volcanic region near Naples, Italy. We are thus surely talking about an extremophilic organism here! Next to the evolutionary and ecological implications of the paper, I am also fascinated by the wide range of genetic, biochemical, computational and biophysical methods which they used. I will not describe these methods here, but rather concentrate on the results and the conclusions which can be drawn from these results. For more information please refer to the actual paper which is named at the end.

I will begin with an introduction of the 3D structure of CS2 hydrolase, which is essential to the paper. Later I will also describe the functional consequences that the evolution of this enzyme had over time. Enjoy!

Fig. 1: X-ray chrystallographic structure of an archeal CS2 hydrolase and overview of the tertiary and quaternary structure of the enzyme. A more detailed description can be found in the text.

In the top left corner the basic dimer structure of the enzyme is depicted. Due to interactions of the N- and C-terminal tails of the monomeric amino acid chains four dimers can combine to assemble a square shaped octamer, which consists out of eight parts in total (four dimers, consisting out of two basic amino acid chains each). This construct is displayed in the top right corner where the colors denote the eight individual basic parts. The bottom left corner shows the final so-called quaternary structure of the hydrolase enzyme which consists out of two intertwined octameric (eight pieced) square “rings”. When summing up the total enzyme is thus a hexadecameric complex which is constructed out of sixteen amino acid chains (primary structure) each possessing a N- and C-terminus. The graph in the bottom right corner is the result of a small-angle X-ray scattering (SAXS) experiment. It shows the X-ray intensity (y-axis) plotted against the scattering angle (x-axis) of a sample containing all bigger and smaller pieces described above. Since the resolution of this method is relatively low, the resulting total scattering curve has to be matched to more accurate sub X-ray or NMR curves of the structures of the individual parts which are in the sample. The scattering of the X-rays can thus give first clues about whether the  tertiary (eight pieced in this case) or the quaternary structure (sixteen pieced) is predominant in a given sample. In this case the red line describes a mix of 83% of the sixteen pieced total structure and 17% of the smaller eight pieced subpart.

Fig. 2: Model of the CS2 hydrolase active site and the channel structure which defines the nature of the molecules which can gain access to the enzymes catalytic site. Again, a more detailed explanation can be found in the text.

The left part of Fig. 2 pictures a detailed model of the active site of the enzyme. One histidine and two cysteine amino acids can clearly be seen. The physiochemical characteristics of these three active site residues hold the catalytic zinc ion in place which is essential for the hydrolysis of CS2 into H2S and CO2. The blue, orange and green cages around the molecules and atoms represent the electron density which confirms the positions of the compounds within the structure. The single zinc ion could be located because its electron density is significantly different from its surroundings. On the right half of Fig. 2 the active site is pictured again, but now more in relation to the overall structure. This enzyme structure allows only one single passage way which compounds can take in order to arrive at the catalytic site (red circle) and is called “tunnel” (green). Large non-polar and uncharged amino acids like the predominant phenylalanine give this tunnel very hydrophobic characteristics which together with the diameter dramatically restricts the amount of compounds which could theoretically reach the catalytic site. In red, blue and grey the association of the hydrophobic amino acids to the different monomers (Fig. 1) is described.

Fig. 3: Mutational sites in the tunnel leading to the active site and the effects of these respective mutations on the hydrolases reaction speed and affinity.

In order to check whether the size and characteristics of the tunnel really influence the catalytic site in terms of specificity or this is purely based on the build-up of the active site itself, seven different mutations where one at a time introduced into the tunnel. The right-handed portion of Fig. 3 denotes the position and sort of these mutations. From the graph on the left it can be concluded that mutations which introduce smaller amino acids or delete one and thus lead to a widening of the tunnel, also lead to increased reaction speed. Inversely, obstructing the tunnel by larger-than-normal amino acids or blocking the active site (position 78) decreases the ability of the enzyme to process its substrates.

In addition a genetic comparison of the present enzyme with related enzymes that catalyze different or the same chemical reactions had shown that the active site of all of these enzymes is relatively conserved (for example between carbonic anhydrase and CS2 hydrolase). The specificity of the active site itself thus seems to be low and the activity universal. So the specificity of such a CS2 hydrolase must originate from somewhere else.

With this paper the authors deliver a possible answer to this question and bring up more evidence that nature (evolution) does not have to change active sites at high costs all the time, but that it is also possible to modify quaternary structures of enzymes in order to make them more specific for another compound which might be more favorable for an organism. By modifying non-catalytic  site residues (tunnel residues in this case) and allowing the passage of other chemical compounds it is therefore possible redesign the functional properties of an enzyme.

Smeulders et al. Evolution of a new enzyme for carbon disulphide conversion by an acidothermophilic archeon. Nature 478, 412-416 (2011).

When mathematics and biology come together, interesting things can happen. For a Radboud University Nijmegen Honours Academy course entitled “Mathematics and Harmony“, given by Dr. Bernd Souvignier, I wrote a piece on the mathematical model that describes the chemical gradients which determine biological pattern formation in nature. This model is mainly based on the British mathematician Alan Turing. He was one of the founders of modern informatics, but shortly before his tragic death, also published one single paper on chemistry and biology. Published in 1952 and entitled “The Chemical Basis of Morphogenesis”, in this paper some easy to understand principles concerning chemical gradient formation that determine pattern formation are described in a relatively complex mathematical manner. In my work on this topic, I mainly concentrated on the conceptional basis behind the mathematics, since my field of study is more biochemically orientated. Nevertheless this early combination of biology, chemistry and mathematics is extremely interesting and is still keeping scientists from a number of disciplines busy.

With the help of reaction-diffusion equations, which describe the interaction and movements of chemical compounds through structures, Alan Turing postulated his hypothesis of pattern formation and morphogenesis. Figure 1 gives an easy-to-understand overview of Turing’s principle.

Figure 1: The elements and meaning of one version of a reaction-diffusion equation which was also used by Alan Turing in his 1952 publication on “The Chemical Basis of Morphogenesis”. A “thank you” to Kele’s Science blog for the “simplification of the complex“.

The above mentioned formulas helps to describe how chemical compounds, now known as transcription factors or morphogens, can interact with each other in relatively simple feed-forward or inhibition loops to create biological patterns such as stripes, spirals and much more from an original homogeneous and uniform begin situation. My small article on the topic, which can be found as a pdf document below, describes some of the implication of Turing’s model on modern-day knowledge of morphogenesis and connects this knowledge to other existing theories. In some sort of timeline research is reviewed which describes applications of Turing’s theories. Finally, an outlook in the future states that Turing’s ideas are also applicable in “real” 3D systems. A critical conclusion on the conformity of his theory with other existing morphogen theories follows as well. The article is in Dutch (all figures have journal references, so it’s also interesting for English speakers) and can be accessed here:


Approximately 250 gram of this chemical in our body give us the power to do what we want. Ok, the “power” (or better lets call it chemical potential energy from now on) we are talking about here is not free or pure. Actually, this potential energy originating from ATP (also called adenosine triphosphate) is stored in the covalent phosphoanhydride bonds which can be seen in Fig. 1 (red circles). I really like this molecules because every day I encounter its enormous significance for biology. In almost every biochemical reaction, every folding, or cell dynamical process there is almost a 100% certainty that ATP will be encountered. Per mol ATP 7.3 calories are released when one of the named phosphoanhydride bonds is broken. Because of the presence of two of these high-energy bonds ATP is present in large amounts in every cell and serves as the energy currency of the cell.

Fig. 1: A molecule of ATP. It is composed of three phosphate groups which are interlinked by phosphoanhydride bonds (red circles), a ribose sugar (circular molecule in the middle of the structure), and the so-called purine base adenosine. The four nucleotides that make up the DNA have actually a similar structure: Only two of the three phosphates are removed, one hydroxyl of the ribose is exchanged for a sole proton, and the bases are varying between purines and pyrimidines.

ATP is so special because some of its subparts. The two most important parts which lend ATP these behaviours are ADP, which is just missing one phosphate, and the other part is, what a surprise, the missing third phosphate called Pi. At neutral pH 7, which is usually is present in living cells, these to parts are strongly negatively charged. Two charges of the same kind do not combine very well. That is why the cell uses a lot of potential electrical energy called the proton-motive force (pmf) across the mitochondrial membrane to combine these charges via a protein called ATPase. Electrons required for the build-up of the pmf are transported across the mitochondrial membrane through the breakage of other energy storing chemical bonds in for example sugars during the process of glycolysis (oxidation reactions). The electron gradient of the pmf thus delivers the energy to let the endergonic reaction occur in which the  ADP and Pi molecules fuse into a newly formed covalent phosphoanhydride bond via ATPase. Since bonds release energy when they are cleaved, the already mentioned 7.3 calories/mol are available for doing work now. Using the chemical energy which was saved within these bonds, ATP can be used to transform this energy via one or more of so-called “energy coupling” reactions into mechanical kinetic energy. Resulting movement is often mediated by protein molecules which can now catalyze new chemical reactions or assist in folding other proteins due to their changed conformational state which “pushes” them into the right native state. But also “normal” movement can be induced, like the famous “power stroke” in which the muscular proteins actin and myosin are involved. Millions of power strokes at the same time finally result in the enormous strength a human leg or arm muscle can have.

All this and much more is based on this beautiful and relatively simple molecule. Fig. 2 depicts a self-made balls-and-sticks 3D model of ATP which is orientated in the same way as in Fig. 1. When observing carefully the same chemical groups which are also described in Fig. 1 can also be traced back here as well.

Fig. 2: A 3D ball-and-stick model of ATP. Atoms, bonds, and spacial forces between atoms can be clearly seen.

All depictions of chemical structures in this article were created by myself by making use of the program ACD/ChemSketch product version 12.01.

An overview for German speaking non-biologists about the research I do at the department of Molecular Microbiology at Rijksuniversiteit Groningen:

Mein Master an der Rijksuniversität Groningen, die mit 25,000 Studenten die dritt größte und zweit älteste des Landes ist, nennt sich „Molecular Biology and Biotechnology“. Um den ersehnten Master am Ende auch zu erhalten muss viel praktische Labor- und Forschungserfahrung gesammelt werden. Zwei Elemente sind hierfür vorgesehen. Diese decken ein Drittel bzw. ein Viertel der ganzen Studienzeit ab. Mit dem längeren Projekt, welches bis Mitte März dauern wird, beschäftige ich mich gerade.

Diese Übersicht habe ich vor allem geschrieben, weil ich es schade finde, dass ich meistens nur mit Leuten über mein Fach rede, die eh schon sehr viel davon verstehen. Menschen deren Fokus nicht so auf der Biologie liegt, bleibt diese spannende Welt oft völlig verschlossen. Ich finde es jedoch wichtig, dass man ein wenig Ahnung davon hat was Gentechnik, Stammzellen und andere Elemente der modernen Biologie eigentlich sind.

Wenn man sich den Namen meines Masters anschaut wird schon ein bisschen deutlich worum es geht. Ein „Molekül“ ist jeder Stoff, der aus mehr als einem Atom besteht. In der Molekularbiologie schaut man sich also die kleinsten Bausteine des Lebens an. Diese sind:

(1)   Proteine

(2)   Zucker

(3)   Fette

(4)   Nukleinsäuren (die DNA könnte man sagen)

Man weiß wie sie aufgebaut sind, aber man weiß in den allermeisten Fällen nicht was sie machen, zumindest im Detail. Kompliziert wird es dann, wenn diese vier Stoffe alle in Interaktion miteinander treten. Und so ist das nun mal im echten Leben. In den Gebieten (1) und (4) wird die allermeiste Forschung betrieben, denn die Vorgänge die bei diesen zwei Molekülen auftreten sind sehr interessant. Sie formen die Grundlage des Lebens und steuern viele Prozesse die uns zu dem machen was wir sind. Diese Prinzipien sind schon uralt und die basalen Elemente sind in allen Lebewesen zurück zu finden. Weil die Forscher noch recht wenig wissen fängt man deswegen nicht beim Menschen an zu forschen. Man würde den Wald vor lauter Bäumen nicht sehen (das gilt übrigens für fast alle Lebewesen). Ein bisschen einfacher machen es uns Bakterien, Schimmel, Hefe, bestimmte Würmer und die Fruchtfliege. Aus diesen unappetitlichen „Tieren“ stammt ca. 90% allen Wissens über das Leben. Als molekularer Mikrobiologe beschäftige ich mich zur Zeit mit den Proteinen (also Molekülgruppe (1)) der Bakterien.

Das zweite wichtige Wort im Namen meines Masters ist „Biotechnologie“. In diesem Teilbereich der Biologie probiert man Erkenntnisse aus der molekularen Biologie, die ich oben kurz beschrieben habe, zu benutzen um daraus (industrielle) Produkte herzustellen. Ein Biotechnologe ist also eine Art Ingenieur. Viele Impfstoffe oder Medizinprodukte wie z.B. Insulin werden schon auf diesem Wege gewonnen. Auch genveränderte Pflanzen, für die man z.B. keine Pestizide mehr benötigt, sind Produkte dieses Zweiges. Die Forschung konzentriert sich derzeit vor allem auch noch auf einen dritten Teilbereich: Die Herstellung alternativer Energieformen. So kann man z.B. Algen nutzen oder manche chemische Reaktionen die Bakterien ausführen. Im Grunde genommen geht es darum, dass man einen Stoff der für den Menschen eher unpraktisch ist, in einen überführt der als Brennstoff nutzbar ist. Es gibt hier einige vielversprechende Ansätze, aber wie man an den Tankstellen sehen kann, hat sich noch nicht wirklich etwas durchgesetzt.

In der ganzen Molekularbiologie und Biotechnologie gibt es allerdings ein riesen Problem, welches man folgendermaßen übersetzten könnte: Nur weil man alle Namen der Schachfiguren und die Spielregeln kennt, ist man noch lange kein guter Schachspieler. Die Namen der Schachfiguren kennt die moderne Biologie so langsam. Bei den Spielregeln fängt man gerade an. Ein guter Schachspieler zu werden wird noch sehr, sehr lange dauern. Glück für mich und meine Mitstudenten: Es wird noch auf Jahrzehnte hinaus nicht langweilig werden uns es gibt noch sehr viel zu entdecken.

Nun wisst ihr also ein bisschen womit sich die Biologie beschäftigt, über die ich die letzten paar Jahre einiges gelernt habe. Nun aber endlich etwas konkretes, denn mein Ziel ist ja zu erklären, was ich eigentlich genau im Labor mache bzw. woran ich arbeite und arbeiten werde. Man kann das alles mit sehr vielen Details aus der Chemie und Physik erklären. Man kann es aber auch lassen. Blitzmerker werden deswegen wahrscheinlich an der einen oder anderen Stelle etwas auszusetzen haben, aber das ist ja egal.

Hierfür ist allerdings einiges an Vorwissen nötig, das alle Leute die vor den 80er Jahren die Schule beendet haben wahrscheinlich nicht haben. Das Wissen gab es nämlich noch nicht. Ich hoffe der Weg zum Ziel ist aber trotzdem spannend. Ich habe ihn in einige Etappen aufgeteilt, sodass man immer mal eine Verschnaufpause einlegen kann und nicht den Faden verliert. Mit jeder Etappe zoomt man ein Stück weiter in die Materie hinein. Man zoomt aber auch in Wirklichkeit immer weiter hinein, denn die Dimensionen werden immer kleiner werden, bis wir fast beim Atom ankommen. Die kleinen „Kapitel“ heißen:

(1)   Was ist eine Zelle und wie ist sie aufgebaut

(2)   Proteine werden in Zellen gemacht

(3)   Form folgt Funktion – Auch bei Proteinen

(4)   Proteine müssen aus der Zelle heraus – Ein Tunnel ist nötig

(1) Was ist eine Zelle und wie ist sie aufgebaut

Das Leben ist eine Zelle. Die meisten Lebewesen auf der Erde bestehen nämlich nur aus einer Zelle und auch wir kommen ursprünglich aus diesem Stadium. Das System „Zelle“ ist nämlich so effektiv und genial, dass die simpelsten Bausteine schon seit mehr als 3.5 Milliarden Jahren bestehen. Auch ein Mensch besteht aus vielen, vielen Zellen. Man kann sie alle in verschiedene Gruppen einteilen wie Leberzellen, Hautzellen, Nervenzellen etc. Alle sehen etwas anders aus und erfüllen natürlich verschiedene Funktionen. Die basalen und wichtigen Prozesse um die es hier geht, sind allerdings überall gleich. Auch der Aufbau ist sehr vergleichbar. Weil ich mit Bakterienzellen arbeite und sie etwas simpler sind als eine menschliche Zelle stelle ich hier auch eine Bakterienzelle vor (Bild 1).Bild 1: Eine Bakterienzelle. Für uns ist das wichtigste an dieser Zelle die DNA, die zusammengerollt in der Mitte der Zelle liegt und alle Lebensinformationen enthält. Zweitens sind die Ribosomen (kleine Punkte) wichtig, die aus der DNA Proteine machen. Proteine werden für wirklich fast alles in und auch außerhalb der Zelle benötigt. Drittens brauchen wir die Zellwand (grün, gelb, rot). Sie schützt die Zelle und ermöglicht ein geschlossenes System, in dem Reaktionen möglich sind die nichts direkt mit der Umwelt zu tun haben. (

Bei Menschen würde diese Zelle etwas anders aussehen. Da sich die meisten menschlichen Zellen nicht bewegen müssen, haben sie keinen Schwanz und die DNA liegt noch mal extra in einem Kern geschützt. Außerdem liegen natürlich noch ganz viele Zellen drum herum, die so z.B. die Haut formen. Eine Zelle produziert also Produkte (meistens Proteine) die vielleicht auch für umgebene Zellen sehr wichtig sind. Das können Hormone oder Enzyme (die chemische Reaktionen schneller verlaufen lassen) sein. Eine Bakterienzelle ist alleine schon komplett. Sie scheidet höchstens Stoffe aus um mit anderen Bakterien zu kommunizieren (ja, wirklich), sich irgendwo festzukleben oder der Person zu schaden in deren Nase sie sich gerade befindet.

In meinem Projekt geht es vor allem darum herauszufinden wie solche Proteine, die in der Zelle gemacht werden, auch aus der Zelle heraus kommen können. Hierfür müssen sie natürlich durch eine Art Kanal. Wie dieser Kanal funktioniert und wie er aufgebaut ist, darum geht es. Zwischen Bakterien und Menschen besteht, was diesen Aspekt betrifft, kein großer Unterschied.

Aber wie werden Proteine jetzt eigentlich gemacht? Das ist sehr wichtig, denn Proteine braucht man für wirklich fast alles. Im nächsten Kapitel dazu mehr.

(2) Proteine werden in Zellen gemacht

Proteine haben viele Funktionen. Sie können beim Ablaufen von chemischen Reaktionen helfen (Enzyme), Botschaften überbringen (Hormone), Strukturen formen (Kanäle, Fasern etc.) und vieles mehr.

Wie Proteine nun wirklich gemacht werden ist aber erst seit den 70er Jahren deutlich. Man nennt den Prozess „Proteinbiosynthese“. Dieses komplizierte Wort beschreibt den Prozess während dem aus DNA Proteine werden. Bild 2 gibt einen Überblick über diesen Prozess (Wenn er zu verwirrend erscheint, kann er auch erstmal Überschlagen werden. Mit meinem Forschungsgebiet hat er nämlich direkt nichts zu tun).

Bild 2: Die Proteinbiosynthese. Via mRNA wird die DNA in Ribosomen zu Proteinen übersetzt. Für eine detaillierte Erklärung siehe unten. (

In der Zelle befindet sich die doppelsträngige DNA. Dies ist ein Molekül aus zwei Fäden Nukleinsäure die die Erbinformation enthält. Diese wird überschrieben in mRNA während die DNA erhalten an ihrem Platz bleibt. mRNA ist eigentlich das gleiche wie DNA, aber ist kürzer und hat nur noch einen Strang aus Nukleinsäure (wie oben zu sehen ist). mRNA codiert nur für die Proteine die gerade benötigt werden. Das Abschreiben der ganzen DNA würde viel zu lange dauern und wäre unpraktisch. mRNA besteht aus vier verschiedenen Nukleinsäuren (die vier Farben). Diese formen immer Dreier Gruppen (Kodon, siehe oben). Es gibt also 43 = 64 mögliche Kombinationen. Diese 64 Möglichkeiten nennt man den genetischen Code.

Im nächsten Schritt kommt neben der mRNA noch die tRNA ins Spiel. Passend zum Code-Stück der mRNA (eines von 64), bietet sie einen Proteinbaustein an. Proteinbausteine nennt man Aminosäuren. Nur ein Typ Aminosäure passt spezifisch auf jedes Code-Stück. Im Schutze des sogenannten Ribosoms (siehe oben) findet dieser Prozess des „sich findens“ statt. Da es chemisch gesehen nur 20 Proteinbausteine gibt, aber 64 Codierungsmöglichkeiten vorhanden sind, sind die meisten Proteinbausteine dreifach codiert (3 x 20 = 60). Die vier übriggebliebenen Stücke (64 – 60 = 4) dienen als Start und Stop Signale.

In dem dieser Prozess hunderte oder tausende Male stattfindet wird aus den codierten Proteinstücken Anhand der Anleitung (mRNA) langsam erst eine Kette und nach dem Prozess des „Faltens“ ein ganzen Protein (siehe oben).

Für diesen Übersetzungsprozess im Ribosom gab es 2009 den Nobelpreis in Chemie. Eine wirklich gute und noch ausführlichere Erklärung gibt es hier.

(3)  Form folgt Funktion – Auch bei Proteinen

Die Funktion eines Proteins hat auch immer Einfluss auf seine äußere Form (oder eben andersherum… wer weiß das schon). Ein kleines Beispiel hierfür ist Bild 3, das einen HIV Virus zeigt mit vielen Proteinen und ihrer Position an oder im Viruspartikel.

Bild 3: Ein Virus (HIV) und seine Proteine. Die Funktionen können vielfältig sein von Kanalprotein bis Rezeptoren und deshalb sind auch die Formen unterschiedlich. (David S. Goodsell, RCSB PDB)

Die Kanal- und Motorproteine an denen ich arbeite sehen auch sehr verschieden aus, weil sie verschiedene Funktionen erfüllen. Im Großen und Ganzen geht es um die Kanalstücke die wiederum andere Proteine aus der Zelle heraus befördern. Diese Proteine wurden wie in Bild 2 dargestellt hergestellt und können Formen haben wie in Bild 3. Im nächsten kleinen Kapitel mehr darüber.

(4)  Proteine müssen aus der Zelle heraus – Ein Tunnel ist nötig

Die Forschungsgruppe von Professor Driessen und ich sind also daran interessiert wie Proteine die Zelle verlassen. Da die Membranen der Zellwand wegen ihrer chemischen Eigenschaften nicht durchlässig für Proteine sind, müssen diese via Kanälen nach draußen.

Details dieses Prozesses zu kennen und zu verstehen ist wichtig, denn ca. 30% aller Proteine verlassen früher oder später die Zelle und ca. 60% (!!) aller Medikamente interagieren mit solchen oder ähnlichen Zellmembrankanälen.

Es gibt also zwei Fragen: Erstens, wie ist der Kanal aufgebaut (ein, zwei, oder mehr Teile) und zweitens durch welchen Motor-Mechanismus erfolgt die Ausscheidung der Proteine (eine Art schieben oder ziehen oder geht es vielleicht von „selbst“?). Hierfür ist es wichtig die atomare Struktur der Kanal-Proteine, aber auch die der Motor-Proteine zu kennen. Sobald man diese kennt, kann man Anhand von chemischen und physikalischen Prinzipien ableiten, wie sich bestimmte übergeordnete Molekülstrukturen bewegen. Ich finde die Biologie auf dieser kleinen Ebene sehr spannend, denn sie erklärt manchmal ganz einfach im Legosteinprinzip wie essentielle Prozesse funktionieren. Bild 4 zeigt wie einzelne Atome ein viel größeres Protein formen, welches man dann weiter untersuchen kann.

Bild 4: Links sieht man die Primärstruktur des Proteins aus den Atomen Wasserstoff (weiß), Kohlenstoff (schwarz), Sauerstoff (rot) und Stickstoff (blau). Jeder dieser, auch in Bild 2 genannten 20 Proteingrundbausteine (Aminosäuren), hat seinen typischen atomaren Aufbau. Die Sekundärstruktur wird nun aus diesen 20 Aminosäuren geformt und bildet entweder einer Helixform oder ein Faltblatt. Welches Konstrukt geformt wird hängt von den elektrischen und atomaren Eigenschaften der Aminosäuren in Wechselwirkung mit dem umgebenden Zellwasser ab. Zusammen ergeben diese Helices (Spiralen) und Faltblätter dann das fertige Protein als Tertiärstruktur. Experimentell festgestellt wird meistens die Tertiärstruktur (via Röntgenchrytallographie). Es kann allerdings auch versucht werden Anhand der DNA auf die Struktur zu schließen. Dies ist allerdings noch sehr schwierig und gelingt nur teilweise. (MaxPlanckForschung 4/2003)

Viele Medikamente greifen die in Bild 4 rechts dargestellte Tertiärstruktur der Proteine an. Durch Bindung oder das Auslösen einer bestimmten chemischen Reaktion wird die Form des Proteins geändert. Da die Form sich verändert hat, kann das Protein nun auch nicht mehr seine ursprüngliche (krankmachende) Funktion ausführen. Um gute Medikamente entwickeln zu können, ist es darum sehr wichtig das Ziel des Medikaments extrem gut und detailreich zu kennen. Ansonsten kommt es zu Nebenwirkungen, da das Medikament auch Proteine angreift die ähnlich aufgebaut sind, aber eben nicht das Ziel sind.

Aber kommen wir zurück zur ersten Forschungsfrage rund um die Kanalproteine. Sie lautet: Wie ist der Kanal aufgebaut? Des Weiteren sind auch ein paar Teilfragen sehr wichtig: Besteht der Kanal aus einem, zwei oder sogar drei Stücken? Und wie ist die dreidimensionale Anordnung des Stücks oder der Stücke zueinander und zur Umgebung? Und: Lassen sich auf Basis dieser Struktur Annahmen über die Funktionsweise machen? Also legen wir los.

Zum Glück wurde in den letzten Jahren schon viel über die Struktur dieser Kanäle heraus gefunden. Es ist deswegen praktisch die ganze Sache einfach an Hand eines Bildes zu erklären (Bild 5). Vielleicht wirkt diese Struktur verwirrend, eigentlich ist es aber nur eine Abwandlung der Tertiärstruktur rechts in Bild 4 die auch Helices (Spiralen) und Faltblätter enthält.

Bild 5: Der SecYEG Kanal und seine Lage in der Zellmembran. Der obere hell gräuliche Teil des Bildes zeigt schematisch die Zellmembran und darin in dunklerem Grau den SecYEG Kanal der die komplette Zellmembran überbrückt. Mithilfe eines Stopfens („plug“) der aus Aminosäuren besteht, ist der Kanal verschlossen wenn er nicht aktiv ist (schwarz dargestellt). Im aktiven Zustand bewegen zwei SecA Proteine (weiße Ovale) den Proteinstrang (rot) durch den Kanal. Im unteren Teil des Bildes befindet sich eine weniger schematische und realistischere Darstellung der gleichen Situation. Wie oben ist auch hier die Membran dargestellt. In ihr befindet sich ebenfalls der Kanal (lila). Ebenso ist der SecA „Motor“ gezeigt, der aus zwei Teilen besteht (blau und grün) und das zu transportierende Protein (nicht dargestellt) durch den Kanal bewegt. Im Kanal selbst ist auch der „Stopfen“ (gelb) zu sehen. (Maillard et al. Protein Movement Across Membranes und Kusters et al. Quaternary Structure of SecA in Solution and Bound to SecYEG Probed at the Single Molecule Level)

In Bild 5 ist eigentlich die Forschung der letzten 25 Jahre dargestellt. Es ist sehr aufwendig diese Tertiärstruktur des Proteins mithilfe von Kristallstrukturen so auszukleiden, sodass später durch Röntgenstrahlung die wirkliche Struktur sichtbar wird (in 3D). Das Bild verrät allerdings schon sehr viel über die Funktion. So ist nun bekannt, dass es eine Art „Stopfen“ im Kanal gibt, der bei Bedarf wegklappt und, dass der Kanal selbst aus zwei Halbkreisen besteht die durch eine Nieten-artige Struktur zusammengehalten werden. Aufgrund der  gewählten 3D Perspektive sind diese drei Teile in Bild 5 leider nur schwer sichtbar. Darüber hinaus stellte man fest, dass der Kanal nicht alleine alle Arbeit vollbringt. Man registrierte immer einen Verbund mit dem sogenannten SecA Protein (in Bild 5 grün und blau dargestellt). Im Laufe der Zeit konnte die Hypothese bestätigt werden, dass dieses SecA Protein unter Energieverbrauch das zu transportierende Protein durch den Kanal befördert.

Somit wäre Forschungsfrage Nummer eins zumindest grundlegend beantwortet.

Die Antwort auf die zweite Forschungsfrage, nämlich wie der Motor SecA das Protein nun durch den Kanal transportiert, ist bedeutend kürzer: Der genaue Ablauf des Prozesses ist noch nicht deutlich. Es ist allerdings deutlich, dass die Proteinkette (rot in Bild 5) durch wechselseitiges Binden und Loslassen an den beiden SecA Hälften bewegt wird. Undeutlich ist allerdings weiterhin, ob die beiden Hälften bei diesem Prozess aneinander gebunden sind oder nicht und ob die Hälften „verbraucht“ werden und deswegen ein Auswechseln mit neuen Hälften nötig ist. Auch sind die Aminosäuren noch unbekannt an denen die Proteinkette wirklich bindet.

Man hat diese Probleme, weil man den oben beschriebenen Komplex immer erst isolieren und „befestigen“ muss, sodass man ihn sich anschauen kann. Hierbei treten oft Beschädigungen auf, die unter Umständen gar nicht als Beschädigungen auffallen, aber zu falschen Schlüssen führen können. Ist der Komplex dann einmal zu sehen, sieht man ihn nur von außen und hat auch immer nur Momentaufnahmen von einem komplizierten dynamischen Prozess zur Verfügung. Erschwerend kommt hinzu, dass der Kanal und Motor Komplex zu klein sind um ihn unter einem normalen Mikroskop wirklich zu sehen. Man betätigt sich deswegen indirekter bildgebender Verfahren aus der Physik. Diese lassen (leider) auch manchmal ein gewisses Maß an Interpretationsspielraum.

Durch diese vielen Schwierigkeiten ist die zweite Forschungsfrage also noch nicht beantwortet. Viele Menschen auf der ganzen Welt und neuerdings auch ich, arbeiten daran sie zu lösen.

Im Detail schaue ich mir verschiedene SecA Varianten von verschiedenen Bakterien an und versuche heraus zu finden in wiefern sie mit den Bekannten Komponenten zusammen passen. In dem man sich viele Varianten anschaut und probiert ob sie zueinander passen, versucht man irgendwo einen Strohhalm zu fassen zu bekommen der das Rätsel ein wenig löst.

Ich habe viele Details ausgelassen und viele Techniken nicht erklärt. Ich hoffe, allerdings trotzdem, dass es nun zumindest ein bisschen deutlich ist womit ich mich so beschäftige. Vor allem ist mir das Wissens- und Theorieumfeld wichtig in dem mein Projekt beheimatet ist. Viele von diesen Dingen klingen sehr abgehoben und scheinen mit dem wirklichen Leben nichts zu tun zu haben. Ganz so einfach ist es aber leider nicht. Alles was ich in diesem Text beschreibe sind grundlegendste Elemente allen Lebens auf der Erde. Sie treten von Bakterien, über Schimmel und Pilze, bis hin zu Säugetieren wie dem Menschen überall auf. Ich finde es sehr spannend diese allgegenwärtige Welt, die doch immer noch so unbekannt ist, weiter zu erkunden.