GIMP my protein

September 8, 2012

While playing around with the open-source GNU Image Manipulation Program (GIMP) I asked myself: Why not transform proteins into a construct that does not resemble the standard Protein Data Base structures? Well and this is the humble result: Another perspective on the famous green fluorescent protein, which has become so important for many projects. The GFP molecule you are seeing above is actually a modified version of the “real” protein. By modifying certain amino acids researchers around Jenny J. Yang (Georgia State University, Department of Chemistry) managed to lend calcium and proteinase detecting properties to GFP. GFPs can then be incorporated into tissues were it is important to monitor calcium concentrations. In many organisms calcium is used to build up gradients to for example transmit chemical “information” as in nerve cells. Therefore, this type of protein, next to its funny looking barrel shape, also offers some handy features to work with and might help to detect diseases such as cardiomyopathy Alzheimer’s on a molecular scale (= earlier).

With the fitting amino acid mutations GFP can not only be used for the detection of calcium ions, but also for the detection of other ions. A very famous ion is the hydrogen ion. The concentration of hydrogens ions determines the pH (yes, that’s the H in pH). The following few lines might be a bit technical, but they explain why GFP is loved by so many scientists. And why it is a perfect example of a biomolecular structure-function relationship. High pH sensitivity and specificity, rapid signal response and good optical properties are important characteristics for a pH sensor. In addition it must be possible to reach  intracellular sites in a non-invasive and non toxic manner. GFP fulfills many of these prerequisites and therefore has been used as intracellular pH sensor. But how does it work? The sensitivity and specificity of GFP mainly seems to be based on the protonation state of the phenolic group of the chromophore (the green thing in the center). In other words the charge of the light emitting parts of the GFP molecule changes. The GFP chromophore 4-(p-hydroxybenzylidene)imidazolidin-5-one (HBI) lies in the center of GFP’s β-barrel structure (made up of the white arrows) and normaly is non-fluorescent due to its unionized phenol group. However, the inward facing amino acid residues Ser65–Tyr66–Gly67 can cause the ionisation of the phenol group, leading to potential fluorescence. During different pHs, different protonation states of the chromophores phenol group are likely to influence its photochemical properties by influencing the ionisation of the phenol group. Different pH values therefore change the charges of the centrally located molecule that is responsible for the green light. A different pH value translates into a different light intensity.

Why light becomes emitted in a different manner/intensity during different protonation states is, however, a topic for a future blog entry. Only this far: The hydroxyl group of the phenol and its protonation states within the chromophore play a role, leading to different relaxation times of the electrons that become excited by the incoming laser…

Check Ulrich Haupt’s and coworkers article if you want to know more.

Building a Single Speed

September 5, 2012

Mine is done. Now construct your own Single Speed bike. Here’s what you’ll need:

  1. Everything starts out with a nice frame. I would suggest a 1970s/80s steel frame, but that’s basically a matter of taste. Buy a new one (expensive!), buy a used one, or use that old dusty frame from your garage. Some things you should care about when deciding for a frame: (A) Think about the size you need. This table gives you an overview. Mentioned is the height in centimeters of the seat tube. (B) Check whether a headset and a bottom bracket is still part of the frame, since both are quite expensive parts and relatively difficult to mount. If you can check/ask for the quality (smooth movements, no slackness). (C) Next thing on the list is to check what kind of back dropouts the frame has. For a single speed bicycle horizontal dropouts are best because they allow you to adjust chain tension. I have a frame with vertical dropouts you’ll need an extra chain tensioner later (which kind of destroys the great look of your new bike). (D) The last thing is to check is in what state the frame finishing/paint is. Make sure there is no rust in the headset and bottom bracket area.
  2. If your frame has a bottom bracket already just buy a fitting crankset. If a chainring is attached already make sure that it’s not too large. A 53-tooth chainring is definitely too large for a single speed bike and pedalling will be quite heavy. For me a 46-tooth chainring is perfect. In case your frame doesn’t have a bottom bracket, you should first find out which standard-type you need.This mainly depends on the diameter and the width. Here’s an overview. For the headset there’s basically only one standard. Mounting both parts into your frame is quite complex and if you are not an experienced bike mechanic you should seek the assistance of your favourite bicycle shop.
  3. Now the hardest part is over and you can start searching for the essential parts of your bike that actually make the frame look like a bike: The wheels. Since a single speed bike is not a fixed-gear bike you can use a freehub. I think it’s a bit more comfortable and safer for a beginner. Best thing is to use 28” (inch) diameter rims. For the back wheel just remove the cassette from the freehub and replace it with a single speed conversion kit. Once you have it it’s about 15 minutes work to set it up. I am using a 18-tooth sprocket in combination with the already mentioned 46-tooth chainring in front which is perfect for flat cities and longer journeys. So choose your sprocket size wisely.
  4. For the likely case that you have horizontal dropouts: Be aware that back wheels with quick release skewer might slip if you are a strong guy. I recommend to replace the quick release axle with a standard axle with nuts. If you can find a solid axle with the same diameter and thread pitch and a couple of nuts it will work. Remember that your  new axle should also be at least 4 cm longer than the old (quick release) hollow axle. You need some special tools for it, but you local bike shop can probably assist you.
  5. Now buy some tires. This might seem a bit early, but it’s good to buy them now because you need to check whether they fit your brakes and frame. I’m using 28 mm wide Continental Grand Prix Four Season tires because I think they are fast and comfortable at the same time. Get some high quality inner tubes as well.
  6. It’s time for brakes now. Mounting them is not difficult, just have a look at an existing bike. However, sometimes some fiddling is necessary to get them parallelly aligned.
  7. Next, organize yourself a seat post and a saddle. It’s very important that the diameter of the seat tube exactly matches the diameter of the seat post. Otherwise, you’ll always have fun with a slightly down-sliding seat post. Also find a handlebar now that fits your needs and you think is comfortable.
  8. A bike is nothing without the chain. I would buy a new chain to prevent slipping issues. A special single speed chain is not necessary. To shorten the chain you need a special tool that you can again find in your favourite bike shop (or borrow one from a friend). Concerning the chain: Shimano offers a system called “Quick Link” which makes closing the chain, after having adjusted the length, very easy.
  9. Last, but not least get some pedals. If you cannot decide whether you want to use bike shoes with cleats or not, just buy pedals with a regular and a cleat side.

Parts you should consider to buy new include bottom bracket and headset, as well as front chain ring, sprocket and a chain. New rims and tires are nice, but not necessary. As I wrote, sometimes you will probably need the assistance of an experienced bike mechanic. Probably you will also have to pay him or her, but I definitely think you can have a very beautiful and good working single speed bicycle for around 200€. Well, I think that’s it. Have fun. For more info there is also the famous single speed/fixed-gear bible by the late Sheldon Brown. His “Bicycle Technical Info” page supplies with everything you need to know. If you are from Groningen, you should also pay a visit to Fietsje, a great bike shop with many useful single speed accessories.