Lipids

Lipids play several roles in the body. The most prominent role is, of course, forming membranes. But lipids are also used for energy storage, and as a means to anchor proteins to membranes.

Lets warm up with a bit of repeat of many things already discussed:

Question 33:

• Mention five essential membranes in eukaryotic cells.
• What kinds of proteins do you expect in those membranes (I mean functionality, not actual names, although the latter is not forbidden).
• Think of a few ways to classify membrane proteins, and search for web pages that use those schemes.
• Think of the steps needed to produce a membrane protein and to get it into the right membrane.
• What is the function of GPCRs, ion-channels, F1ATPases, ABC transporters, porins, tyrosine kinases?
• How do membrane proteins differ from water soluble proteins?
• How many different membrane protein structures have been solved at atomic resolution?

Figure 42. Bacteriorhodopsin is a transmembrane protein. In the electron diffraction structure you can see a series of lipid molecules. As lipids tend to be very flexible, it is not possible to see very many of them.

Lets first look at protein - lipid interactions in a membrane. We have looked at bacteriorhodopsin before. This time, get the complete, raw file 2brd from the PDB and read it in in YASARA. Display the protein as stick model and the lipids (and retinal if you want) with full atoms. Answer the first two bacteriorhodopsin questions.

In the next figure all Lys and Arg have been indicated. Do that too in your Yasara session. Do you see an asymmetry? I.e. at what side of the membrane does bacteriorhodopsin have more positive residues?

Figure 43. One monomer of bovine opsin.

Now look at bovine opsin monomer (the exercise file: OPSIN.pdb). And again, look at the distribution of positive residues. Now answer the related question listed at the bottom of the page.

Look at the distribution of aromatic residues. Where roughly do aromatic residues seem suspiciously absent? Which other residue types do you see instead between the helices at the cytosolic side? Now figure out how this molecule functions. What kind of motions are needed for this functioning? Do you see a relation between residue type and characteristics on the one hand, and function on the other?

The membrane

The membrane is a very dynamic thing, and it is very leaky and absorbing. Many molecules (also water) leak through the membrane at a slow pace. Many molecules (noticeably hydrophobic hormones and medicines) dissolve in membranes. Look at the Bilayer Structure page of Stephen White's group pages. Explain the figures 1 and 2 on this page. How does this 'explain' the leakiness of membranes? (Feel free to look at my explanation: ).

Transmembrane helix prediction

In a way, it seems easier to predict a transmembrane helix than a 'normal' water soluble helix. Why? Think of two very different recipes for the prediction of transmembrane helices; one method based on your knowledge of the amino acids and one method based on statistics/pattern-recognition. Do a search for transmembrane helix prediction software. Summarize for 5 programs in maximally 10 words each how they work (and be prepared to answer the question what each term means that you copy from the WWW)! What accuracy do these programs claim? How large is the data-set of proteins with known (i.e. experimentally determined by X-ray) transmembrane helices? So do you trust these accuracy claims?

Send the OPSIN.pdb file to the WHAT IF server that returns "Secondary Structure, symmetry and accessibility" (sits in Protein Analysis). Now take the sequence of OPSIN.pdb (obtainable via some "Administration" server) and send it to several transmembrane helix prediction servers. Answer the questions about transmembrane helix prediction at the bottom of the page.

Which program is 'the best'?

Question 34:

• The lipids aren't packing all around the bacteriorhodopsin. Why not?
• I can find only one non-hydrophobic residue that packs against the lipid.
• You too? And which one is it?

Question 35: Look at Arg 175 and Asn 176. These residues illustrate the so-called 'snorkel effect'. Any idea what is meant with this name?

Transmembrane prediction questions

Question 36: Look up the 'positive-inside' rule. What does it say? And who or what is responsible for the existence of this effect?

Question 37: Look up for the following algorithms how they work:

• (Artificial) Neural Networks
• Profile alignments
• Kyte and Doolittle plot
• Hidden Markov model

And find for each of these technologies one transmembrane helix prediction method that directly or indirectly is based on it.

Question 38: We are using the sequence of bovine opsin to test several transmembrane helix prediction servers. What is fundamentally wrong with this approach?

Question 39: And now an example of a typical exam question: The small cytochrome C17 from the bacillus geronticus is dangerous for Emus (big birds that taste well, and stick their head in the sand when in danger). These birds can get an anaphylactic shock when cytochrome C17 enters their blood-stream. Not only does that kill them, but on top of that the bird farmers cannot sell the dead animals as they are no longer suitable for human consumption. So, we need a vaccine. Obviously, we cannot just inject the birds with this protein. Given the sequence:

SSWNSHINANESDADHMAIGLLFVWVAALLCAAMALVAWSPFAQPKR
AVKYRQTTALLMAFHSGRMVPMVKNNAPYDLLENAKIVEVLKSLNAL
PFAFFAAATENPDARWIEPWSDAAKFGQKQQAFNNNIVKLSLVADAG
EMEKLFAAQGDSAGVCKACSDHYRK

Question 40: The protein CO18 is a splice variant of COX18. CO18, however, goes to the cell membrane where phosphorylation on a serine is one essential step in activation. It has as sequence: