Play with model kit. Due Fri 31 Oct 2008 (beginning of class)

As part of the exercise, I want you to be able to create the whole document as a PDF file, as this skill is essential for submitting papers to conferences and journals, and for putting information on the web. There are scanners in most of the ITS computer labs (not in Baskin 105, but next door in Baskin 109), if you want to convert something hand-drawn into a digital image. There is also a scanner behind the Faculty Services office in E2. See http://ic.ucsc.edu/labs/hardware.shtml for a list of which ITS labs have which equipment. You can also use Chemsite, ChemDraw, or similar software (see http://ic.ucsc.edu/labs/software.shtml for what software is in the instructional computing labs). So far as I know, we do not have any of the chemistry drawing programs on the SoE machines.

Note: I will only be accepting PDF files, *not* Microsoft word files. Because this assignment requires some pictures, plain text will not be adequate. Note: Acrobat is supposedly available in all Mac and PC labs run by Instructional Computing, so PDF should be fairly easy to produce. Acrobat Distiller is also available on School of Engineering SUN Sparc computers (as "distill"), but not on the Linux machines or Macs (run distill on "sundance", "moondance", or "apache" from an SoE account). We have ps2pdf and pstill on the Linux machines, but they do not do as good a job of compression as distill. The Mac OS X machines can produce PDF from any program that can print.

1. Build a protein backbone, at least 6 (and preferably 9) amino acids long, following the instructions I wrote in http://www.soe.ucsc.edu/~karplus/bme205/f05/darling-model/build-peptides.html. Make sure you get the chirality right---this is determined by which of the two available bond positions on C-alpha is used for the hydrogen and which is used for the sidechain. The CORN mnemonic (CO-R-N clockwise around the alpha carbon when looking at the alpha hydrogen) helps get chirality right. Fold the backbone into an alpha helix, remembering that there should be a hydrogen bond between O_i and N_i+4. Remember that the peptide plane with O_i has N_i+1. Some things to check: do all the attachment points for side chains point out of the helix? Is the helix fairly rigid? Have you got the twist going the right way (if you hold the helix axis vertically, the backbone should be slanted like the middle line of a Z)?

   Measure the distances between C-alpha atoms separated by 1, 2, 3, ... The scale of the Darling models is about 2 inches (or 5cm) to an Angstrom. (Turn in the table of distances in Angstroms between the centers of C-alpha atoms.)

   Add a C-beta carbon to each C-alpha, and measure the distances for C-beta atoms separated by 1, 2, 3, ... (Turn in the table of distances in Angstroms between the centers of C-beta atoms.)

   Extra credit: use Rasmol, Pymol, or other molecular visualization software to look at and measure the same properties of an alpha helix in a protein with a good alpha helix (say 1i4y). On the Linux machines in the BME labs, the programs are installed in /projects/compbio/bin/i686/rasmol and /projects/compbio/bin/i686/pymol. You might also want to use the pdb-get script at /projects/compbio/bin/pdb-get to download PDB files from the master copies at RCSB. I usually use the command "rasmol `pdb-get 1i4y`" to fetch and display a pdb file.

2. Make a proline residue and add it to the C-terminal end of the helix. Note how the lack of a hydrogen on the backbone nitrogen prevents the proline from participating in the hydrogen bonds that stabilize the helix. Try putting the proline at the N-terminal end of the helix. Are there problems with the H-bond at this end? Can the helix extend back before the proline? How far back can it extend? That is, can the proline be the first, second, third, ... position in an alpha helix?) I am not looking for a distance here, but a count of the number of residues. Stated yet another way, if proline is residue "i", can i-1 be part of the helix, i-2, ... ?

   Try converting the peptide of the proline from trans to cis conformation. There are two ways to do this: taking apart the peptide plane and twisting the omega angle from 180^o to 0^o, or changing which carbon you think of as C_alpha and which as C_delta, changing which one the carbonyl carbon bonds to. The first method is probably closer to what happens with a cis-trans-isomerase, but the second is a lot easier to do with the models.

3. Remove the proline, and add a serine side chain to the first residue of the helix (remember that "first" means N-terminal). Make a hydrogen bond between the O of the OH group and the HN of fourth residue of the helix (so OG of residue i to N of residue i+3). Try the same cap with a threonine instead of a serine (also fairly common). This is a common N-cap motif for helices---about 1/4 of all helices start with a serine or threonine.

   Look for an example in a PDB file. For example, in 1fr2A, S29-L33 is a good region to look at, as is T223-I227 of 1kskA Note: PDB files all have 4-alphameric characters as names. When I give a 5-letter code, the 5th letter is the chain within the pdb file, so 1epuA means chain A in PDB file 1epu.

4. Undo your helix H-bonds and split your protein backbone into two chains. Align the chains as parallel beta strands, and arrange the H bonds correctly. Which Hbonds are formed? Measure and report the distance between the closest C-beta atoms from one strand to the other. What is the distance from one C-beta to the next one along the strand? To the C-beta that is two away on the strand?
5. Now rearrange the strands to be anti-parallel. Again, which H-bonds are formed, and what is the distance between the closest C-beta atoms?
6. Join the two anti-parallel strands with an aspartic acid (D) or asparagine (N) followed by a glycine. If you want, you can also make the two residues on either side be lysine (K), so that you have the sequence KDGK or KNGK. This sequence makes a tight bend about 41% of the time, XXK[DN]GXX forms a hairpin about 36% of the time. A good example of this structure can be found in 1b77A K143 through K146.

   In past years, I suggested XPDG, which produces a tight bend about 45% of the time, but XXXPDGXX only forms a hairpin about 2% of the time. Note that the K[DN]GK turn is going to be a type I' turn (See http://www.cryst.bbk.ac.uk/PPS95/course/6_super_sec/super1.html by J. Cooper for an explanation of type I' and II' turns.

   Come up with a pattern of hydrogen bonds that holds this hairpin together. Hand in a sketch of the Hbonds. Make sure that you take into consideration that the atoms are actually space-filling—many of the conformations obtainable with the models correspond to physically impossible ones.

   Also, make sure that the lysine side chains are both on the same face of the hairpin. Note that to stabilize the hairpin as an antiparallel beta sheet, there need to be at least 2 hydrogen bonds holding the strands together.

7. Build a pair of complementary DNA bases (either AT or CG) and find the Hbonds. Note that the Hbonds do not come straight out from the double-bonded oxygen (as in the proteins) but may be at closer to a 120 degree angle. For the pair of bases you chose, make a sketch or photo of the structure showing the Hbonds, and indicate the angles for carbon-donor-acceptor (CDA) and donor-acceptor-carbon (DAC). Note: the nitrogen (or whatever the hydrogen is covalently bound to) is the donor of the hydrogen for the hydrogen bond, and the oxygen (or whatever electronegative atom is not covalently bonded to the hydrogen) is the acceptor.

   The CDA angle has its vertex at the donor atom, one ray to the acceptor, and the other ray to the carbon that the donor is covalently bonded to (there may be multiple such carbons, and so multiple CDA angles). Similarly the DAC angle has its vertex at the acceptor, and the rays to the donor and to a carbon covalently bonded to the acceptor.

   Note: no one outside the Karplus lab uses the CDA and DAC angles, preferring to work with angles around the hydrogen atom.

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   Kevin Karplus
   Biomolecular Engineering
   University of California, Santa Cruz
   Santa Cruz, CA 95064
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