Zrusso Biol 368 week 9

HIV Structure Project

Bobby Arnold's Homepage

Alex Cardenas' Homepage

Using first and last visits for rapid progressors and last visit for our control subjects. Going to select a clone randomly from each visit to compare due to the fact that each visit has many clones for each visit
- Subject 1: Visit 1 and 5
- Subject 3: Visit 1 and 6
- Subject 4: Visit 1 and 4
- Subject 10:Visit 1 and 6
- Subject 11: Visit 1 and 4
- Subject 15: Visit 1 and 4
- Control Subject 6 (moderate progressor): Visit 9
- Control Subject 13 (non-progressor): Visit 5
- Media:HIV_Structure_Subject_and_Visit_Data.txt
Convert your DNA sequences into protein sequences.
- I would do this using the tools on NCBI to translate each DNA sequence into each of its three reading frames, but there is no way to know which one is actually correct.
- Bedrock problem space has all the amino acid sequences so can just use those for our sequences instead of translating the DNA sequences
Perform a multiple sequence alignment on the protein sequences.
- There are fewer differences in the amino acid sequence compared to the nucleic acid sequence. This is most likely due to the fact that since there are three bases to each amino acid, there are literally more positions to line up. Also there is more than one three base codon for each amino acid so it can erase some base pair substitutions that mess up the alignment.
- BioWorkbench not only shows complete conservation of residues, but also conservation of 'strong' vs 'weak' groups which it defines separately. However we are only concerned with absolute changes in the sequence, not conservation of activity so any change of a residue is important to us.
Which of the procedures from Chapter 6 that you ran on the entire gp120 sequence are applicable to the V3 fragment you are working with now?
- Finding transmembrane domains of the V3 region is kind of useless considering that the entire region is definitely outside the surface of the virion, so ProtParam would most likely be the most useful tool. If I were to use it I would look at each clone from each subject and see if the number of positively and negatively charged residues were different and if there was a greater percentage of the total V3 region that was more or less of a specific amino acid.
Chapter 11 contains procedures to use for working with protein 3D structures. Find the section on "Predicting the Secondary Structure of a Protein Sequence" and perform this on both the entire gp120 sequence and on the V3 fragment that we are now working with. You will compare the predictions with the actual structures.
- pages listed in book don't work, so used [1]
- Found amino acid sequence of gp120 after following one of the links below and scrounging around for a bit, [2]
  - Media:Gp120_prediction_results_from_Kwong_paper.pdf
Download the structure files for the papers we read in journal club from the NCBI Structure Database.
- Here is the Kwong 1GC1 structure:
- Here is the Stanfield 1999 1F58 structure:
- Here is the Stanfield 1999 2F58 structure:
- Here is the Stanfield 2003 1NAK structure:
We are going to use StarBiochem to look at these structures, found here. Star Biochem program to do this portion of your work. Answer the following:
- Find the N-terminus and C-terminus of each (poly)peptide structure.
  - 1GC1 has three peptides, gp120 domain G has its N-terminus at THR at position 90 and its C-terminus at GLU at position 492. gp120 domain C has its N-terminus at LYS at position 1 and its C-terminus at LYS at position 181. The light chain of the antibody has its N-terminus at GLU at position 1 and C-terminus at ARG at position 213. Image found here
  - 1F58 mostly focuses on the antibody with only the V3 region loop from the gp120 and its image with its N and C termini highlighted can be found here
  - 2F58 also mostly focuses on the antibody with only a small section of the V3 loop. Its image can be found here
  - 1NAK
- Locate all the secondary structure elements. Do these match the predictions you made above?
- Locate the V3 region and figure out which sequences from your alignment are present in the structures and which sequences are absent.
Once you have oriented yourself, analyze whether the amino acid changes that you see in the multiple sequence alignment would affect the 3D structure and explain why you think this.
The journal club papers we read are quite old already for a fast-moving field. Using the Web of Science (or PubMed or Structure) databases, find at least one more recent publication that has a structure of gp120 (V3) in it and download the structure file to view. What additional information has been learned from this new paper?
Your presentation for Week 10 will be formatted similarly to the previous HIV Evolution Project. In this case, you will want to work on creating structure figures that illustrate what result you are trying to show.
- Your presentation will be 15 minutes long (approximately 15 slides, one per minute). Include:
  - Title slide
  - Outline slide
  - Background that led you to ask your research question
  - Your question
  - How you answered your question, method/results
  - Interpretation of your results; answer to your question
  - Discussion and interprettion of your results in light of the new paper you found.
- Upload your slides to the OpenWetware wiki by the Week 9 journal assignment deadline. You may make changes to your slides in advance of your presentation, but you will be graded on what you upload by the journal deadline.
  - Use these Presentation Guidelines when preparing your PowerPoint slides.
  - Your presentation will also be graded on the following Guide to critiquing talks.