Zrusso Biol 368 week 9

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zrusso13 qwerty

Contents

HIV Structure Project

Bobby Arnold's Homepage

Alex Cardenas' Homepage

  1. 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
  2. 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
  3. 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.
  4. 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.
  5. 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.
  6. Download the structure files for the papers we read in journal club from the NCBI Structure Database.
    • Here is the Kwong 1GC1 structure: Image:1-1GC1.png
    • Here is the Stanfield 1999 1F58 structure: Image:1-1F58.png
    • Here is the Stanfield 1999 2F58 structure: Image:1-2F58.png
    • Here is the Stanfield 2003 1NAK structure: Image:1-1NAK.png
  7. 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 seems to also focus on the antibody but there are far more domains in this structure than any of the previous ones. It's image can be found here
    • Locate all the secondary structure elements. Do these match the predictions you made above?
      • The prediction from PSIPRED is pretty good, some of the residues however were associated with different secondary structures, however the overall ordering of secondary structure as well as the central residues in the secondary structures were intact, with issues only arising on residues at the edges of the sequence responsible for any specific secondary structure.
    • Locate the V3 region and figure out which sequences from your alignment are present in the structures and which sequences are absent.
      • V3 region from Kwong et. al PDB: 1GC1 is not present due to the fact that it was excised to make crystallizing the whole gp120 structure easier. Amino acids 300G-328G are missing and replaced with the 'gars' substitution. In between the coil (blue) and sheet (yellow) in the following picture is where the V3 region should be. Image:V3Should 1GC1.png
      • V3 region for secondary structure 1F58 is found 313P-325P. Image:V31F58.png
      • V3 region for secondary structure 2F58 is found 315P-324P. Image:V3 2F58.png
      • V3 region for secondary structure 1NAK is found 312P-323P. Image:V3 1NAK.png
  8. 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.
Subject 1 Before and After AIDS Alignment
S1V5-4        EVVIRSENITNNAKIIIVQLNESVAISCTRPNNNIKQRIMHIRPGRAFYTK-DITEDIRQ
S1V1-2        EVVIRSENFTNNAKIIIVQLNESVEINCTRPNNNTR-KSIHIRPGRAFYTTGDIIRDIRQ
              ********:*************** *.******* : : :**********. ** .****

S1V5-4        AYCNISRTAWNNTLKQIVKKLREHFVNKTIVFNHSS
S1V1-2        AYCNISRAEWNNTLKQIVIKLREHFRNKTIVFNHSS
              *******: ********* ****** **********
Subject 3 Before and After AIDS Alignment
S3V6-6        DIVIRSANFSDNAKTILVQLNETVVMNCTRPGNNTRKRVTLGPGRVYYTTGQIIGDIRKA
S3V1-3        DVVIRSANFTNNAKTILVQLNETVVMNCTRPGNNTRKRVTLGPGKVYYTTGQIIGDIRKA
              *:*******::*********************************:***************

S3V6-6        HCNLSRAGWNSTLERIAIKLREQFQNKTIAFNQSS
S3V1-3        HCNLSRADWNNTLKRIAIKLREQFQNKTIAFNQSS
              *******.**.**:*********************
Subject 4 Before and After AIDS Alignment
S4V4-2        EVVIRSENFTNNAKIIIVQLNESVEINCTRPDNHTVRKIPIGPGRSFYTTGIVGDIRQAH
S4V1-1        EVVIRSENFTNNAKIIIVQLNKSVEINCTRPNNNTIRRIPIGPGRAFYTTGRIGDIRPAH
              *********************:*********:*:*:*:*******:***** :**** **

S4V4-2        CNISKTKWNNTLKLIVNKLREQFGNKTIIFNQSS
S4V1-1        CNISRTKWNNTLKLIVNKLREQFRNKTIIFNQSS
              ****:****************** **********
Subject 10 Before and After AIDS Alignment
S10V6-1       EVVIRSENFTDNAKTIIVHLNKSVEINCTRPNNNTRRSINMGPGRAFYATGEIIGDIRQA
S10V1-7       EVVIRSENFTDNAKTIIVQLNKSVEINCTRPNNNTRRSINMGPGRAFYTTGEIIGDIRQA
              ******************:*****************************:***********

S10V6-1       HCNLSRTKWNDTLKQVVAKLREQFRNKTIIFTQSS
S10V1-7       HCNLSRTKWNDTLKQVVDKLGEQFRNKTIIFNQSS
              ***************** ** **********.***
Subject 11 Before and After AIDS Alignment
S11V4-5         EVIIRSENFSNNAKNIIVQLNESVVINCTRPDNTIKQRIIHIGPGRPFYTTGIKGNIRQA
S11V1-3[4]      EVIIRSENFSNNAKNIIVQLNESVVINCTRPDNTIKQRIIHIGPGRPFYTTGIKGNIRQA
                ************************************************************

S11V4-5         HCNVSRGQWNKTLEQVVRKLREQYGPNKTIVFKQPI
S11V1-3[4]      HCNVSRGQWNKTLEQVVRKLREQYGLNKTIVFKQPI
                ************************* **********
Subject 15 Before and After AIDS Alignment
S15V4-7       EVVIRSENFTNNAKIIIVHLNESVVINCTRPNNNTRRKIHIGPGKTFYTGDIIGNIRQAH
S15V1-10      GVVIRSENFTNNAKIIIVQLKEAVRINCIRPNNNTRRRIPIGPGSAFYTTGIIGDIRQAH
               *****************:*:*:* *** ********:* ****.:*** .***:*****

S15V4-7       CNISGSKWNNTLKQIVNKLREQFGNKTIVFNQSS
S15V1-10      CNISGSKWNSTLKQIVNKLREQFVNKPIIFNQSS
              *********.************* **.*:*****
All Rapid Progressor Subjects Post-AIDS Alignment
S11V4-5       EVIIRSENFSNNAKNIIVQLNESVVINCTRPDNTIKQRIIHIGPGRPFYTT-GIKGNIRQ
S1V5-4        EVVIRSENITNNAKIIIVQLNESVAISCTRPNNNIKQRIMHIRPGRAFYTK-DITEDIRQ
S15V4-7       EVVIRSENFTNNAKIIIVHLNESVVINCTRPNNNTR-RKIHIGPGKTFYTG-DIIGNIRQ
S4V4-2        EVVIRSENFTNNAKIIIVQLNESVEINCTRPDNHT-VRKIPIGPGRSFYTT-GIVGDIRQ
S10V6-1       EVVIRSENFTDNAKTIIVHLNKSVEINCTRPNNNTR-RSINMGPGRAFYATGEIIGDIRQ
S3V6-6        DIVIRSANFSDNAKTILVQLNETVVMNCTRPGNNTR-KRVTLGPGRVYYTTGQIIGDIRK
              :::*** *:::*** *:*:**::* :.****.*    : : : **: :*:   *  :**:

S11V4-5       AHCNVSRGQWNKTLEQVVRKLREQYGPNKTIVFKQPI
S1V5-4        AYCNISRTAWNNTLKQIVKKLREHF-VNKTIVFNHSS
S15V4-7       AHCNISGSKWNNTLKQIVNKLREQFG-NKTIVFNQSS
S4V4-2        AHCNISKTKWNNTLKLIVNKLREQFG-NKTIIFNQSS
S10V6-1       AHCNLSRTKWNDTLKQVVAKLREQFR-NKTIIFTQSS
S3V6-6        AHCNLSRAGWNSTLERIAIKLREQFQ-NKTIAFNQSS
              *:**:*   **.**: :. ****::  **** *.:. 
Control Subjects 6 & 13 Alignment
S13V5-1[4]      EIVIRSENFTNNAKIIIVQLKESVEINCTRPGNNTRRSINIGPGRAFYASRGIIGDIRQA
S6V9-3          EVVIRSANLTDNAKIIIVHLNESVEMNCTRPNNNTRKGIHIGPGRAFYATGEIIGNIRQA
                *:**** *:*:*******:*:****:*****.****:.*:*********:  ***:****

S13V5-1[4]      YCNISKAKWDNTLGQVAAKLREQFRNATIVFNQSS
S6V9-3          HCNLSRAPWNDTLKRIAIKLREQFKNKTIAFNQSS
                :**:*:* *::** ::* ******:* **.*****
Alignment of Control Subject 13 Sequence to Post-AIDS Sequences from all Subjects
S13V5-1[4]      EIVIRSENFTNNAKIIIVQLKESVEINCTRPGNNTR-RSINIGPGRAFYASRGIIGDIRQ
S15V4-7         EVVIRSENFTNNAKIIIVHLNESVVINCTRPNNNTR-RKIHIGPGKTFYTG-DIIGNIRQ
S1V5-4          EVVIRSENITNNAKIIIVQLNESVAISCTRPNNNIKQRIMHIRPGRAFYTK-DITEDIRQ
S10V6-1         EVVIRSENFTDNAKTIIVHLNKSVEINCTRPNNNTR-RSINMGPGRAFYATGEIIGDIRQ
S4V4-2          EVVIRSENFTNNAKIIIVQLNESVEINCTRPDNHTV-RKIPIGPGRSFYTT-GIVGDIRQ
S11V4-5         EVIIRSENFSNNAKNIIVQLNESVVINCTRPDNTIKQRIIHIGPGRPFYTT-GIKGNIRQ
S3V6-6          DIVIRSANFSDNAKTILVQLNETVVMNCTRPGNNTR-KRVTLGPGRVYYTTGQIIGDIRK
                :::*** *:::*** *:*:*:::* :.****.*    : : : **: :*:   *  :**: 

S13V5-1[4]      AYCNISKAKWDNTLGQVAAKLREQFR-NATIVFNQSS
S15V4-7         AHCNISGSKWNNTLKQIVNKLREQFG-NKTIVFNQSS
S1V5-4          AYCNISRTAWNNTLKQIVKKLREHFV-NKTIVFNHSS
S10V6-1         AHCNLSRTKWNDTLKQVVAKLREQFR-NKTIIFTQSS
S4V4-2          AHCNISKTKWNNTLKLIVNKLREQFG-NKTIIFNQSS
S11V4-5         AHCNVSRGQWNKTLEQVVRKLREQYGPNKTIVFKQPI
S3V6-6          AHCNLSRAGWNSTLERIAIKLREQFQ-NKTIAFNQSS
                *:**:*   *:.**  :. ****::  * ** *.:. 
Alignment of Control Subject 6 Sequence to Post-AIDS Sequences from all Subjects
S6V9-3        EVVIRSANLTDNAKIIIVHLNESVEMNCTRPNN-NTRKGIHIGPGRAFYATGEIIGNIRQ
S11V4-5       EVIIRSENFSNNAKNIIVQLNESVVINCTRPDNTIKQRIIHIGPGRPFYTT-GIKGNIRQ
S1V5-4        EVVIRSENITNNAKIIIVQLNESVAISCTRPNNNIKQRIMHIRPGRAFYTK-DITEDIRQ
S4V4-2        EVVIRSENFTNNAKIIIVQLNESVEINCTRPDN-HTVRKIPIGPGRSFYTT-GIVGDIRQ
S15V4-7       EVVIRSENFTNNAKIIIVHLNESVVINCTRPNN-NTRRKIHIGPGKTFYTG-DIIGNIRQ
S10V6-1       EVVIRSENFTDNAKTIIVHLNKSVEINCTRPNN-NTRRSINMGPGRAFYATGEIIGDIRQ
S3V6-6        DIVIRSANFSDNAKTILVQLNETVVMNCTRPGN-NTRKRVTLGPGRVYYTTGQIIGDIRK
              :::*** *:::*** *:*:**::* :.****.*  . : : : **: :*:   *  :**: 

S6V9-3        AHCNLSRAPWNDTLKRIAIKLREQFK-NKTIAFNQSS
S11V4-5       AHCNVSRGQWNKTLEQVVRKLREQYGPNKTIVFKQPI
S1V5-4        AYCNISRTAWNNTLKQIVKKLREHF-VNKTIVFNHSS
S4V4-2        AHCNISKTKWNNTLKLIVNKLREQFG-NKTIIFNQSS
S15V4-7       AHCNISGSKWNNTLKQIVNKLREQFG-NKTIVFNQSS
S10V6-1       AHCNLSRTKWNDTLKQVVAKLREQFR-NKTIIFTQSS
S3V6-6        AHCNLSRAGWNSTLERIAIKLREQFQ-NKTIAFNQSS
              *:**:*   **.**: :. ****::  **** *.:. 
    • While all the rapid progressors showed only a difference of 1 to 17 aa difference between their first and last visits, showing rather good consensus within a certain subject.
    • When compared to each other the Control Subjects 6 and 13 showed moderate conservation with a deviation of only 28 aa out of the 95.
    • Comparing all the rapid progressor AIDS sequences to each other, only get ~40 aa that are completely conserved across the entire region.
    • When comparing all rapid progressor AIDS sequences to each control sequence separately, as seen above, there is not a large difference in conservation seen. This indicates to me that the changes in the V3 region do not seem to help or hinder the pathogenicity of the gp120 protein on the virion coat.
    • The sequences that are conserved when comparing across all the rapid progressors with AIDS are conserved across practically all sequences, both before and after AIDS and on the control subjects
    • We decided not to use the short V3 region sequences from the Stanfield papers due to the fact that the Markham sequences are 95 aa long compared to 12-15 aa for the Stanfield papers so they are not long enough to do a good comparison of conservation.
  1. 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?
  2. 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 interpretation 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.


POWERPOINT

Updated ppt with work from Alex and Zeb

updated ppt in .ppt form

Slides from Svicher article

Final powerpoint

Links for Biol 368

Biol 368 Homepage

Zeb Russo's Homepage

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Assignment Pages

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BIOL368/F11:Week 8

BIOL368/F11:Week 9

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