1. Loyola Marymount University
Analyzing Differences in Protein Sequences Between Subjects with Varying T Cell Counts
J’aime Moehlman
Amanda Wavrin
Loyola Marymount University
March 22, 2010

2. Outline
Introduction
Results
Discussion
Further Research
References

3. Introduction to the HIV-1 Structure
The site on gp120 that binds to the CD4 receptor is vulnerable to neutralizing antibodies.
However, most antibodies that interact with the site cannot neutralize HIV-1.
There are many features that help the gp120 protein escape the immune system of the host such as:
Variable Loops
N- Linked Glycosylation
Confomational Flexibility

4. Our Proposed Question: Will there be specific differences between the protein sequences in subject 10 and 12 that results in different protein structures, which changes the function of the virus?
Hypothesis: There will be specific amino acids that cause differences between the protein sequences in S10 and S12.

5. Subject’s 10 and 12 were selected based on their Annual Rate of CD4 T cell decline

6. The Phylip’s Drawtree shows that Subject 10 and 12 are not closely related.

7. BOXSHADE Sequences for our Representative Visits

8. Predicting the Secondary Structure of Subject 10’s Protein Sequence Using PSIPRED
H: Helical
E: Extended
C: Random Coil
Conf:
Pred:CEEEECCCCCCCCEEEEEECCCCEEEECCCCCCCCCCCCCCCCCCEEEECCCEECHHHHH
AA: EVVIRSENFTDNAKTIIVQLNKAVEINCTRPNNNTRRRISMGPGRVLYTTGEIIGDIRQA
Conf:
Pred: HHCCCHHHHHHHHHHHHHHHHHHHCCCEEEEECCC
AA: HCNLSRTKWNDTLKQVVDKLREQFRNKTIIFNQSS

9. Graphical Representation of the Secondary Structure of Subject 10’s Protein Sequence

10. Predicting the Secondary Structure of Subject 12’s Protein Sequence Using PSIPRED
H: Helical
E: Extended
C: Random Coil
Conf:
Pred:CEEEECCCCCCCCEEEEEECCCCEEEECCCCCCCCCCCCCCCCCCHHEECCCEECHHHH
AA:EVVIRSKNFTDNAKIIIVQLNETVEINCTRPNNNTRKSIPIGPGRAFYTTGEIIGDIRQA
Conf:
Pred: HHCCCHHHHHHHHHHHHHHHHHHHCCCEEEECCCC
AA: HCNLSGAKWNETLKQIVIKLKEQFRNKTIVFSPSS

11. Graphical Representation of the Secondary Structure of Subject 12’s Protein Sequence

12. Locating the V3 Loop of the gp120 Protein
EVVIRSVNFTDNAKTTIIVQLNTSVEINCTGAGHCNISRAKWNNTLKQIASKLREQFGNNKTIIFKQSSGGDPEIVTHSFNCGGEFFYCNSTQLFNS
By using the Kwong et. al article, we were able to identify the V3 region of the gp120 protein using the two representative clones.

13. The V3 Loop of the gp120 Protein

14. Boxshade of Subjects 10 and 12 to find differences within their sequences

15. Compared Our Secondary Structure’s with the Kwong et al. 3-D Structure
In the Kwong Structure:
At location 15 there is a threonine that resulted in an extended sheet.
At location 38 there is an arginine that resulted in a helical structure.
At location 66 there is a glutamine that resulted in an extended sheet.
At location 78 there is a serine that resulted in an extended sheet.
At location 93 there is a leucine that resulted in a random coil.

16. Comparison Continued with Subject 10
In our secondary structures we found:
Subject 10:
At amino acid 15, the predicted structure, an extended sheet, was found
At amino acid 38, the predicted structure was a random coil, which is not consistent with the actual structure.
At amino acid 66, the predicted structure was helical, which is not consistent with the actual structure.
At amino acid 78, the predicted structure was helical, which is not consistent with the actual structure.
At amino acid 93, the predicted structure, a random coil, was found.

17. Comparison Continued with Subject 12
In our secondary structures we found:
Subject 12:
At amino acid 15, the predicted structure, an extended sheet, was found.
At amino acid 38, the predicted structure was a random coil, which is not consistent with the actual structure.
At amino acid 66, the predicted structure was helical, which is not consistent with the actual structure.
At amino acid 78, the predicted structure was helical, which is not consistent with the actual structure.
At amino acid 93, the predicted structure, a random coil, was found.

18. Subject 10 Subject12 V3 Loop 15 38 66 78 93 Position
T:Threonine, polar
I:Isoleucine, nonpolar-hydrophobic 38
R:Arginine, basic, polar, hydrophilic
S:Serine, polar,
(uncharged), hydrophilic 66
G:Glycine, hydrophobic
Q:Glutamine, polar 78
D:Aspartic acid, acidic
S: Serine, Polar, 93
P:Proline, hydrophobic
L: Leucine, hydrophobic

19. I:Isoleucine, nonploar-hydrophobic
Position
Subject 10
Subject12
V3 Loop 15
T:Threonine, polar
I:Isoleucine, nonploar-hydrophobic
The closest side chain on the blue domain is a glutamine, which is polar.

20. R:Arginine, basic, hydrophilic
Position
Subject 10
Subject12
V3 Loop 38
R:Arginine, basic
S:Serine, polar
R:Arginine, basic, hydrophilic
Serine is a lot smaller than arginine.

21. G:Glycine, hydrophobic
Position
Subject 10
Subject12
V3 Loop 66
R:Arginine, basic
G:Glycine, hydrophobic
Q:Glutamine, polar

22. Subject 10 Subject12 V3 Loop 78 Position D:Aspartic acid, acidic
I:Isoleucine, nonpolar-hydrophobic
S: Serine,
polar

23. Subject 10 Subject12 V3 Loop 93 Position Q:Glutamine, polar
P:Proline, hydrophobic
L: Leucine, hydrophobic

24. Our hypothesis was rejected based on our findings.
There were specific amino acid changes between the subjects, but they did not result in structural changes.
There were differences in the amino acids between our secondary structure and that of the actual V3 structure (from Kwong et al).
This resulted in structural differences between them.

25. References
Markham RB, Wang WC, Weisstein AE, Wang Z, Munoz A, Faradegan H, and Yu XF. Patterns of HIV-1 evolution in individuals with differing rates of CD4 T cell decline. Proc Natl Acad Sci U S A 1998 Oct 13; 95(21)
Kwong PD, Wyatt R, Robinson J, Sweet RW, Sodroski J, and Hedrickson WA. Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody. Nature 1998 Jun 18; 393(6686)
Chen L, Kwon YD, Zhou T, Wu X, O’Dell S, Cavacini L, Hessell AJ, Pancera M, Tang M, Xu L, Yang ZY, Zhang MY, Arthos J, Burton DR, Dimitrov DS, Nabel GJ, Posner MR, Sodroski J, Wyatt R, Mascola JR, Kwong PD. Structural basis of immune evasion at the site of CD4 attachment on HIV-1 gp120. Science Nov 20;326(5956):