Download Will there be specific differences between the

Analyzing Differences in Protein
Sequences Between Subjects with
Varying T Cell Counts
J’aime Moehlman
Amanda Wavrin
Loyola Marymount University
March 22, 2010
Outline
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Introduction
Materials & Methods
Results
Discussion
Further Research
References
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
•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.
Subject’s 10 and 12 were selected based on
their Annual Rate of CD4 T cell decline
The Phylip’s Drawtree shows that Subject 10
and 12 are not closely related.
BOXSHADE Sequences for our Representative
Visits
Materials & Methods
• Ran multiple sequence alignments for all
clones from visits 4 and 5.
• Used the Boxshade tool to show the locations
of the amino acid differences.
• Used the PSIPRED tool to generate the
secondary structures.
• Analyzed and compared 3-D structures.
Predicting the Secondary Structure of Subject
10’s Protein Sequence Using PSIPRED
H: Helical
E: Extended
C: Random Coil
Conf:947814567777306999858867895368998735651458871002107441055763
Pred:CEEEECCCCCCCCEEEEEECCCCEEEECCCCCCCCCCCCCCCCCCEEEECCCEECHHHHH
AA: EVVIRSENFTDNAKTIIVQLNKAVEINCTRPNNNTRRRISMGPGRVLYTTGEIIGDIRQA
10
20
30
40
50
60
Conf: 20547877765799999999976189447760589
Pred: HHCCCHHHHHHHHHHHHHHHHHHHCCCEEEEECCC
AA: HCNLSRTKWNDTLKQVVDKLREQFRNKTIIFNQSS
70
80
90
Graphical Representation of the Secondary
Structure of Subject 10’s Protein Sequence
Predicting the Secondary Structure of Subject
12’s Protein Sequence Using PSIPRED
H: Helical
E: Extended
C: Random Coil
Conf:94782456777730699985885789636899874565045887100210733105676
Pred:CEEEECCCCCCCCEEEEEECCCCEEEECCCCCCCCCCCCCCCCCCHHEECCCEECHHHH
AA:EVVIRSKNFTDNAKIIIVQLNETVEINCTRPNNNTRKSIPIGPGRAFYTTGEIIGDIRQA
10
20
30
40
50
60
Conf: 10557878765799999999976189447750689
Pred: HHCCCHHHHHHHHHHHHHHHHHHHCCCEEEECCCC
AA: HCNLSGAKWNETLKQIVIKLKEQFRNKTIVFSPSS
70 80 90
Graphical Representation of the Secondary
Structure of Subject 12’s Protein Sequence
Locating the V3 Loop of the gp120
Protein
EVVIRSVNFTDNAKTTIIVQLNTSVEINCTGAGHCNISRAKWNNTLKQIA
SKLREQFGNNKTIIFKQSSGGDPEIVTHSFNCGGEFFYCNSTQLFNS
• By using the Kwong et. al
article, we were able to
identify the V3 region of the
gp120 protein using the two
representative clones.
The V3 Loop of the gp120 Protein
Boxshade of Subjects 10 and 12 to find
differences within their sequences
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.
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.
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.
Position
Subject 10
Subject12
V3 Loop
15
T:Threonine, polar
I:Isoleucine,
nonploarhydrophobic
T:Threonine, polar
38
R:Arginine, basic
S:Serine, polar
R:Arginine, basic
66
R:Arginine, basic
G:Glycine,
hydrophobic
Q:Glutamine,
polar
78
D:Aspartic acid,
acidic
I:Isoleucine,
nonpolarhydrophobic
S: Serine,
polar
93
Q:Glutamine, polar
P:Proline,
hydrophobic
L: Leucine,
hydrophobic
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.
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) 12568- 73.
• 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) 648-59.
• 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. 2009 Nov 20;326(5956):11237.