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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
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
• The functions of the gp120 protein are influenced by
the structure within the V3 region.
•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 in
the protein structures between subjects
10 and 12.
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
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,
hydrophilic
I:Isoleucine,
nonpolarhydrophobic
T: Threonine,
polar, hydrophilic
38
R:Arginine, basic,
polar, hydrophilic
S:Serine, polar,
(uncharged),
hydrophilic
R:Arginine, basic,
polar, hydrophilic
66
R:Arginine, basic,
polar, hydrophilic
G:Glycine,
hydrophobic
Q: Glutamine,
polar, hydrophilic
78
D: Aspartic acid,
acidic, polar,
hydrophilic
I:Isoleucine,
nonpolarhydrophobic
S: Serine, Polar,
(uncharged),
hydrophilic
93
Q: Glutamine,
polar, hydrophilic
P:Proline,
hydrophobic
L: Leucine,
hydrophobic
Position
Subject 10
Subject12
V3 Loop
15
T:Threonine,
polar, hydrophilic
I:Isoleucine,
nonpolarhydrophobic
T:Threonine,
polar, hydrophilic
•The closest side chain on
the blue domain is a
glutamine, which is polar.
•Between the two subjects
there is a change between
a hydrophobic and
hydrophilic amino acid.
Position
Subject 10
Subject12
V3 Loop
38
R: Arginine, basic,
polar, hydrophilic
S: Serine, polar,
hydrophilic
R: Arginine, basic,
polar, hydrophilic
•Arginine is greater in
size than Serine.
•They are all hydrophilic
amino acids.
Position
Subject 10
Subject12
V3 Loop
66
R:Arginine, basic,
polar, hydrophilic
G:Glycine,
hydrophobic
Q:Glutamine,
polar, hydrophilic
•In subjects 10 and 12;
the amino acid changes
from hydrophilic to
hydrophobic causing a
potential structural
difference.
•Glutamine is larger in
size than Glycine, but is
closer in size to
Arginine.
Position
Subject 10
Subject12
V3 Loop
78
D: Aspartic acid,
acidic, polar,
hydrophilic
I: Isoleucine,
nonpolar,
hydrophobic
S: Serine,
Polar, hydrophilic
•There is a difference
between nonpolar and
polar properties.
•Isoleucine is larger in
size than the other two
amino acids.
•Within the two subjects
there is a change based
off of hydrophobic and
hydrophilic properties
which has the potential
to cause a structural
difference.
Position
Subject 10
Subject12
V3 Loop
93
Q:Glutamine, polar,
hydrophilic
P:Proline,
hydrophobic
L: Leucine,
hydrophobic
•Both Proline and
Leucine are hydrophobic
and Glutamine is
hydrophilic, which can
cause a change in the
structure.
•Structurally, Leucine
and Glutamine are
similar, while Proline has
a cyclical structure.
There will be specific amino acids that cause
differences in the protein structures of subjects
10 and 12
• Based off of our results, we accept our hypothesis for the
tertiary structures of the proteins.
• There were specific amino acid changes between the
subjects, but they did not result in predicted secondary
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.
Opportunities for Further Research
• Analyze an individual subjects amino acid
sequence and protein structure to potentially
create a neutralizing antibody for the HIV-1 virus.
• 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
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.