Download Examination of Amino Acid Differences as a Means

Examination of Amino Acid Differences as
a Means of Determining Functional
Changes in HIV-1 Protein Sequences
Chris Rhodes and Isaiah Castaneda
Loyola Marymount University
Department of Biology
BIOL 368 11/2/11
Outline
• Amino acid sequence is related to protein function through
structure and sidechain interactions
• Previous experiments indicate possible links between
phylogenetic trees and Subjects’ immune response pattern
• Conserved residue changes between individual visits indicate
potential functional differences in the protein sequences
• Observed functional differences between visits agree with
previous conclusions. Subject 7 shows a Best Fit immune
response and Subject 13 shows a Broad immune response
• According to Ogert et al. (2009) certain residue changes can
result in immune cell resistance to infection by HIV-1 strains
Protein function is dependent on numerous
structural effects of the amino acid sequence
• Protein structure and therefore function is affected by:
• Amino Acid Type
– Acidic, Basic, Polar Uncharged, or Nonpolar
• Side Chain Interactions
– Hydrophillic/Hydrophobic Interactions
– Ionic Bonding
– Cysteine Disulfide Bonding
• Significant changes in the amino acid sequence will have an
effect on the function of the protein
Our previous study shows a link between patterns of
phylogenetic trees and patterns of Subjects’ immune response
• Based on interpretations of the phylogenetic
trees of the Subjects’ viral clones the pattern
of each Subjects’ immune response was
hypothesized
• Subject 13 showed a Broad immune
response:
• Low Diversity and Low Divergence
• Little Branching
• Strong Selection Against Change
• Subject 7 showed a Best Fit immune
response
• High Diversity and High Divergence
• One Prominent Branch
• Strong Selection Against Change from
Best Fit Sequence
• Phylogenetic trees of amino acid sequences
Experimental sequences were chosen based on
phylogenetic trees of amino acid sequences
• Major divergence of amino acid
sequences indicates possible functional
differences between sequences
• Sequences were chosen in order to
maximize possible functional differences
and to be relevant to the previous study
• The chosen sequences for Subject 7 are:
• All visit 3 sequences
• Visit 4 sequences 1, 2, 3, 4, and 6
• Visit 5 sequences 1, 3, 6, 7, 8, and 9
Experimental sequences were chosen based on
phylogenetic trees of amino acid sequences
• Divergence of amino acid sequences
indicates possible functional differences
between sequences
• Subject 13 is hypothesized to show
strong selection against change in
function
• Due to the divergence and disappearance
of the Visit 4 sequences it can be
assumed that Visit 4 may constitute a
functional change
• The Visit 5 sequences were chosen to act
as a control representing the root
sequences
• The chosen sequences for Subject 13 are:
• All of Visit 4 sequences
• All of Visit 5 sequences
Outline
• Amino acid sequence is related to protein function through
structure and sidechain interactions
• Previous experiments indicate possible links between
phylogenetic trees and Subjects’ immune response pattern
• Conserved residue changes between individual visits indicate
potential functional differences in the protein sequences
• Observed functional differences between visits agree with
previous conclusions. Subject 7 shows a Best Fit immune
response and Subject 13 shows a Broad immune response
• According to Ogert et al. (2009) certain residue changes can
result in immune cell resistance to infection by HIV-1 strains
A comparison of Subject 13’s Visit 4 and 5 amino acid
sequences shows two major conserved changes
• Conserved changes are residue
changes the are consistent
throughout all the sequences of a visit
• Conserved changes are most likely to
indicate functional differences
between different visits
• Between Visits 4 and 5 there are two
conserved residue changes:
1. GR
2. RG
RG and GR changes constitute
major structural differences
Arginine
(R)
Glycine (G)
• Arginine contains a large, hydrophilic, and positively charged side
chain
• Glycine contains a very small, hydrophobic, and uncharged side
chain
• The difference between the two amino acids would have a major
effect on protein structure and therefore function
A comparison of the selected Subject 7 amino acid
sequences shows multiple conserved changes
•Between the selected sequences there are a total of eleven conserved
changes
•Of these eleven conserved changes nine of them are major changes
and constitute potential functional change in the protein
Multiple conserved residue changes of Subject
7’s alignment show functional significance
•
•
•
Visit 4 Conserved Changes:
1. LP: Proline is a helix breaker and is inflexible compared to Leucine
2. AT: Hydrophobic Nonpolar to Hydrophillic Polar Uncharged
3. KN: Hydrophillic Positive Charge to Hydrophillic Polar Uncharged
4. SP: Small Hydrophillic Polar Uncharged to Hydrophobic Nonpolar
5. ND: Hydrophillic Polar Uncharged to Hydrophillic Negative Charge
6. KQ: Hydrophillic Positive Charge to Hydrophillic Polar Uncharged
7. EG: Polar Negatively Charged to Nonpolar Hydrophobic
Visit 5 Conserved Changes
1. VI: Hydrophobic Nonpolar to Hydrophobic Nonpolar
– No functional change
2. SP: Small Hydrophillic Polar Uncharged to Hydrophobic Nonpolar
Visit 3 Conserved Changes
1. TS: Hydrophillic Polar Uncharged to Hydrophillic Polar Uncharged
– No functional change
2. AT: Hydrophobic Nonpolar to Hydrophillic Polar Uncharged
Outline
• Amino acid sequence is related to protein function through
structure and sidechain interactions
• Previous experiments indicate possible links between
phylogenetic trees and Subjects’ immune response pattern
• Conserved residue changes between individual visits indicate
potential functional differences in the protein sequences
• Observed functional differences between visits agree with
previous conclusions. Subject 7 shows a Best Fit immune
response and Subject 13 shows a Broad immune response
• According to Ogert et al. (2009) certain residue changes can
result in immune cell resistance to infection by HIV-1 strains
Conserved changes in Subject 13’s amino acid sequences
indicate functional differences between Visits consistent
with a Broad immune response pattern
• Based on the RG and GR changes between the Visit 4 and 5
sequences it is likely that the sequences of Visit 4 have a different
function than those of Visit 5
• The disappearance of Visit 4 in the phylogenetic tree could thus be
explained by the functional difference between Visits 4 and 5
• This agrees with the previous findings that Subject 13 uses a Broad
immune response pattern that is selective against functional
change
• Since the Visit 4 sequences represent a change in function from the
Visit 5 sequences they were eradicated by the immune system
Conserved changes in Subject 7’s amino acid sequences
indicate functional differences between Visits consistent
with a Best Fit immune response pattern
• The multiple conserved changes between the Visit 4 sequences and
the other two Visits indicate Visit 4 has a drastically different
function than both the Visit 3 and Visit 5 sequences
• This agrees with the previous findings that Subject 7 uses a Best Fit
immune response where Visit 4 represents the best fit sequence
• Since the Visit 3 sequences had such a divergent function from the
Visit 4 sequences they were wiped out by the immune system
which is selective against drastic change from the best fit sequence
• Based on this is it likely we would also see a disappearance of the
divergent Visit 5 clones in a hypothetical Visit 6 sample
Outline
• Amino acid sequence is related to protein function through
structure and sidechain interactions
• Previous experiments indicate possible links between
phylogenetic trees and Subjects’ immune response pattern
• Conserved residue changes between individual visits indicate
potential functional differences in the protein sequences
• Observed functional differences between visits agree with
previous conclusions. Subject 7 shows a Best Fit immune
response and Subject 13 shows a Broad immune response
• According to Ogert et al. (2009) certain residue changes can
result in immune cell resistance to infection by HIV-1 strains
Research by Ogert et al. (2009) indicates that specific
amino acid changes in the V3 loop corresponds to viral
infectivity potential
• The function of the V3 loop can be affected by six specific
amino acid changes
• When all six changes are present in the V3 sequence the virus
cannot infect CD4 cells and replicate
• These findings show the link between amino acid residue
change and viral functionality
• It is possible that minute changes in the amino acid sequence
can render a protein non-functional
• For our experiment we assume all proteins are functional but
it is possible that this is not the case
• If not, the disappearance of certain visits may then be due to
mutations causing non-functionality
Summary
• Previous research shows a link between phylogentic patterns
and Subject immune response patterns
• Changes in the amino acid sequence of a protein can affect
functionality
• When comparing amino acid sequences conserved changes
can be considered to be functionally relevant
• Functional differences between observed sequences confirm
previous hypothesis about Subjects’ immune response type
• Subject 7 shows a Best Fit immune response pattern
• Subject 13 shows a Broad immune response pattern
Acknowledgements
Kam D. Dahlquist, Ph. D
Ogert, Robert, Yan Hou, Lei Ba, Lisa Wojcik, Ping Qiu, Nicholas Murgolo, Jose
Duca, Lisa Dunkle, Robert Ralston, and John Howe. "Clinical resistance to
vicriviroc through adaptive V3 loop mutations in HIV-1 subtype D gp120
that alter interactions with the N-terminus and ECL2 of CCR5." Virology.
400.1 (2009): 145-55.
Markham, Richard, Wei-Cinn Wang, Anton Weisstein, Zhe Wang, Alvaro
Munoz, Alan Templeton, Joseph Margolick, David Vlahov, Homayoon
Farzadegan, AND Xiao-Fang Yu. "Patterns of HIV-1 evolution in individuals
with differing rates of." Medical Sciences. 95. (1998): 12568–12573.