How does HIV status (diagnosed, progressing or non-trending) affect the structure of the V3 protein region?
Hypothesis
We hypothesize that diagnosed groups will express greater variability in the V3 region in their protein structure, in comparison to the non-trending groups. Initial comparisons show that diagnosed groups and progressing groups expressed greater genetic variability than non-trending groups. These changes may affect the third variable region, affecting the host's ability to adapt to the changes and generate sufficient immune response.
Subject Data
According to the BEDROCK HIV Sequence Data Table, I was able to determine which of the subjects used within my study actually developed aids. All 3 AIDS diagnosed were confirmed with the disease by their final visit. In the AIDS progressing groups, subjects developed AIDS within 1 year after their final visit. The Non-Trending groups all maintained high CD4 T Cell Counts above the threshold, even after the study was conducted. Sequences were for each visit and subject were chosen using a Random Integer Generator, to eliminate selection bias.
The following sequences was taken from the BEDROCK HIV Problem Space Database, from the Markham et al. (1998) study. Table 1: Sequences analyzed
Group
Subject
Visit
Sequences
AIDS Diagnosed
3
10
15
1 6
1 6
1 4
1, 2, 4 3, 4, 5
3, 6, 7 2, 4, 8
2, 3, 4 5, 8, 10
AIDS Progressing
7
8
14
1 5
1 7
1 9
2, 3, 9 2, 8, 9
1, 4, 5 1, 6, 7
2, 3, 4 9, 10, 11
No Trend
5
6
13
1 5
1 9
1 5
1, 3, 8 4, 5, 2
1, 2, 3 6, 7, 9
1, 3, 4 3, 5, 4
Working with Protein Sequences In-class Activity
Chapter 4: Reading a SWISS-PROT entry
Source: Bioinformatics for Dummies pp.110-123
Using the database UniProt KB which has two subsections: Swis-Prot and TrEMBL.
If you search on the keywords "HIV" and "gp120", how many results do you get?
180,227 results.
General Information about the entry
Entry name: 9HIV1
Primary Accession Number :Q75760
Secondary Accession Numbers :N/A
Intergrated into Swiss-Prot on :November 1, 1996
Sequence was last modified on : November 1, 1996
Annotations were last modified on : October 1, 2014
Fasta format of Envelope glycoprotein gp160 obtained from UniProt KB was placed in the in the imput box of NCBI Open Reading Frame Finder . Six parallel horizontal bars were present with integers correlating to the reading frame.
Putting in DNA sequences: Subject 1 visit 1, clone 1:
The protein with the open reading frame was in the ORF Finder database was projected to be #2, or #3. Looking at the exPASy database 5’3” Frame 3 had an open reading frame with no stop codons interrupting /truncating the lengh of the DNA.
Chapter 6:Working with a single protein sequence
Source: Bioinformatics for Dummies pp.159-195
Predicting the main physico-chemical properties of a protein
Used the program ExPasy ProtParam for computation of physcial and chemical parameters of a given protein
Imput the Swiss-Prot/TrEMBL accession number Q75760. Then click compute parameters button >Submit
Corresponds to the HIV gp120 sequence that was used for the crystal structure for the Huang et al. (2005) paper.
The amino acid scale Hphob. / Kyte & Doolittle was preselected
Changed the Window size to 19 because best window value for viewing trans membrane regions
Covert the image to GIF format
Save file
Interpreting ProtScale results
To confirm use Hphob. / Eisenberg et al.scale, set threshold to 1.6
Piece of paper to over results
lower paper to strongest peaks visible Error creating thumbnail: File missingFigure 4. Identifying transmembrane domains using the TMHMM database. Transmembrane proteins are denoted in red.
Keep lowering threshold as long as you keep seeing sharp peeks
4 Sharp Peaks, 4 Transmembrane domains
Running TMHMM
UseTMHMM Q75760 sequence was inputted in FASTA format>Submit
Changed input sequence format to SwissProtID or AC Error creating thumbnail: File missingFigure 5. Coils output for Q75760, using the coils server at EMBnet. Look at regions between 600-700.
Predicting post-translational modifications in your protein
Slide mouse over color rectangles to see name displayed, click to receive more information
the list of segments contain patterns within protein; numbers indicate position and capital letters are specified and lowercase letters are unspecified by patterns
~be careful with short patterns
Weak signals add up to give strong signals –two related patterns at close distance, significant
InterProScan, imput fasta format of Q75760 sequence>submit
Error creating thumbnail: File missingFigure 7. Domain determined for glycoprotein gp160 using InterProScan when comparing sequences with domain databases.
Used Predict Protein. In order to use program had to make an account. Input amino acid for S1V1-1.
Error creating thumbnail: File missingFigure 10. The results using Predict Protein database for S1V1-1. Red denotes a helix, Blue are portions that are exposed, and yellow means buried
Comparison of crystal structure of gp120
From the NCBI website the Hunag et al. (2005) paper was downloaded via CN3D file.To make it more visible I used a secondary shortcut to make the helices (green) and beta sheets (yellow) so they could be better differentiated and analyzed. The structure of the protein form NCBI website favors a composition favors a composition where there are more beta sheets present with random coil in comparison to the prediction from PsiPred that had them being in equal abundance.
