Courtney L. Merriam Week 9

Purpose

This experiment was designed to foster my ability to develop a research question and then conduct proper research, in this instance based around specific amino acid sequences in depth to develop a better understanding of how they function, relate, differ, and interact.

Methods and Results

Defining Your HIV Structure Research Project

What is your question?
- Is there a correlation between amino acids mutation from the first to the last visit and CD4 cell count from the first to the last visit for each subject selected?
Make a prediction about the answer to your question before you begin your analysis.
- The more mutations seen in the amino acids between the first and the last visit will result in a lower CD4 cell count.
Which subjects, visits, and clones will you use to answer your question?
- The first and the last visit for all the clones of subjects 1-3
Justify why you chose the subjects, visits, and clones you did.
- What to look at the differences between the beginning of the study and the end of the study and how the mutations changed or progressed over the two years.

HIV Structure In-Class Activity

Convert your DNA sequences into protein sequence using NCBI Open Reading Frame Finder
- How do you know which of the six frames is the correct reading frame?
  - Frame 3 would be the true reading frame because it is the only one with no stop codon. We know we do not have the entire DNA sequence so we know we will not see a start codon and we know that the stop codon should come later so the correct ORF would be the one with no stop codon.
- Once you have done one example, you can obtain the rest of the protein sequences from the BEDROCK HIV Problem Space.
Find out what is already known about the HIV gp120 envelope protein in the UniProt Knowledgeable (UniProt KB) .
1. If you search on the keywords "HIV" and "gp120", in the main UniProt search field, how many results do you get?
  - 723,401 for HIV and 207,983 for gp120
2. Use the entry with accession number "P04578" which corresponds to the reference entry for HIV gp120.
3. What types of information are provided about this protein in this database entry?
  - Function
  - Names and taxonomy
  - subcellular location
  - Pathology and biotech
  - PTM/processing
  - Interactions
  - Structure
  - Family and domains
  - Sequence
  - Cross-references
  - Entry information
  - Similar proteins
We are going to use the PredictProtein server to analyze just the V3 region from Markham et al. (1998).
- Paste one of the amino acid sequences from Markham et al. (1998) into the input field and submit.
- Explore the types of information provided. How does this information relate to what is stored in the UniProt database?
  - It seems like there is way more information, research findings, and visual aids on this rather than the UniProt database.
  - Amino Acid Composition, sequence length
  - Secondary structure and solvent accessibility
  - Transmembrane helices
  - Protein Disorder and flexibility
  - Disulphide bridges
  - Effects of point mutations
  - Gene ontology terms
  - Subsellular localization
  - Binding sites
Download the structure file for the paper we read in journal club from the NCBI Structure Database
The files can be opened with the Cn3D software site. Answer the following:
1. Find the N-terminus and C-terminus of each polypeptide tertiary structure.
2. Locate all the secondary structure elements. Do these match the predictions made above?
3. Locate the V3 region and figure out the location of the Markham et al. (1998) sequences in the structure.

Data and Files

No files or data was used for this assignment.

Conclusion

Before beginning the actual lab, our first task was to develop a research question, essentially, clarifying exactly what we want to know, and then from the question, creating a purpose, prediction, and then research methods for determine if our prediction was true. The research question was based around how the HIV virus evolves and changes with different amino acid sequences. Once this was developed, I began the class activity that required the translation of various DNA sequences. From doing this I learned how to use the exPASY tool for DNA and amino acid translation. After using Unipot Knowledgeable, I moved to PredictProtein.org to further analyze the amino acid sequences, which may be helpful in further experiments and projects.

Acknowledgments

I collaborated with Shivum A Desai in class on this assignment. While I worked with the people noted above, this individual journal entry was completed by me and not copied from another source.

References

Markham, R.B., Wang, W.C., Weisstein, A.E., Wang, Z., Munoz, A., Templeton, A., Margolick, J., Vlahov, D., Quinn, T., Farzadegan, H., & Yu, X.F. (1998). Patterns of HIV-1 evolution in individuals with differing rates of CD4 T cell decline. Proc Natl Acad Sci U S A. 95, 12568-12573. doi: 10.1073/pnas.95.21.12568 Nguyen, S., Ramos, A., Chang, J., Li, B., Shanmugam, V., Boeras, D., ... & Ellenberger, D. (2015). Monitoring the quality of HIV-1 viral load testing through a proficiency testing program using dried tube specimens in resource-limited settings. Journal of clinical microbiology, 53(4), 1129-1136. Dos: 10.1128/JCM.02780-14

NCBI Open Reading Frame Finder

BEDROCK HIV Problem Space

UniProt Knowledgebase (UniProt KB)

PredictProtein server

NCBI Structure Database

Cn3D software site