- Kwong PD, Wyatt R, Robinson J, Sweet RW, Sodroski J, and Hendrickson WA. . pmid:9641677.
- Stanfield R, Cabezas E, Satterthwait A, Stura E, Profy A, and Wilson I. . pmid:10368281.
- Stanfield RL, Ghiara JB, Ollmann Saphire E, Profy AT, and Wilson IA. . pmid:14592768.
Working with Protein Sequences In-class Activity
- This week we will begin to learn how to analyze protein structures. For today, we will be using the Bioinformatics for Dummies book extensively, so be sure to bring it to class. We will be using some bioinformatics tools to analyze the structure of the gp120 envelope protein.
- Chapter 2: Retrieving Protein Sequences/Retrieving a list of Related protein sequences (pp. 42-51 in second edition). The example worked through in the book uses the sequence of an enzyme called dUTPase. Follow the book example yourself and then work through the example again, this time using the HIV gp120 envelope protein instead.
- Chapter 4: Reading a SWISS-PROT entry (pp. 110-123 in the second edition). The example worked through in the book is the epidermal growth factor receptor. Work through this example and then do it again with the HIV gp120 envelope protein instead.
- Chapter 5: ORFing your DNA sequence (pp. 146-147 in second edition). In the previous section of the course, we were working with DNA sequences from the HIV gp120 envelope protein. Take one of your DNA sequences and follow the instructions to find the open reading frames in the sequence. Since you were working with just a portion of the entire envelope protein, you may get some strange results. Compare your results with the SWISS-PROT entry you found for the protein above to decipher what the output means. Besides the NCBI Open Reading Frame Finder described in the book, ExPASy also has a translation tool you can use, found here.
- Chapter 6: Working with a single protein sequence (pp. 159-195 in second edition). Work through the following examples in this chapter using the entire HIV gp120 envelop protein sequence that you obtained from SWISS-PROT. We will then compare the results of these analyses with the actual structure of the gp120 protein obtained by X-ray crystallography.
- Looking for transmembrane segments
Preparation for Week 8 Journal Club
The class will be divided into groups of 3 or 4. Each group will create and present a Journal Club (in class in Week 8) on the Kwong et al. (1998), Stanfield et al. (1999), or Stanfield et al. (2003) articles. The groups will be:
- Kwong et al. (1998):
- Stanfield et al. (1999):
- Stanfield et al. (2003):
In preparation for the Journal Club, each individual will do the following assignment on their individual Week 7 Journal page.
- Make a list of at least 10 biological terms for which you did not know the definitions when you first read the article. Define each of the terms. You can use the glossary in any molecular biology, cell biology, or genetics text book as a source for definitions, or you can use one of many available online biological dictionaries (links below). List the citation(s) for the dictionary(s) you use, providing a URL to the page is fine.
- Write an outline of the article. The length should be the equivalent of 2 pages of standard 8 1/2 by 11 inch paper. Your outline can be in any form you choose, but you should utilize the wiki syntax of headers and either numbered or bulleted lists to create it. The text of the outline does not have to be complete sentences, but it should answer the questions listed below and have enough information so that others can follow it. However, your outline should be in YOUR OWN WORDS, not copied straight from the article.
- What is the main result presented in this paper? (Hint: look at the last sentence of the introduction and restate it in plain English.)
- What is the importance or significance of this work?
- What were the limitations in previous studies that led them to perform this work?
- What were the methods used in the study?
- Briefly state the result shown in each of the figures and tables.
- How do the results of this study compare to the results of previous studies (See Discussion).
- Journal Club presentations in class.
HIV Structure Research Project
Today you will begin your HIV gp120 Structure Research Project.
- For this project, you can choose to work with the same sequences you used for the HIV Evolution Project, or you may choose different sequences. You will reframe your question from the HIV Evolution Project to make it a structure→function question. Instead of looking at how the evolution of variation of the viral DNA sequence affects the different patient groups, you will look at how variations in the viral sequence affect the structure and, therefore, function of the virus. To answer your question, you will need to do the following:
- Convert your DNA sequences into protein sequences.
- How will you do this?
- How will you know that it was done correctly?
- Perform a multiple sequence alignment on the protein sequences.
- Are there more or fewer differences between the sequences when you look at the DNA sequences versus the protein sequences?
- How do you account for this?
- Which of the procedures from Chapter 6 that you ran on the entire gp120 sequence are applicable to the V3 fragment you are working with now?
- Chapter 11 contains procedures to use for working with protein 3D structures. Find the section on "Predicting the Secondary Structure of a Protein Sequence" and perform this on both the entire gp120 sequence and on the V3 fragment that we are now working with. You will compare the predictions with the actual structures.
- Download the structure files for the papers we read in journal club from the NCBI Structure Database. These files can be opened with the Cn3D software site that is installed on the computers in the lab (this software is free, so you can download it and use it at home, too.) Familiarize yourself with the software features (rendering and coloring) with both the gp120 peptide and ternary complex structures. (The Dummies book has some information on this program as well). Answer the following:
- Find the N-terminus and C-terminus of each (poly)peptide structure.
- Locate all the secondary structure elements. Do these match the predictions you made above?
- Locate the V3 region and figure out which sequences from your alignment are present in the structures and which sequences are absent.
- Once you have oriented yourself, analyze whether the amino acid changes that you see in the multiple sequence alignment would affect the 3D structure and explain why you think this.
- The journal club papers we read are quite old already for a fast-moving field. Using the Web of Science (or PubMed or Structure) databases, find at least one more recent publication that has a structure of gp120 (V3) in it and download the structure file to view. What additional information has been learned from this new paper?
- Your presentation will be formatted similarly to the previous HIV Evolution Project. In this case, you will want to work on creating structure figures that illustrate what result you are trying to show.
- Project work session
- Prepare for Project presentations in Week 10
- Project presentations in class.