Matthew R Allegretti Week 10

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Week 10 Assignment

Purpose

To familiarize ourselves with protein sequencing and analysis tools. To understand how changes in amino acids and therefore changes in protein structure affect the function of gp120.

Methods and Results

The previous steps were performed in Week 9

  1. These structure 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.) Alternately, you may choose to use the Star Biochem program to do this portion of your work. Answer the following:
    • Find the N-terminus and C-terminus of each polypeptide tertiary structure.
      • The C and N termini are highlighted in yellow
      • Matthew R Allegretti Mmdb 1GC1 CNTerminus.PNG
    • Locate all the secondary structure elements. Do these match the predictions made by the PredictProtein server?
      • By comparing PredictProtein data and the structure model, the predictions made do match with the secondary structure at those locations.
    • Locate the V3 region and figure out the location of the Markham et al. (1998) sequences in the structure.
      • Matthew R Allegretti Mmdb 1GC1 Markham Region.PNG
      • The region studied by Markham et. al (1998) is highlighted in yellow.
  2. Perform a multiple sequence alignment on the protein sequences that you and your partner are analyzing for your project using the Biology Workbench.
    • Are there more or fewer differences between the sequences when you look at the DNA sequences versus the protein sequences?
      • There are more differences looking at the DNA than looking at the protein sequences.
    • How do you account for this?
      • Some base pair changes can result in synonymous mutations that do not effect the protein that is coded for within the reading frame.
  3. 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.
    • Instead of using Cn3D or StarBiochem to do this, you could use the program ConSurf.
      • While the region of the structure that would be affected by the changes in amino acids is limited to a 95 amino acid region, I believe that many of these changes would in fact affect the structure of the protein in this region. Many of the amino acid substitutions resulted in drastically different amino acid type or size. Few of the substitutions replaced one amino acid with a different one of a similar size or group.

Data and Files

Conclusion

  • The purpose of this lab was to familiarize ourselves with protein sequencing tools, and to understand how changes in amino acids and therefore changes in protein structure affect the function of gp120. By performing the exercises, I was exposed to a variety of protein sequencing and analyzing tools, and have gained a greater understanding of how to examine the structure of a protein. Through our research project and presentation, we will examine the effects that amino acid substitutions cause within the gp120 protein and how those changes affect its functional success.

Research Project

Subject 4

Visit 1

  • 3 Clones

Visit 4

  • 13 Clones

Subject 9

Visit 1

  • 5 Clones

Visit 4

  • 11 Clones

Subject 11

Visit 1

  • 7 Clones

Visit 4

  • 9 Clones

Subject 14

Visit 1

  • 6 Clones

Visit 4

  • 10 Clones
  • 64 Total Clones

Variability

  • We observed the positions at which amino acid substitutions occurred, and variability can be measured either by:
    1. How many different amino acids are present at each position
    2. How many clones are affected by substitutions at that position

Method 1

  • Single variability: 1 amino acid among all clones, but different from reference sequence
  • Low variability group: 2 different amino acids present at each position among clones
  • High variability: More than 2 different amino acids present at each position among clones
Subject 4:
  • Single variability
    • Positions 275, 283, 289, 301, 305, 308, 339, 343, 346, 358, 361
  • Low variability
    • Positions 290, 299, 300, 302, 311, 313, 314, 321, 322, 324, 333, 335, 337, 345
      • 321 is a technicality as some clone sequences did not have an amino acid for that position
  • High variability
    • 304, 306, 320, 327, 334, 353
      • 320 is a technicality as some clone sequences did not have an amino acid for that position

Data and Files

Acknowledgments

  • Anu Varshneya
    • We communicated electronically and collaborated for our presentation.
  • Will Fuchs and Matthew Oki
    • Collaborated with Will and Matt on our methods of comparing protein sequences.

While I worked with the people noted above, this individual journal entry was completed by me and not copied from another source.

  • Dr. Dahlquist

Methods section pulled heavily from Dr. Dahlquist's Week 10 instructions

References

Useful links

Course Home Page