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Task

Instructions for the Final Take-home Project: Analysis of protein structure/function

1. Step from assignment 1 !!! Identify a protein (minimum polypeptide length of 300 amino acids; shorter sequences are not permitted) from Mouse (mus musculus) (no other species permitted) from PDB (http://www.rcsb.org/) that has a known or predicted 3D structure. You will have to submit your selected protein and at least one protein ID online in the sakai forum listed as “Assignment 1 protein claim”. Provide the full name of your protein, the URL to the PDB of your protein and a very brief (1 paragraph) summary of its cellular function and structural features – in your own words. Do not copy paste the protein description from RCSB and use proper sources. Note that using the wrong species or length of protein will result in a grade of zero. Please ensure you are not using a protein that is not the appropriate length, from the appropriate species, or already claimed by another student. 

Note that many times RCSB will have a fragment of your protein rather than the entire protein. For example “protein A” may be 400 a.a. in length however on RCSB there may be various entries for “protein A” that have only small sections of the protein crystalized (eg: residues 1-80, a binding domain from residue 100-300, etc). The length requirement for the assignment is in reference to the full protein, not the length of the sequence in your pdb file (ie: the fragment). Also note that the RAR/RXR proteins – including all subtypes – are not permitted as they were used in the assignment demonstration. Use Pubmed to investigate the basic structure/function of your chosen protein. Use the paper(s) you’ve found as a guide to carry out an original analysis on how the protein structure influences and determines its function(s). Note that your goal is not to repeat the work that has already been done, but instead use this as a foundation in which to build and continue the structure/function study of your protein. In the report provide background information about your protein and discuss the rationale for the analysis you have performed. Use Deep View or Pymol to illustrate your analysis and conclusions. Prepare a formal report no longer than 9 double spaced pages (excluding references and title page), discussing and illustrating this link.  Further details will be provided in a video lecture.

The paper(s) reference and a brief outline (1/2 page max) of the proposed protein and your planed analysis must be posted to the assignment 2 forum no later than Nov 5 at 5pm (no email, no hardcopies). Proposals which are submitted after this date will not be accepted and a penalty of 10% will be given to the final project.

Note that as with assignment 1 no two students can write on the same protein, even if the intended projects are different or the protein IDs are unique. Students can however use the same protein they used for assignment 1 if they wish to do so, but must claim it in the new forum. Proteins must be claimed on a first come, first serve basis. As with assignment 1, you will be required to claim your protein with a forum post under “assignment 2 protein claim/proposal”. You can claim your protein at any time, and do not need to submit a project outline/proposal until Nov 5 at 5pm, which you can do by adding to your original forum post where you claimed your protein.

The final project is due for submission on Nov 21 by 5pm. Submit a copy of your report to turnitin.com (details to be released in an announcement on Sakai). There is no hard copy, email copy, forum post, etc. which needs to be submitted, only an upload to turnitin.com.  Late penalties of 15% per day will be given for submissions after this deadline. Early submissions of the proposal or final project are welcomed and strongly encouraged. Details as to how to submit the assignment will be released in an announcement on Sakai.

Examples of a potential original analysis:

1. Hypothesize the effect on function of changes in primary amino acid sequence, in the context of disease or in the process of developing novel functions.

2. Modeling of the potential tertiary structure of a protein based on the known structure of similar proteins as for example modeling the structure of an alternative splice variant protein based on the structure of the wild type protein.

3. Analysis of the function of conserved amino acids in a family of homologous proteins.

General outline of report:

Your goal in this report to carry out an original analysis on how the protein structure influences and determines its function(s). This will first require a brief introduction to your protein, what it does and how it works. This should not take more than a page. Importantly, we are interested in the link between structure and function, not the “selling” of the reader on how important your protein is to a cell, in a disease state, etc. In other words we are not looking to have a literature review performed nor a long winded discussion on how the chosen protein is involved in various diseases, cellular functions, etc….we are purely concerned with the structure/function relationship, not about the perceived “importance” of the chosen protein. If you are working with a protein that is altered in a disease state then this is fine to indicate, however the length of this background information should be kept quite minimal.

The task of compiling an original analysis on how the protein structure influences and determines its function(s) is the main focus of this report. In the same line that we are not looking for a literature review or sales pitch towards the importance of your protein, this assignment is also not looking to summarize work that has already been done in the field. The Deep View and/or PyMol programs offers dozens of customizable tools and modifications that can be made to any PDB file and as such nearly anything can be done to the protein of your choice. Rather than show what has been done by others, the goal of this assignment is to get you to use Deep View or PyMol to carry out your own independent analysis. Think of this as being able to custom modify or build, tweak, distort, etc. your protein of interest. Every change you make will have the capacity to impact the function and structure of your protein. The video lecture on the assignment showed and spoke of a number of things you can do to start. Briefly, tools that are readily available to you with little to no practice learning the program are below. These are just a few starting areas you may be interested in looking at…there are dozens more and you are expected to spend some time going through the Deep View or PyMol programs trying out different modifications/tools. You don’t have to use all of these, but the following are good to get you started.

1. change residues to anything you want

2. show charges on sections of the protein (ie: electron clouds)

3. measure distances between key atoms/residues

4. measure angles between key atoms/residues

5. calculate the energy of folding of a protein before/after any modification

6. physically rearrange and change the shape of a protein

7. calculate hydrogen bonds

8. show surface topology

9. show surface charge distribution

10. change the orientation of single residues

The write up of your report will consist of a brief introduction followed by a lab report style “results” section. In this section you will discuss and show with figures a number of changes you have made to the protein. You must be able to illustrate how the change you made has affected the structure of the protein. From there you will then hypothesize what the resulting change in function is within your protein. For example changing a small residue “A” located in a turn between two helices to something larger might lead to a tighter turn between the two helices and cause one helix to swing out of position, expanding an active site area (show this with a figure or two) . From this you could then predict that the widened active site may make the protein more accommodating for a variety of ligands (and offer a few suggestions). Importantly you must be able to support your hypothesis/prediction with hard evidence. Deep View and PyMol can be used to validate your predictions in a number of ways. For example if you are predicting that your active site is enlarged you could provide data for this by measuring distances between key residues known to be important in the active site (from the literature). You could measure the distances before and then after your modification. By doing this you are showing data proving that your active site is now enlarged, adding support to your hypothesis that the protein function may now change. Moreover you could calculate the energy of folding which, as long as it is a negative number of any value, would indicate that your predicted structural change could be actually seen in nature and is properly stabilized (vs something completely unable to fold). This analysis gives support and strength to your predicted change(s) in function and is key in this report. Without this your predictions are simply untested and unsupported opinions. Similarly, if your active site was mutated from a negative core to a positive core then showing charges and/or electron clouds (which are colour coded to show charges) would provide evidence of the change you made. You can use as many figures as you like but they must be able to clearly articulate the point you are trying to make. Images should be cropped so as not to include the deep view or pymol program, just the protein in the viewing window. Use the control & print screen buttons on the keyboard to take screenshots, then crop and modify as needed in paint. The use of colour coding, labels, arrows to highlight key areas, etc. are highly recommended; the reader must be able to clearly understand your figures. Figures must be integrated into the body of your text (rather than put in the back of the report or an appendix) and properly formatted for a formal report (ie figure titles, captions, references to them in text….just like a scientific paper).

Please note that your assignment is entirely open ended; you can perform whatever analysis you would like however I would caution you to think over the changes you are making. Part of the assignment will assess your proficiency with using Deep View or PyMol to perform a number of different modifications to the protein and perform different types of analysis on your changes.  Showing an amino acid change from negative to positive at one residue in an active site is fine. If you then show this for 15 other amino acids, all at different sites, or show hydrophobic to hydrophilic changes, small residue to large residue changes, etc. you have not really demonstrated a good depth of analysis as you have really just performed the same technique many times over and over again. Showing (for example) an amino acid change, then a large scale structural change which leads to modifications of hydrogen bonds and angles/distances between key residues, etc. provides a far more meaningful depth of analysis and offers a more detailed look into structure and function; at least 4 different tools and levels of analysis were used.  You are expected to have at least 3 different experimental approaches to offer this type of multi-faceted analysis. Note that having 3 approaches should be considered a starting point rather than an end point; the quality of what you’ve done with these (at bare minimum) three approaches will determine your grade, not the fact that 3 approaches were used.

Lastly you will end your report with a brief (~1-2 paragraphs) conclusion wrapping up your findings from the various analyses you performed and summarize the key link of structure and function with your protein.