Protein to be analyzed :Crystal structure of human P-cadherin (ss-X-dimer)
URL for protein:
https://www.rcsb.org/structure/4ZMQ
resources and guide for assignment:
https://echo360.ca/media/6778cbbe-ad04-4172-9fca-bbe7bc625009/public?autoplay=false&automute=false&startTimeMillis=0
https://echo360.ca/media/9dffd97e-1452-4c36-9cfc-2fb4315cead9/public?startTimeMillis=0
Instructions for the Final Take-home Project: Analysis of protein structure/function Choose at least one recent paper investigating a topic in protein structure and function with a protein of interest specifically from human with a polypeptide length of at least 300 amino acids (incorrect length or species will result in a grade of zero). Note that the RAR/RXR proteins – including all subtypes – are not permitted as they were used in the assignment demonstration. Use only primary literature; no reviews. Use the chosen paper(s) as a guide to carry out an original analysis on how the protein structure influences and determines its function(s). 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 June 11 at 5pm (no email, no hardcopies). Proposals which are submitted after this date, or proposals with the wrong protein length or species will receive a penalty of 15% 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 June 11 at 5pm, which you can do by adding to your original forum post where you claimed your protein. Ensure you are not working with a protein that is already claimed as duplicate protein projects will receive a grade of zero. The final project is due for submission on June 25th 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 pymol programs offer 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 on Deep View and/or pymol 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 program 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 11. use the Ramachandran plot convert sheets to helices, or at least rearrange/modify the protein backbone 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 and/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. 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.