Course No. 4 "Protein Structural Analysis: Binding Sites to Distant Homologs"

  Problem 2

We will follow these steps to analyze Clostridium perfringens str. 13 protein, BAB81623, currently annotated as conserved hypothetical protein in the Entrez Protein database:

A. Identify conserved domain(s) present in it
B. Search for other proteins containing similar domain(s)
C. Explore a 3D modeling template for the conserved domain
D. View and annotate the protein structure
E. Find distant sequence homologs that may not be identified by BLAST
F. To obtain a 3D modeling template based on the sequence similarity

NCBI's Conserved Domain Search allows you to match your protein sequence to a library of conserved protein domains, generate a multiple sequence alignment based on this match, and explore 3D modeling templates for your sequence. Since this protein is in NCBI's Entrez Protein database, retrieve the protein entry and click on the "Conserved Domains" link to access information about the conserved domain(s) in the protein. (If the protein is not the Entrez Protein database, you can run the search against the CDD database using the CD-Search tool.

A. What is the domain present in this protein?

B. Obtain a list of proteins with a similar domain architecture by clicking on the "Search for similar domain architectures" button. To display the records, click on the link + sign and from there on the "Look up Sequences in Entrez". Change the display from "Summary" to "FASTA".

C. Go back to the CD-Search results page. Generate a multiple sequence alignment for the top 10 sequences representative of the conserved domain hit by clicking on the "Specific hits" graphic of the domain. Identify amino acids in your query protein that correspond to the annotated features such as the pour gating glutamate residue.  Invoke Cn3D with a display of a 3D modeling template and a multiple sequence alignment including your query sequence by pressing the "Structure View" button. The structures of the Escherichia coli and Salmonella typhimurium Clc chloride channel proteins and their sequence alignment with our query protein are displayed. Use File--Realign Structures, if necessary. Click on the “Show Annotations” button in the CDD Descriptive Items window. Highlight the amino acids annotated in the features by selecting the feature and selecting the Highlight button.

D. To view the complete structure (not just the conserved domain part), click on the 1KPL_A link, then on the structure image and finally on the "Structure View in Cn3D” button. For a better view of the backbone, remove the side chains globally (Style--Edit global style--Protein side chains). Double click on the chloride ion. Identify the residues within 4 angstroms of this ion by using the Select--Select by distance menu. Display the sidechains of these residues (Use Style--Annotate--New--Edit Style). Change the protein backbone Rendering to Tubes, Color Scheme to User Selection and User Color to choose the color for the highlighted residue; for example red. Repeat these steps for the Protein Sidechains row and click the Protein Sidechains on. Click on the "Done" button. Use Select--Selected residues to display only the highlighted region.  In the sequence viewer window, un-highlight the residues by clicking on the white area in the sequence window while holding the control key.

E. To obtain the structural neighbors for Salmonella typhimurium chloride channel protein, first click on the chain A graphic in the MMDB structure summary page. Change the List menu to non-identical sequences if desired. Change the Graphic display to Table and click on the List button. Select one or more of the check boxes next to the structure neighbors and download the structures by clicking on the "View 3D Alignment" button.

F. To obtain the structural template for the protein based on the sequence similarity against the PDB database, either run a BLASTp search against PDB or, since the query sequence is already in the Entrez Protein database, retrieve the entry and access the template by using the Related Structures link. Change the View option, for example, to non-identical, if desired and select the Go button. Mouse over the structure images to identify the structure. Click on the arrow graphic next to structure image and then on the "View structure and alignment in Cn3D" button.
 
 

Questions, Comments:  Medha Bhagwat, PhD