MCB3- Investigation of a novel protein

Introduction

From the course essay you will be aware of the importance of searching sequence databases to identify a gene product. The aim of this project is to give you access to the same tools that practising molecular cell biologists use to do this. We also want you to report your results using PowerPoint. This will teach you how to convey information in a succinct, graphical manner and to learn some elementary skills in using presentation software (all transferable skills).

Aims and Outcomes

Understand the principles of a "pull down" experiment to identify other proteins that interact with a protein of interest
Understand the principles by which a novel protein can be identified by MALDI-TOF analysis of its tryptic peptides
Use a on-line database to identify the novel protein
Find any annotated domains within the identified protein.
Find the pI and MW of the protein.
Report on the function of the identified protein.
Create a PowerPoint slide to report your findings.
Upload your Powerpoint slide onto the course Web site.

Overview

Many interesting problems in cell biology involve protein complexes. Indeed, a systematic analysis of protein interactions in S.cerevisiae showed that lone proteins are the exception. On average, each protein has five interactors.

The link below explains the pulldown technique and how MALDI-TOF analysis of tryptic peptides from the interactor can be used to identify it.
Subsequently the amino-acid sequence can be analysed for the signature of protein domains, such as SH2 and SH3 domains, which sometimes aid efforts to work out the function of the protein.

For this exercise you are presented with a research scenario, common in many aspects of mocleular cell biology, which takes you through this process and should allow you to propose a model for how the system might work.

Your understanding of this exercise will be tested in the ICA test. You will receive individual feedback and, on successful completion of the task, a grade of 1 will appear in your marks. This mark is not itself part of your ICA, but it does signify that you have successfully completed the task as part of your DP for the course.

The Scenario - Investigation of interacting proteins with Anyport, a Drosophila protein involved in Axonal Guidance.

You are studying a Drosophila protein, Anyport, which consists of three SH3 domains and one SH2 domain. One of SH3 domains of Anyport protein was shown to bind and activate a kinase, Pak, which regulates the actin cytoskeleton. Interestingly a mutant lacking Anyport shows axon guidance defects in Drosophila embryos. Axon guidance requires correct recognition of the target and regulation of axon growth.

As SH2 domain is known to bind a short sequence containing a phosphorylated tyrosine, you thought identification of proteins which bind to this SH2 domain would be crucial to understand a role of Anyport in axon guidance.

One night you had a brilliant idea. You made a plasmid expressing a protein which contains the SH2 domain of Anyport fused with pentahistidine. You transfected this plasmid to Drosophila cultured cells and pull down this fusion protein using nickel beads which binds to pentahistidine.

Results of pulldown experiment

You do a suitable control experiment (C). In both experiments you spin down the beads and subject them to SDS polyacrylamide gel electrophoresis. To visualise bands that bind to Anyport you overlay the gel with a purified construct that expresses radioactive SH2 domain of Anyport.

MALDI-TOF analysis

You cut this band out and subject it to trypsin hydrolysis. The peptides are subjected to MALDI-TOF analysis. Some of the m/z masses found are:

m/z
833.47
834.49
836.44
839.38
843.52
855.50
857.47
872.52
873.47

Gathering Information

(1) Identifying protein

Select the m/z values for the tryptic peptides derived from the novel protein. Drag the mouse over the values in the table above while depressing the left mouse button. Copy it either by right clicking and then selecting Copy in the menu, or hit Ctrl-C, or in the top tool bar, hit Edit and then Copy in the drop-down menu.

Then click the following button to access a web-server that interrogates a database of precomputed tryptic peptides to identify any unique signature that may be your protein ( http://prospector.ucsf.edu/prospector/4.0.7/html/msfit.htm)

Scroll down to "Data Paste Area". You need to delete the values that are there. Either put your cursor in the area, hit Ctrl-A, then delete or drag the cursor over all the values, and then finally hit delete.
Now paste your values either by hitting Ctrl-V, or in the top tool bar, hit Edit and then Paste in the drop-down menu, or right-click and select Paste from the drop-down menu.
Set the "Database" to be "SwissProt Date" where Date is the release date for the database. You will more likely have a more uptodate database than when this is written, so we don't give it here. Do not select "SwissProt Date.random" etc.
Set the masses of the peptides to "Monoisotopic", not "Average" .
To speed up the search we may limit the sizes of the protein that the server must search. Look at the pulldown experiment. What is the size of the protein we are looking for? Click the "+" sign next to "Pre-Search Parameters". At "Protein MW (Da)" change the limits to more sensible values. Note that the mw from SDS- Polyacrylamide electrophoresis is never precise and that some proteins may run anomalously. So don't make your limits too narrow ( Hint: there are not many proteins larger than 100,000 Da).
Set "Instrument" to "MALDI-Q-TOF"
Hit "Start Search"

Results

Look for the result with the highest percentage of input peptides that are matched, not the MOWSE score ( see (i) in the example). Open the link to the sequence (see example below) Note the pI and MW of the protein found and its Sequence Code. Select the single letter sequence and copy it. ( Don't worry about including the sequence numbers - subsequent programs will remove them). See example for an unrelated protein .

(2) Finding mammalian proteins similar to your protein

Then click the following button to access the BLAST sequence searching program at http://www.ebi.ac.uk/blastall/

Paste your sequence into the box following "Enter or Paste a Protein Sequence in any format". Then hit Run Blast

Wait until the answers are loaded into your page. Then scroll down the page until you reach "Sequences producing High-scoring Segment Pairs: ". Search for any mammalian proteins, particularly Human and Mouse, to which your protein has similarity.

(3) Identifying domains in the protein through sequence similarity

Open SMART web-server (Simple Modular Architecture Research Tool) http://smart.embl-heidelberg.de/
Paste your sequence into the sequence window. Then hit "Sequence Smart". Hit "Print Screen" on your keyboard to capture an image of the domain structure that the server finds. This image can be pasted into PowerPoint (see below)

Click the domains shown in the diagram to learn more about them and record their putative functions.

Making a PowerPoint slide

You must make a single PowerPoint Slide summarising your results. You may use the example file shown in the course book as a template. It can be downloaded

Your slide should include

The image of the gel, annotated to highlight the band binding to Anyport and notes on design of the control lane.
The image of the domain structure of the interactor that was identified, showing its sequence database accession number, pI and MW and identify one similar mammalian protein (only the best human match, otherwise only the best mouse match).
A model for how the system may be working

Instructions for using PowerPoint are

Instructions for uploading the PowerPoint Slide are

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