KE0026 Biochemistry Labs

Computer lab: Protein design

In this computer lab, the concept and scope of protein design will be illustrated using Green Fluorescent Protein (GFP).

Facts about GFP:
Small protein found in marine life forms like jellyfish and hydras. GFP is exited by blue light and emits green light. GFP has lately become very popular as a tool for molecular biologists that use GFP, i.e. the gene that codes for GFP, as a genetic marker. The fluorophore of GFP is an integral part of the protein that is formed by an autocatalyzed crosslinking between residue Serine 65 and Glycine 67.

Problem:
GFP is exited by blue light. Blue light is suboptimal as a tool used on biological samples since:

1) High autofluorescence in living matter at short wavelengths. This hides the measured signal in background noise.
2) Blue light is harmful for cells (Why?)

Solution:
Design a GFP that is exited (and emits) at longer wavelengths!
(Does one gain anything else with a second coloured GFP?)
Are there other ways to do this than with site directed mutagenesis?

Materials:
Coordinates for GFP.
Schematic drawing of the fluorophore surrounding.
Swiss PDB Viewer

Instructions:

Download the GFP coordinates from the PDB WWW Home Page (See instructions further down.)
Orient yourself in the structure and try to find the fluorophore.
Develop a strategy to that will result in an altered GFP that could have the characteristics that you desire. Think of what determines the absorption and emission wavelengths of a molecule.
Identify suitable residues.
Use the mutagenesis tool in Swiss PDB Viewer, and change the residue/residues that you decided upon. Check for steric clashes, possible H-bonds etc. Do you think the mutation will affect the folding of the protein? Why/Why not?

Downloading GFP from the PDB database:

Log into PDB using Netscape
Go into the browser and search for Green Fluorescent Protein
(Entry code 1ema).
Select the entry and fetch it.
Choose to down load the whole document.
Save as source on your disc area.

Lab report

guidelines

Introduction: What is GFP used for? Why does one want to engineer GFP? Why could a second or third colour be useful? Can a colour of a protein be changed? How? What determines the energy levels of an excited state?
Methods: State the choice of method/methods that you would use to alter GFP. How will you engineer GFP to meet your demands, i.e., what was the basis for selecting mutations that might give the desired effect?
Results: Did the selected mutation(s) fit in the protein? Give the important atomic distances to other close-by residues. Are there any hydrogen bonds formed? Are there any steric clashes? Pictures showing a closeup of your mutation(s) and the surroundings are very useful to illustrate what you did.
Discussion: What does one need to think about when designing mutations? Are the mutations you looked at likely to change the colour of GFP or not, i.e. would you recommend mutating GFP according to your specifications?

Lab index

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