"
Team:EPF-Lausanne/Results/Mutations
From 2009.igem.org
m (→Motivation) |
m (→Active site) |
||
| Line 59: | Line 59: | ||
The selected residues for mutations are ILE427 in purple on the next video, LEU453 in orange and VAL416 in yellow. | The selected residues for mutations are ILE427 in purple on the next video, LEU453 in orange and VAL416 in yellow. | ||
<html><center><object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/m-eLb7OV00Q&hl=fr&fs=1&"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/m-eLb7OV00Q&hl=fr&fs=1&" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object></center></html> | <html><center><object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/m-eLb7OV00Q&hl=fr&fs=1&"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/m-eLb7OV00Q&hl=fr&fs=1&" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object></center></html> | ||
| + | |||
| + | Another video using the surf function shows better the steric interactions between those residues. | ||
| + | <html><center><object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/C-FtGXfeJTE&hl=fr&fs=1&"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/C-FtGXfeJTE&hl=fr&fs=1&" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object></center></html> | ||
=ILE427 to PHE= | =ILE427 to PHE= | ||
Revision as of 11:50, 21 October 2009
Motivation
Cys450 side chain is involved in light state in bond formation with the cofactor. Cys450 can assume two conformational states, called here ON and OFF, and corresponding respectively, to the Sg being near or far from FMN.
The more populated conformer is the OFF, as showed by X-ray structures (OFF/ON=90/10 at low temperature and =70/30 at room temperature) but for the bond to form the Cys should switch the less favoured ON. The ON-OFF equilibrium is believed to be influenced by nearby residues. Moreover the steric hindrance of some nearby apolar residues is thought to have an effect also in the process of bond splitting. By looking at the structure and by examining previous mutation analysis we hypothesized that mutation favouring the OFF conformation of Cys450 in the dark state would likely result in favouring the splitting of the covalent adduct. On the other way round a strategy to increase the lifetime of covalent adduct could consist in mutating residue as to stabilize the ON conformation.
Since our simulation are capable of reproducing the experimental OFF/ON conformational equilibrium in the dark state (about 70/30) we decided to perform in-silico mutations, and to compute the OFF/ON ratios of mutants from MD simulations, as to have a guide to experimentally perform the mutations leading to lower OFF/ON ratios. Of course the OFF/ON conformational equilibrium is only indirectly connected to the energy barrier of the bond splitting, but some experimental evidences suggest a link between Cys conformational propensity and the splitting reaction.
We planned to perform QM calcualtions as to directly study the effect of specific mutations on the splitting reaction rate, but since those calcualtions require much more time we have at our disposal, we used for our mutations only chemical intuition and the results of the simulations of mutated systems.
Active site
The selected residues for mutations are ILE427 in purple on the next video, LEU453 in orange and VAL416 in yellow.
Another video using the surf function shows better the steric interactions between those residues.
ILE427 to PHE
LEU453 to GLY
VAL416 to ILE
