Rational Stabilization Of A Nitrilase Through Optimization Of Protein's Surface Charge Distribution

Our goal is to investigate contribution from the charge-charge interactions on protein's surface to protein thermo-stability. In this paper, a hypothesis that one can improve protein thermo-stability through optimize the protein charge distribution on protein surface was made and it had been proved to be feasible.A nitrilase (NCBI code:AJ248287) was cloned and built model based on the template protein:PDB code 1J31 with the sequence identity of 84 in percentage. The Verify Score of model, named abyssi.B99990005, had a value of 249.05, which was less than 248.12 in template. It meant the model was reliable. Then program Macrodox was used to screen the ionizable residue on protein surface. the mutation sites GLU15, GLU24, GLU51, GLU58, GLU67, ASP93, GLU125, GLU200, GLU227, ASP236 were presented. In each site, the residue's electronegativity was reversed and mutated to be neutral. Between these mutations, three mutation's thermo-stability has proved to be changed with activity retained by experiment, which prove this method can find way to change the protein's thermo-stability without great loss of activity.A program was written based on the Tanford-Kirkwood model to investigate the charge-charge interactions on protein surface. In this program, theΔGqq value was used to measure the contribution of the charge-charge interactions to the protein's thermo-stability. the three mutation'sΔGqq were calculated and proved to be fit the initial assumption. It proved that this program can calculate the contribution from the charge-charge interactions to the protein thermo-stability quantificationally...