Study On The Relationship Between Amino Acid Side-Chain Groups And The Salt Adaptation Of Ubiquitin-Like Proteins

At present,researches on the mechanism of protein salt-adaptation mostly focuse on the qualitative aspects.They lack details while some contradict each other.Elabrate studies,such as those on quantitative effects of different positions and types of amino acids on protein salt adaptation,have not been reported.Previous studies have found that Samp2,a ubiquitin like protien(Ubl)from halophilic archea,does not fold as well in low salt as it does in high salt,while the single-point mutant Samp2-D31G can be fully folded in low salt.According to sequence analysis,eukaryotic ubiquitin like proteins have G(Glycine)amino acids at the structural sites corresponding to the residue 31 of Samp2.Therefore,Samp2 and other ubiquitin like proteins are taken as the objects of our study on the mechanism of protein salt adapation.This study aims to,by investigation on the mechanism,provide theoretical basis for rational modificaiton or design of proteins to make them better adapt to the application environments.In this study,using site-directed mutagenesis conbined with far-ultraviolet circular dichroism,intrinsic fluorescence,and size exclusion(SEC)etc,the effects of different amino acid positions and types on the salt adaptation of Samp2 and other ubiquitin like proteins were investigated.Firstly,mutants of different amino acid types at site 31 of Samp2 were recombinantly prepared.Intrinsic fluorescence results showed that all the tertiary structures of Smap2-D31 mutants in high and low salt environments were identical,and SEC results denoted that all the quaternary structures of the mutants were identical in high and low salt respectively.However,by CD,it was found that the mutations of D to positively charged or uncharged amino acid at site 31 made the mutants folded better and more stable in low salt environments than mutations to negatively charged amino acids.These indicated that the salt adaptation of proteins is related to the charge of amino acids.In addition,our data showed that mutation of D/E to G within loop regions made the mutants fold better under low salt conditions than the sme mutations within secondary structure regions,implying the relation between the salt adapation of Samp2 and the flexibility or solvent accessibility of the sites negatively charged residues located at.Based on the above results,the Gly residues in Trypanosoma brucei ubiquitin like proteins structurally corresponding to the D31 of Samp2 were mutated to Asp,and the salt adaptation of the mutants were measured to verify the role of the two types of amino acid in salt adaption and resolution.The results showed that,though most mutants had the same secondary and tertiary structures in high salt as in low salt,and both of the structures were the same as those of the wide type protein,Tb-Nedd8-G36D showed higher degree of fold in high salt than in low salt,supporting negatively charged amino acid facilitate protein adapting to high salt environments.However,both the stabilities of Tb-UB-G35D in low salt and high salt were lower than those of Tb-UB,inconsistent with the argument.These data denote the relation between the type of amino acid and the salt adaption of protein is sophisticated,which needs more in depth investigations.Our study on the salt adaptation of Samp2 and other Ubiquitin like proteins is helpful for our understanding of the mechanism of protein salt adaptation,and contributes to elucidation of the mechanisms of protein evolution and protein folding.