title: Probing protein stability and proteolytic resistance by loop scanning: a comprehensive mutational analysis.
abstract: Improvement in protein thermostability was often found to be associated with increase in its proteolytic resistance as revealed by comparative studies of homologous proteins from extremophiles or mutational studies. Structural elements of protein responsible for this association are not firmly established although loops are implicated indirectly due to their structural role in protein stability. To get a better insight, a detailed study of protein wide mutants and their influence on stability and proteolytic resistance would be helpful. To generate such a data set, a model protein, Bacillus subtilis lipase was subjected to loop scanning site-saturation mutagenesis on 86 positions spanning all loops including termini. Upon screening of ~16,000 clones, 17 single mutants with improved thermostability were identified with increment in apparent melting temperature (Tm(app) ) by 1-6°C resulting in an increase in free energy of unfolding (ΔG(unf) ) by 0.04-1.16 kcal/mol. Proteolytic resistance of all single mutants upon incubation with nonspecific protease, Subtilisin A, was determined. Upon comparison, post-proteolysis residual activities as well as kinetics of proteolysis of mutants showed excellent correlation with ΔG(unf) , (r > 0.9), suggesting that proteolysis was strongly correlated with the global stability of this protein. This significant correlation in this set, with least possible sequence changes (single aa substitution), while covering >60% of protein surface strongly argues for the covariance of these two variables. Compared to studies from extremophiles, with large sequence heterogeneity, the observed correlation in such a narrow sequence space (ΔΔG(unf) = 1.57 kcal⁻¹) justifies the robustness of this relation.
authors.affiliation: Centre for Cellular and Molecular Biology (Council of Scientific and Industrial Research), Uppal Road, Hyderabad 500007, India.
authors.forename: Shoeb
authors.initials: S
authors.lastname: Ahmad
authors.forename: Virender
authors.initials: V
authors.lastname: Kumar
authors.forename: K Bhanu
authors.initials: KB
authors.lastname: Ramanand
authors.forename: N Madhusudhana
authors.initials: NM
authors.lastname: Rao
mesh_terms: D001412:Bacillus subtilis / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D008049:Lipase / Q000737:chemistry / Q000235:genetics; D008958:Models, Molecular; D016296:Mutagenesis; D055550:Protein Stability; D058767:Protein Unfolding; D059748:Proteolysis; D013696:Temperature; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D008049:Lipase