Mutation, stability effects

Temperature-sensitive mutations usually arise from a single mutation s effect on the stability of the protein. Temperature-sensitive mutations make the protein just unstable enough to unfold when the normal temperature is raised a few degrees. At normal temperatures (usually 37°C), the protein folds and is stable and active. However, at a slightly higher temperature (usually 40 to 50°C) the protein denatures (melts) and becomes inactive. The reason proteins unfold over such a narrow temperature range is that the folding process is very cooperative—each interaction depends on other interactions that depend on other interactions. 

The contribution of Asp to maintaining His in such an orientation that can accept a proton from Ser is considered to be of minor importance since the electrostatic contribution to the difference in free energy between native and mutant serine proteinases accounts for most of the calculated effects of the mutations (Asp to Asn in trypsin, Asp to Ala in subtilisin). The other stabilizing effect is of the oxyanion hole on the negatively charged TI, which is also electrostatic in nature. 

Khurana, J., R. Singh, and J. Kaur. 2011. Engineering of Bacillus Lipase by Directed Evolution for Enhanced Thermal Stability Effect of Isoleucine to Threonine Mutation at Protein Surface. Molecular Biology Reports 38 (5) 2919-2926. 

Khurana J, Singh R, Kaur J. Engineering of Bacillus lipase by directed evolution for enhanced thermal stability effect of isoleucine to threonine mutation at protein surface. Mol Biol Rep 2010 1-8. 

In Figure 7b, the data are plotted as AG yielding a linear function. Extrapolation to 2ero denaturant provides a quantitative estimate of the intrinsic stability of the protein, AG, which in principle is the free energy of unfolding for the protein in the absence of denaturant. Comparison of the AG values between mutant and wild-type proteins provides a quantitative means of assessing the effects of point mutations on the stability of a protein. 

Both types of mutations have been made in T4 lysozyme. The chosen mutations were Gly 77-Ala, which caused an increase in Tm of 1 °C, and Ala 82-Pro, which increased Tm by 2 °C. The three-dimensional structures of these mutant enzymes were also determined the Ala 82-Pro mutant had a structure essentially identical to the wild type except for the side chain of residue 82 this strongly indicates that the effect on Tm of Ala 82-Pro is indeed due to entropy changes. Such effects are expected to be additive, so even though each mutation makes only a small contribution to increased stability, the combined effect of a number of such mutations should significantly increase a protein s stability. 

Alber, T. Mutational effects on protein stability. Annu. Rev. Biochem. 58 765-798, 1989. 

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