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Influence of point mutations on the stability, dimerization, and oligomerization of human cystatin C and its L68Q variant.

ABSTRACT: Human cystatin C (hCC) is a small but very intriguing protein. Produced by all nucleated cells is found in almost all tissues and body fluids where, at physiological conditions, plays a role of a very potent inhibitor of cysteine proteases. Biologically active hCC is a monomeric protein but during cellular trafficking it forms dimers, transiently losing its inhibitory activity. In vitro, dimerization of cystatin C was observed for the mature protein during crystallization trials, revealing that the mechanism of this process is based on the three dimensional swapping of the protein domains. In our work we have focused on the impact of two proposed "hot spots" in cystatin C structure on its conformational stability. Encouraged by promising results of the theoretical calculations, we designed and produced several hCC hinge region point mutation variants that display a variety of conformational stability and propensity for dimerization and aggregation. A similar approach, i.e., rational mutagenesis, has been also applied to study the amyloidogenic L68Q variant to determine the contribution of hydrophobic interactions and steric effect on the stability of monomeric cystatin C. In this overview we would like to summarize the results of our studies. The impact of a particular mutation on the properties of the studied proteins will be presented in the context of their thermal and mechanical stability, in vitro dimerization tendency as well as the outcome of crystallization. Better understanding of the mechanism and, especially, factors affecting conformational stability of cystatin C and access to stable monomeric and dimeric versions of the protein opens new perspectives in explaining the role of dimers and the domain swapping process in hCC oligomerization, as well as designing potential inhibitors of this process.

SUBMITTER: Szymanska A

PROVIDER: S-EPMC3406405 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Influence of point mutations on the stability, dimerization, and oligomerization of human cystatin C and its L68Q variant.

Szymańska Aneta A Jankowska Elżbieta E Orlikowska Marta M Behrendt Izabela I Czaplewska Paulina P Rodziewicz-Motowidło Sylwia S

Frontiers in molecular neuroscience 20120727

Human cystatin C (hCC) is a small but very intriguing protein. Produced by all nucleated cells is found in almost all tissues and body fluids where, at physiological conditions, plays a role of a very potent inhibitor of cysteine proteases. Biologically active hCC is a monomeric protein but during cellular trafficking it forms dimers, transiently losing its inhibitory activity. In vitro, dimerization of cystatin C was observed for the mature protein during crystallization trials, revealing that ...[more]

PMID: 22866027

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Project description:BACKGROUND: A basic question of protein structural studies is to which extent mutations affect the stability. This question may be addressed starting from sequence and/or from structure. In proteomics and genomics studies prediction of protein stability free energy change (DeltaDeltaG) upon single point mutation may also help the annotation process. The experimental DeltaDeltaG values are affected by uncertainty as measured by standard deviations. Most of the DeltaDeltaG values are nearly zero (about 32% of the DeltaDeltaG data set ranges from -0.5 to 0.5 kcal/mole) and both the value and sign of DeltaDeltaG may be either positive or negative for the same mutation blurring the relationship among mutations and expected DeltaDeltaG value. In order to overcome this problem we describe a new predictor that discriminates between 3 mutation classes: destabilizing mutations (DeltaDeltaG<-1.0 kcal/mol), stabilizing mutations (DeltaDeltaG>1.0 kcal/mole) and neutral mutations (-1.0</=DeltaDeltaG</=1.0 kcal/mole). RESULTS: In this paper a support vector machine starting from the protein sequence or structure discriminates between stabilizing, destabilizing and neutral mutations. We rank all the possible substitutions according to a three state classification system and show that the overall accuracy of our predictor is as high as 56% when performed starting from sequence information and 61% when the protein structure is available, with a mean value correlation coefficient of 0.27 and 0.35, respectively. These values are about 20 points per cent higher than those of a random predictor. CONCLUSIONS: Our method improves the quality of the prediction of the free energy change due to single point protein mutations by adopting a hypothesis of thermodynamic reversibility of the existing experimental data. By this we both recast the thermodynamic symmetry of the problem and balance the distribution of the available experimental measurements of free energy changes. This eliminates possible overestimations of the previously described methods trained on an unbalanced data set comprising a number of destabilizing mutations higher than stabilizing ones.

Dataset Information

Influence of point mutations on the stability, dimerization, and oligomerization of human cystatin C and its L68Q variant.

Publications

Influence of point mutations on the stability, dimerization, and oligomerization of human cystatin C and its L68Q variant.

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