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Molecular Dynamics as a Powerful Tool in Design of Thermostable Protein Mutants

Contemporary biotechnology requires new enzymes, which could catalyze reactions, retain native conformation and ensure high product yield under high temperature. These enzymes are widely used in molecular biology, food and starch processing, and so forth. As a result, one often faces the problem of obtaining a more thermally stable mutant of some well-studied protein. There is a number of approaches to determining single-point mutations increasing the protein thermostability. Most of them use random mutagenesis or homology based predictions. Here, we introduce a new approach to predict single-point mutations increasing protein thermostability. The method is based on molecular dynamics (MD) simulations of a protein with known spatial structure. MD results at normal and high temperature (300K and 373K, respectively) permit delineation of those parts of protein molecule, which are more sensitive to temperature growth (i.e., thermolabile parts). Root-mean-square fluctuations (RMSF) of selected elements with respect to their mean values were taken as a measure of flexibility.

To test the suggested methodology we used two homologous proteins - thioredoxins from mesophile E. coli (Trx) and thermophile Bacillus acidocaldarius (BacTrx). Their mutants with higher and lower thermostability (Trx, Glu85Arg and BacTrx, Arg82Glu, respectively) have been experimentally studied earlier. The mutations are localized in similar positions of both molecules (fig. 1). 2.5 ns MD simulations of the wild type proteins and their mutants at 300K and 373K permit delineation of several thermolabile protein regions which are similar in all molecules. In each case, one of such regions contains the mutation site. In addition, these mutations affect the temperature-dependent flexibility of the following regions: Y49-L53, K82-V86, D104-A108 in TrxE85R, and H46-V50, K79-P83, A101-Q105 in BacTrxR82E (fig. 2, 3).

Fig. 1 3D structure of Trx, BacTrx from NMR data. Termolabile parts of proteins are marked in red. Mutation sites are indicated with arrows.

Fig. 2 Flexibility at 300K (A) and 373K (B). Secondary structure: red - a-helix, blue - b-sheet.

Fig. 3 Influence of mutations on temperature dependent flexibility changes in considered protein regions.

The thermolabile areas reveal a network of strong ionic interactions in each protein. It is shown that Glu85Arg and Arg82Glu mutations affect these interactions and make them more efficient in one case and less efficient in another. As a result, increasing of temperature leads to stabilization of flexible regions in TrxE85R and to their destabilization in BacTrxR82E.

To summarize, the suggested approach allows delineation of thermolabile proteins areas, which could be essential for thermostability of the entire molecule. Our current studies focus on testing of this method for pairs of mesophile/thermophile mutants of other proteins.

(Authors: Anton Polyansky, Yuri Kosinsky, Roman Efremov).

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