Jan Brezovský

Nanosecond Time-Dependent Stokes Shift at the Tunnel Mouth of Haloalkane Dehalogenases

The tunnel mouths are evolutionally the most variable regions in the structures of haloalkane dehalogenases originating from different bacterial species, suggesting their importance for adaptation of enzymes to various substrates. We decided to monitor the dynamics of this particular region by means of time-resolved fluorescence spectroscopy and molecular dynamic simulations. To label the enzyme specifically, we adapted a novel procedure that utilizes a coumarin dye containing a halide-hydrocarbon linker, which serves as a substrate for enzymatic reaction. The procedure leads to a coumarin dye covalently attached and specifically located in the tunnel mouth of the enzyme. In this manner, we stained two haloalkane dehalogenase mutants, DbjA-H280F and DhaA-H272F. The measurements of time-resolved fluorescence anisotropy, acrylamide quenching, and time-resolved emission spectra reveal differences in the polarity, accessibility and mobility of the dye and its microenvironment for both of the mutants. The obtained experimental data are consistent with the results obtained by molecular dynamics calculations and correlate with the anatomy of the tunnel mouths, which were proposed to have a strong impact on the catalytic activity and specificity of the examined mutants. Interestingly, the kinetics of the recorded time-dependent Stokes shift is unusual slow; it occurs on the nanosecond time-scale, suggesting that the protein dynamics is extremely slowed down at the region involved in the exchange of ligands between the active-site cavity and bulk solvent.

PredictSNP2: A Unified Platform for Accurately Evaluating SNP Effects by Exploiting the Different Characteristics of Variants in Distinct Genomic Regions.

An important message taken from human genome sequencing projects is that the human population exhibits approximately 99.9% genetic similarity. Variations in the remaining parts of the genome determine our identity, trace our history and reveal our heritage. The precise delineation of phenotypically causal variants plays a key role in providing accurate personalized diagnosis, prognosis, and treatment of inherited diseases. Several computational methods for achieving such delineation have been reported recently. However, their ability to pinpoint potentially deleterious variants is limited by the fact that their mechanisms of prediction do not account for the existence of different categories of variants. Consequently, their output is biased towards the variant categories that are most strongly represented in the variant databases. Moreover, most such methods provide numeric scores but not binary predictions of the deleteriousness of variants or confidence scores that would be more easily understood by users. We have constructed three datasets covering different types of disease-related variants, which were divided across five categories: (i) regulatory, (ii) splicing, (iii) missense, (iv) synonymous, and (v) nonsense variants. These datasets were used to develop category-optimal decision thresholds and to evaluate six tools for variant prioritization: CADD, DANN, FATHMM, FitCons, FunSeq2 and GWAVA. This evaluation revealed some important advantages of the category-based approach. The results obtained with the five best-performing tools were then combined into a consensus score. Additional comparative analyses showed that in the case of missense variations, protein-based predictors perform better than DNA sequence-based predictors. A user-friendly web interface was developed that provides easy access to the five tools’ predictions, and their consensus scores, in a user-understandable format tailored to the specific features of different categories of variations. To enable comprehensive evaluation of variants, the predictions are complemented with annotations from eight databases. The web server is freely available to the community at http://loschmidt.chemi.muni.cz/predictsnp2.

Site-specific analysis of protein hydration based on unnatural amino acid fluorescence.

Hydration of proteins profoundly affects their functions. We describe a simple and general method for site-specific analysis of protein hydration based on the in vivo incorporation of fluorescent unnatural amino acids and their analysis by steady-state fluorescence spectroscopy. Using this method, we investigate the hydration of functionally important regions of dehalogenases. The experimental results are compared to findings from molecular dynamics simulations.

Are time-dependent fluorescence shifts at the tunnel mouth of haloalkane dehalogenase enzymes dependent on the choice of the chromophore?

Time-dependent fluorescence shifts (TDFS) of chromophores selectively attached to proteins may give information on the dynamics of the probed protein moieties and their degree of hydration. Previously, we demonstrated that a coumarin dye selectively labeling the tunnel mouth of different haloalkane dehalogenases (HLDs) can distinguish between different widths of tunnel mouth openings. In order to generalize those findings analogous experiments were performed using a different chromophore probing the same region of these enzymes. To this end we synthesized and characterized three new fluorescent probes derived from dimethylaminonaphthalene bearing a linker almost identical to that of the coumarin dye used in our previous study. Labeling efficiencies, acrylamide quenching, fluorescence anisotropies, and TDFS for the examined fluorescent substrates confirm the picture gained from the coumarin studies: the different tunnel mouth opening, predicted by crystal structures, is reflected in the hydration and tunnel mouth dynamics of the investigated HLDs. Comparison of the TDFS reported by the coumarin dye with those obtained with the new dimethylaminonaphthalene dyes shows that the choice of chromophore may strongly influence the recorded TDFS characteristics. The intrinsic design of our labeling strategy and the variation of the linker length ensure that both dyes probe the identical enzyme region; moreover, the covalently fixed position of the chromophore does not allow for a major relocalization within the HLD structures. Our study shows, for the first time, that TDFS may strongly depend on the choice of the chromophore, even though the identical region of a protein is explored.

Interaction of organic solvents with protein structures at protein-solvent interface

The effect of non-denaturing concentrations of three different organic solvents, formamide, acetone and isopropanol, on the structure of haloalkane dehalogenases DhaA, LinB, and DbjA at the protein-solvent interface was studied using molecular dynamics simulations. Analysis of B-factors revealed that the presence of a given organic solvent mainly affects the dynamical behavior of the specificity-determining cap domain, with the exception of DbjA in acetone. Orientation of organic solvent molecules on the protein surface during the simulations was clearly dependent on their interaction with hydrophobic or hydrophilic surface patches, and the simulations suggest that the behavior of studied organic solvents in the vicinity of hyrophobic patches on the surface is similar to the air/water interface. DbjA was the only dimeric enzyme among studied haloalkane dehalogenases and provided an opportunity to explore effects of organic solvents on the quaternary structure. Penetration and trapping of organic solvents in the network of interactions between both monomers depends on the physico-chemical properties of the organic solvents. Consequently, both monomers of this enzyme oscillate differently in different organic solvents. With the exception of LinB in acetone, the structures of studied enzymes were stabilized in water-miscible organic solvents.

FireProt: web server for automated design of thermostable proteins

Abstract There is a continuous interest in increasing proteins stability to enhance their usability in numerous biomedical and biotechnological applications. A number of in silico tools for the prediction of the effect of mutations on protein stability have been developed recently. However, only single-point mutations with a small effect on protein stability are typically predicted with the existing tools and have to be followed by laborious protein expression, purification, and characterization. Here, we present FireProt, a web server for the automated design of multiple-point thermostable mutant proteins that combines structural and evolutionary information in its calculation core. FireProt utilizes sixteen tools and three protein engineering strategies for making reliable protein designs. The server is complemented with interactive, easy-to-use interface that allows users to directly analyze and optionally modify designed thermostable mutants. FireProt is freely available at http://loschmidt.chemi.muni.cz/fireprot.

Computer-assisted engineering of hyperstable fibroblast growth factor 2

Fibroblast growth factors (FGFs) serve numerous regulatory functions in complex organisms, and their corresponding therapeutic potential is of growing interest to academics and industrial researchers alike. However, applications of these proteins are limited due to their low stability. Here we tackle this problem using a generalizable computer‐assisted protein engineering strategy to create a unique modified FGF2 with nine mutations displaying unprecedented stability and uncompromised biological function. The data from the characterization of stabilized FGF2 showed a remarkable prediction potential of in silico methods and provided insight into the unfolding mechanism of the protein. The molecule holds a considerable promise for stem cell research and medical or pharmaceutical applications.