Previous experiments on substitution of the residue Phe290 to Asp, Asn and Ser in NAD(+)-dependent formate dehydrogenase from soya Glycine max (SoyFDH) showed important role of the residue in enzyme thermal stability and catalytic properties (Alekseeva et al. Prot. Eng. Des. Sel., 2012a; 25: :781-88). In this work, we continued site-directed mutagenesis experiments of the Phe290 and the residue was changed to Ala, Thr, Tyr, Glu and Gln. All amino acid changes resulted in increase of catalytic constant from 2.9 to 3.5-4.7 s(-1). The substitution Phe290Ala led to KM (NAD+) decrease from 13.3 to 8.6 μM, and substitutions Phe290Tyr and Phe290Glu resulted in decrease and increase of KM (HCOO-) from 1.5 to 0.9 and -2.9 mM, respectively. The highest improvement of catalytic properties was observed for SoyFDH Phe290Ala which showed 2-fold higher catalytic efficiency with both substrates. Stability of mutants was examined by study of thermal inactivation kinetics and differential scanning calorimetry (DSC). All five amino acids provided increase of thermal stability of mutant SoyFDH in comparison with wild-type enzyme. Mutant SoyFDH Phe290Glu showed the highest improvement-the stabilization effect was 43 at 56°C. The DSC data agree with results of thermal inactivation kinetics. Substitutions Phe290Tyr, Phe290Thr, Phe290Gln and Phe290Glu provided Tm value increase 0.6°-6.6°. SoyFDH Phe290Glu and previously prepared SoyFDH Phe290Asp show similar thermal stability as enzymes from Candida boidinii and Mycobacterium vaccae N10 and have higher catalytic efficiency with NAD(+) compared with all described FDHs. Therefore, these mutants are very perspective enzymes for coenzyme regeneration in processes of chiral synthesis with dehydrogenases.

We used differential scanning calorimetry (DSC) and circular dichroism (CD) to investigate thermal unfolding of recombinant fibroblast isoforms of alpha-tropomyosin (Tm) in comparison with that of smooth muscle Tm. These two nonmuscle Tm isoforms 5a and 5b differ internally only by exons 6b/6a, and they both differ from smooth muscle Tm by the N-terminal exon 1b which replaces the muscle-specific exons 1a and 2a. We show that the presence of exon 1b dramatically decreases the measurable calorimetric enthalpy of the thermal unfolding of Tm observed with DSC, although it has no influence on the alpha-helix content of Tm or on the end-to-end interaction between Tm dimers. The results suggest that a significant part of the molecule of fibroblast Tm (but not smooth muscle Tm) unfolds noncooperatively, with the enthalpy no longer visible in the cooperative thermal transitions measured. On the other hand, both DSC and CD studies show that replacement of muscle exons 1a and 2a by nonmuscle exon 1b not only increases the thermal stability of the N-terminal part of Tm, but also significantly stabilizes Tm by shifting the major thermal transition of Tm to higher temperature. Replacement of exon 6b by exon 6a leads to additional increase in the alpha-Tm thermal stability. Thus, our data show for the first time a significant difference in the thermal unfolding between muscle and nonmuscle alpha-Tm isoforms, and indicate that replacement of alternatively spliced exons alters the stability of the entire Tm molecule.

Thermal denaturation and aggregation of beta(L)-crystallin from bovine lens have been studied using differential scanning calorimetry (DSC) and dynamic light scattering (DLS). According to the DLS data, the distribution of the beta(L)-crystallin aggregates by their hydrodynamic radius (R(h)) remains monomodal to the point of precipitating aggregates (sodium phosphate, pH 6.8; 100 mM NaCl; 60 degrees C). The size of the start aggregates (R(h,0)) and duration of the latent stage (t(0)) leading to the formation of the start aggregates have been determined from the light scattering intensity versus the hydrodynamic radius plots and the dependences of R(h) on time. The R(h,0) value remains constant at variation of the beta(L)-crystallin concentration, whereas the t(0) value increases with diminishing beta(L)-crystallin concentration. The suppression of beta(L)-crystallin aggregation by alpha-crystallin is connected with the decrease in the R(h,0) value and increase in the t(0) value. In the presence of alpha-crystallin the aggregate population is split into two components. The first component is represented by stable aggregates whose size remains constant in time. The aggregates of the other kind grow until they reach the size characteristic of aggregates prone to precipitation. The DSC data show that alpha-crystallin has no appreciable influence on thermal denaturation of beta(L)-crystallin.