Chimeric enzymes were constructed to elucidate the differences in physicochemical properties of two related bacterial RNases, barnase and binase. Chimeras (Ba26Bi, Ba73Bi, Ba26Bi73Ba and Bi73Ba) contain six to thirteen residue substitutions relative to barnase, which are beyond the active site. The catalytic activity of RNases toward GpU, GpC and poly(I), as well as conformational distinctions and heat denaturation parameters, were studied. Thermal denaturation of binase, barnase and chimeric RNases is a two-state transition. The mutation-induced changes in the free energy of unfolding of barnase deduced from thermal and urea denaturation nearly coincide. The kinetic parameters for GpU and GpC demonstrate that the chimeras fall into two groups: barnase-like and binase-like. This division is determined by the origin of their C-terminal part (residues 73-110) which is also responsible for their thermostability at pH 2.4. An inverse linear dependence was found between kcat for poly(I) and denaturation temperature of RNases at pH 5.5, which points out that certain lability of the protein molecule appears to be necessary for efficient polynucleotide cleavage.

New "reference" circular dichroism spectra of a helix, 0-structure (both parallel and antiparallel), 0-bends, and the unordered form are obtained from circular dichroism spectra and x-ray data for six proteins (myoglobin, lysozyme, lactate dehydrogenase, papain, ribonuclease, and subtilisin BPN'). Circular dichroism spectra for a-helix and antiparallel 0-structure are similar to those for p o l y (~-lysine). The circular dichroism spectrum of the parallel 0-structure is qualitatively similar to that theoretically calculated by Madison and Schellman. The circular dichroism spectrum of @-bends is qualitatively similar to that theoretically calculated by Woody. The spectrum of the unordered form is close to that of the denaturated proteins. These "reference" circular dichroism spectra used for the analysis of the secondary structure of ten globular proteins (besides the six reference proteins D-glyceraldehyde 3-phosphate dehydrogenase, concanavalin A, cytochrome c, and insulin).

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A series of 5'-monophosphates of 5-substituted 2'-deoxyuridine analogs, which recently demonstrated in vitro substantial suppression of two strains of Mycobacterium tuberculosis growth (virulent laboratory H37Rv and multiple resistant MS-115), has been synthesized and evaluated as potential inhibitors of M. tuberculosis thymidylate synthases: classical (ThyA) and flavin dependent thymidylate synthase (ThyX). A systematic SAR study and docking revealed 5-undecyloxymethyl-2'-deoxyuridine 5'-monophosphate 3b, displaying an IC50 value against ThyX of 8.32 μM. All derivatives lack activity against the ThyA. It can be assumed that the mechanism of action of 3b may be partially associated with the inhibition of the ThyX.