Victor P. Kutyshenko

Sequential mechanism of refolding of carbonic anhydrase B

The kinetics of refolding of bovine carbonic anhydrase B was studied by a variety of methods over a wide range of times (from milliseconds to hours). It has been shown that protein refolding proceeds through three stages. At the first stage (t ½≈0.03 s) hydrophobic clusters and a compact state of the chain are formed. At the second stage (t ½≈140 s) hydrophobic clusters are desolvated and the rigid native‐like hydrophobic core is formed. At the third stage (t ½≈600 s) the native active protein is formed.

The major mRNP protein YB-1: Structural and association properties in solution

YB-1 is a major mRNP protein participating in the regulation of transcription and translation of a wide range of eukaryotic genes in many organisms probably due to its influence on mRNA packing into mRNPs. While the functional properties of YB-1 are extensively studied, little is known about its structural properties. In the present work we focused on studying its secondary structure, rigidity of its tertiary structure, compactness, and oligomerization in vitro by using far UV-CD, DSC, one-dimensional (1)H NMR, SAXS, sedimentation and FPLC. It was shown that only the cold shock domain within the entire YB-1 chain has a well-packed tertiary structure undergoing cooperative heat and cold denaturation transitions. In contrast, the rest of the YB-1 molecule is not rigidly packed and consists of PP II-like helical secondary structure elements and coil-like regions. At the same time, the overall dimension of the protein molecule is unexpectedly small. The polypeptide chains of YB-1 have a high tendency to form oligomers at neutral pH, while the extent and structural organization of the oligomers depend on protein concentration and ionic strength varying from compact monomeric units up to high molecular weight oligomers. These oligomers in solution are unstable and dissociate upon protein concentration decrease.

Analysis of the Metabolites in Apical Area of Allium cepa Roots by High Resolution NMR Spectroscopy Method

Elucidation of the molecular mechanisms determining the formation of various tissues and organs is one of the central problems of cell biology. High-resolution NMR spectroscopy was applied for the analysis of the metabolites produced at the various areas of the apical part of the onion Allium cepa roots. To this end, three samples were extracted from the root apex (the root cap, the meristem region and the cell elongation zone). These samples were noticeably different in the number of mitoses and the sets of metabolites. Furthermore, the complete stasis of the plant roots and tops growth was registered in heavy water. Comparison of the morphological and NMR data revealed their perfect agreement with the cellular processes occurring in the root apex. The root cap sample was characterized by the greatest mitotic activity reflected in the great variability of the chemical compounds extracted from this area, the high level of energy consumption, and the increased synthesis of the phosphocholines needed for the cell fission. Sample containing the cell elongation zone possessed the high sugar content, which is required for the cell-wall growth. Therefore, our data show that high-resolution NMR spectroscopy can be used for the identification of chemical compounds in the various regions of the onion root apical area.

Phospholipid membranes affect tertiary structure of the soluble cytochrome b5 heme-binding domain.

The influence of charged phospholipid membranes on the conformational state of the water-soluble fragment of cytochrome b5 has been investigated by a variety of techniques at neutral pH. The results of this work provide the first evidence that aqueous solutions with high phospholipid/protein molar ratios (pH 7.2) induce the cytochrome to undergo a structural transition from the native conformation to an intermediate state with molten-globule like properties that occur in the presence of an artificial membrane surface and that leads to binding of the protein to the membrane. At other phospholipid/protein ratios, equilibrium was observed between cytochrome free in solution and cytochrome bound to the surface of vesicles. Inhibition of protein binding to the vesicles with increasing ionic strength indicated for the most part an electrostatic contribution to the stability of cytochrome b5-vesicle interactions at pH 7.2. The possible physiological role of membrane-induced conformational change in the structure of cytochrome b5 upon the interaction with its redox partners is discussed.

High-resolution NMR structure of a Zn2+-containing form of the bacteriophage T5 L-alanyl-D-glutamate peptidase

This paper represents the spatial solution structure of the Zn2+-containing form of the bacteriophage T5 L-alanyl-D-glutamate peptidase (EndoT5-Zn2+). The core of this α + β protein is formed by three α-helices (residues 7–15, 20–30, and 87–104) and a β-sheet containing three β-strands (residues 35–39, 71–76, and 133–135). The protein has two short loops (residues 16–19 and 31–34), a medium-length loop (residues 77–86) containing a short β-hairpin (residues 77–82), and two long loops (residues 40–70 and 105–132). The long loops include a stable 310-helix (residues 66–68) and labile α-helices 46–53 and 113–117. Catalytic Zn2+-binding site is represented by three amino acid residues, His66, Asp73, and His133. The cation-binding His residues are located near the foundations of the long loops, whereas Asp73 is positioned in the middle of the core β-sheet. The catalytic center localization contributes to the stabilization of the entire molecule, with Zn2+-binding playing a key role in the folding of this protein.

Dancing retro: solution structure and micelle interactions of the retro-SH3-domain, retro-SHH-‘Bergerac’

A protein with the reversed direction of its polypeptide chain, retro-SHH, was analyzed by several spectroscopic techniques including circular dichroism and high-resolution NMR to understand its solution structure and structural consequences of interaction with the micelles formed by the zwitterionic detergent dodecylphosphocholine (DPC). This analysis revealed that retro-SHH does not contain rigid 3-D structure, but is characterized by the presence of residual secondary structure. Intriguingly, interaction with the DPC micelles affected the structures of SHH and retro-SHH very differently. In fact, micelles induce pronounced folding of retro-SHH, whereas micelle-bound SHH was noticeably disordered. Finally, we performed a disorder prediction with the PONDR-FIT algorithm and discovered that the reversal of the chain direction almost does not affect the propensity of a polypeptide for intrinsic disorder, since the disorder plot for retro-SHH was almost a mirror image of that for the normal SHH.

Structure and dynamics of the retro-form of the bacteriophage T5 endolysin

Using high-resolution NMR spectroscopy we conducted a comparative analysis of the structural and dynamic properties of the bacteriophage T5 endolysin (EndoT5) and its retro-form; i.e., a protein with the reversed direction of the polypeptide chain (R-EndoT5). We show that structurally, retro-form can be described as the molten globule-like polypeptide that is easily able to form large oligomers and aggregates. To avoid complications associated with this high aggregation propensity of the retro protein, we compared EndoT5 and R-EndoT5 in the presence of strong denaturants. This analysis revealed that these two proteins possess different internal dynamics in solutions containing 8M urea, with the retro-form being characterized by larger dimensions and slower internal dynamics. We also show that in the absence of denaturant, both forms of the bacteriophage T5 endolysin are able to interact with micelles formed by the zwitterionic detergent dodecylphosphocholine (DPC), and that the formation of the protein-micelle complexes leads to the significant structural rearrangement of polypeptide chain and to the formation of stable hydrophobic core in the R-Endo T5.

Evidence for the residual tertiary structure in the urea-unfolded form of bacteriophage T5 endolysin.

Using high-resolution NMR spectroscopy, we studied peculiarities of the unfolding process of the bacteriophage T5 endolysin (EndoT5) by strong denaturants. It was shown that in the absence of zinc ions this protein is mostly unfolded in the solution of 8 M urea or 6 M guanidine hydrochloride. However, in the presence of zinc ions EndoT5 unfolding can be achieved only in acidic solutions (at pH < 4.0), whereas at pH > 4.0 NMR spectra of the metal-bound protein (Zn2+–Ca2+–EndoT5 or Zn2+–EndoT5 complexes) exhibit a few chemical shifts characteristic of the native or native-like proteins. Our data, including the pH–titration curve with the pK of ~5, suggested involvement of the zinc-binding histidines in the stabilization of this protein. Up-field signals that appear in the NMR spectra of apo-EndoT5 in the presence of high concentrations of strong denaturants are probably derived from the amino acid residues included in the formation of structured hydrophobic cluster, which likely corresponds to the 81–93 region of EndoT5 and contains some residual tertiary structure. It is possible also that this hydrophobic fragment serves as a foundation for the formation of structured cluster in the unfolded state.

Looking at microbial metabolism by high-resolution 2H-NMR spectroscopy

We analyzed the applicability of high-resolution 2H-HMR spectroscopy for the analysis of microbe metabolism in samples of mitochondrion isolated from rat liver and from aqueous extracts of homogenates of rat liver and other organs and tissues in the presence of high D2O contents. Such analysis is possible due to the fast microbe adaptation to life in the heavy water. It is also shown that some enzymatic processes typical for the intact cells are preserved in the homogenized tissue preparations. The microbial and cellular metabolic processes can be differentiated via the strategic use of cell poisons and antibiotics.