Dmitry A. Prokhorov

Molecular mechanisms of the anomalous thermal aggregation of green fluorescent protein.

The peculiarities of thermal denaturation and interaction with water of the cycle-3 mutant of green fluorescent protein (GFP) were analyzed by NMR techniques and compared with those of bovine carbonic anhydrase II (BCA-II). Irreversible thermal denaturation was accompanied by massive GFP aggregation with no detectable accumulation of soluble denatured protein. Analysis of the spin diffusion data suggested that the internal part of the GFP β-can is involved in intensive interactions with water molecules. As a result, at high temperatures, the GFP structure does not unfold but rather breaks, consequently leading to enhanced protein aggregation. This is very different from typical BCA-II behavior.

Dynamics of oligomer formation by denatured carbonic anhydrase II

Aggregation and subsequent development of protein deposition diseases originate from conformational changes in corresponding amyloidogenic proteins. Many proteins unrelated to amyloidoses also fibrillate at the appropriate conditions. These proteins serve as a model for studying the processes of protein misfolding, oligomerization and fibril formation. The accumulated data support the model where protein fibrillogenesis proceeds via the formation of a relatively unfolded amyloidogenic conformation. The urea-induced unfolding of bovine carbonic anhydrase II, BCA II, is characterized by a combination of high-resolution NMR, circular dichroism spectroscopy and small angle X-ray scattering. It is shown that the formation of associates of protein molecules in complex with solvent (water and urea), APS, takes place in the presence of 4-6 M urea. The subsequent increase in urea concentration to 8 M is accompanied by a disruption of APS and leads to a complete unfolding of a protein molecule. Analysis of BCA II self-association in the presence of 4.2 M urea revealed that APS are relatively large mostly beta-structural blocks with the averaged molecular mass of 190-220 kDa. This work also demonstrates some novel NMR-based methodological approaches that provide useful information on protein self-association.

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.

Carbonic Anhydrase B Interactions with Water and Urea

High-resolution NMR spectroscopy has been used to study native carbonic anhydrase B unfolding with urea at pH 5.75 and T = 298 K. The rigidity parameter reflecting the effectiveness of spin diffusion (SD) displays a sigma-like dependence on urea concentration, which is characteristic of denaturing processes. The ratio between the integral intensities of urea and protein signals measured in SD spectra and normal 1D spectra are the same. This suggests the absence of a predominant interaction between urea and protein molecules. The concentration of large protein-solvent complexes rapidly increases at urea concentrations of 4.2–6.2 M, which is apparently related to the transition of the protein into the molten globule state. If the urea concentration is increased to 6.6 M, these complexes dissociate, and the polypeptide chain of carbonic anhydrase B becomes completely unfolded.

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.

NMR structure and dynamics of the chimeric protein SH3-F2

In order to further elucidate structural and dynamic principles of protein self-organization and protein-ligand interactions, a new chimeric protein was designed and a genetically engineered construct was created. SH3-F2 amino acid sequence consists of polyproline ligand mgAPPLPPYSA, GG linker, and the sequence of spectrin SH3 domain circular permutant S19-P20s. Structural and dynamic properties of the protein were studied with high-resolution NMR. According to NMR data, the tertiary structure of the chimeric protein SH3-F2 has a topology that is typical for SH3 domains in the complex with the ligand forming polyproline type II helix located in the conservative region of binding in the orientation II. The polyproline ligand closely adjoins with the protein globule and is stabilized by hydrophobic interactions. However, the interactions of the ligand and the part of globule related to SH3 domain is not too large, because the analysis of protein dynamical characteristics points to the low amplitude, high-frequency ligand tumbling relative to the slow intramolecular motions of the main globule. The constructed chimera allows carrying out further structural and thermodynamic investigations of polyproline helix properties and its interaction with regulatory domains.

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.