Solid Xenon Carrier Based on α-Cyclodextrin: Properties, Preparation, and Application.

The inert gas xenon (Xe) is increasingly used in medicine as a universal anesthetic, a regulator of cellular metabolism, and a broad-spectrum organoprotector. Commonly utilized Xe inhalation requires expensive equipment that is not universally available. Here we describe the production process and physical characteristics of a solid, highly stable xenon carrier based on α-cyclodextrin (α-CD), developed for oral administration. It was found, that the interaction of α-CD with Xe in an aqueous solution and elevated pressure leads to precipitation of the α-CD-Xe complex. We have discovered three new properties of the resulting complex that promote long-term storage and oral delivery of Xe. (i) At temperatures below 0 °C, the precipitated α-CD-Xe complex containing water is so stable that it allows the removal of water by vacuum freeze-drying (lyophilization). (ii). Lyophilized α-CD-Xe remains stable for months at room temperature. (iii) Upon contact with water, α-CD-Xe rapidly releases gaseous Xe. As revealed in the forced swim test, after oral administration of lyophilized α-CD-Xe to rats, the duration of swimming was significantly increased. The obtained data open up prospects for the development of drugs based on the lyophilized α-CD-Xe complex suitable for storage, transportation, and medical use, including outside the hospital.

Comparative analysis of the active sites of orthologous endolysins of the Escherichia lytic bacteriophages T5, RB43, and RB49.

The methods of solution NMR, circular dichroism (CD), and differential scanning calorimetry (DSC) were used to study two zinc-containing L-alanyl-D-glutamate peptidases - endolysins of the pseudo T-even myoviruses RB43 and RB49 (EndoRB43 and EndoRB49, respectively), which are orthologous to the EndoT5, which is a zinc-containing L-alanyl-D-glutamate peptidase of the T5 siphovirus. The spatial conservation of the Zn2+-binding sites for the enzymes EndoT5, EndoRB43, and EndoRB49 was established, and the key role of Zn2+ ions in the stabilization of the spatial structures of these three peptidases was confirmed. We are showing here that the binding of the Zn2+ ion in the active center of EndoRB49 peptidase causes conformational rearrangements similar to those observed in the EndoT5 peptidase upon binding of Zn2+ and Ca2+ ions and lead to the formation of a catalytically active form of the enzyme. Therefore, the binding of the Zn2+ ion to the active site of EndoRB49 peptidase is a necessary and sufficient condition for functioning of this protein.

On the roles of calcium and zinc ions in the formation of a catalytically active form of the metalloenzyme, l-alanyl-d-glutamate peptidase of the bacteriophage T5 (EndoT5).

Structural consequences of the binding of metal ions (regulatory Ca2+ and catalytic Zn2+) to the metalloenzyme l-alanyl-d-glutamate peptidase of the bacteriophage T5 (Endo T5) and some of its analogues containing single amino acid substitutions in the active center were analyzed by nuclear magnetic resonance (NMR), circular dichroism (CD) and calorimetry. Analyses revealed that the native EndoT5 undergoes strong structural rearrangements as a result of Zn2+ binding. This structural rearrangement resulting in the formation of an active enzyme is completed by the Ca2+ binding. In this case, the NMR spectra uncover the tautomerism of the NH protons of histidine imidazoles responsible for the Zn2+ coordination. For the EndoT5 analogues with point substitutions in the Ca2+-binding site, similar conformational rearrangements are observed upon Zn2+ binding. However, no characteristic changes in the NMR spectra associated with the Ca2+ binding were detected. The roles of the proton exchange in the process of Ca2+-induced activation of the enzymatic activity of EndoT5 is discussed.

Can a retro-polypeptide fold into a globule?

Communicated by Ramaswamy H. Sarma.

Investigation of the calcium-induced activation of the bacteriophage T5 peptidoglycan hydrolase promoting host cell lysis.

Peptidoglycan hydrolase of bacteriophage T5 (EndoT5) is a Ca2+-dependent l-alanyl-d-glutamate peptidase, although the mode of Ca2+ binding and its physiological significance remain obscure. Site-directed mutagenesis was used to elucidate the role of the polar amino acids of the mobile loop of EndoT5 (111-130) in Ca2+ binding. The mutant proteins were purified to electrophoretic homogeneity, the overall structures were characterized by circular dichroism, and the calcium dissociation constants were determined via NMR spectroscopy. The data suggest that polar amino acids D113, N115, and S117 of EndoT5 are involved in the coordination of calcium ions by forming the core of the EF-like Ca2+-binding loop while the charged residues D122 and E123 of EndoT5 contribute to maintaining the loop net charge density. The results suggest that Ca2+ binding to the EndoT5 molecule could be essential for the stabilization of the long mobile loop in the catalytically active "open" conformation. The possible mechanism of Ca2+ regulation of EndoT5 activity during bacteriophage T5's life cycle through the Ca2+ concentration difference between the cytoplasm and the periplasm of the host bacteria cell has been discussed. The study reveals valuable insight into the role of calcium in the regulation of phage-induced bacterial lysis.

Effect of C-terminal His-tag and purification routine on the activity and structure of the metalloenzyme, l-alanyl-d-glutamate peptidase of the bacteriophage T5.

In this work, we studied the effect of the C-terminally attached poly-histidine tag (His-tag), as well as the peculiarities of the protein purification procedure by the immobilized metal affinity chromatography (IMAC) on the activity and structure of the metalloenzyme, l-alanyl-d-glutamate peptidase of bacteriophage T5 (EndoT5), whose zinc binding site and catalytic aspartate are located near the C-terminus. By itself, His-tag did not have a significant effect on either activity or folding of the polypeptide chain, nor on the binding of zinc and calcium ions to the protein. However, the His-tagged EndoT5 samples had low shelf-life, with storage of these samples resulting in an increased propensity for protein self-association and decreased enzymatic activity of EndoT5. Furthermore, disastrous effects on the activity of the enzyme were exerted by the presence of imidazole and nickel ions accompanying metal chelate chromatography. The activity of the protein can be restored by thorough washing off of these low molecular impurities via the prolonged dialysis of the His-tagged EndoT5 samples at the specifically elaborated conditions.

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.

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.

Membrane-induced changes in the holomyoglobin tertiary structure: interplay with function.

The effect of anionic phospholipid membranes on holomyoglobin (holoMb) conformation and deoxygenation was studied. HoloMb structural changes and behavior in the presence of membranes were monitored by a variety of techniques including far UV and near UV circular dichroism, tryptophan (Trp) fluorescence, absorbance in the Soret region, differential scanning calorimetry, (1)H-NMR spectroscopy, size exclusion chromatography, and macroscopic diffusion. Kinetics of deoxygenation was monitored by absorption at 581 nm. The results gave evidence that proximity to a negatively charged membrane surface can cause destabilization of the structure of holomyoglobin, which delivers oxygen (O2) to mitochondria. It was shown that holoMb undergoes the native-to-intermediate-state transition in the presence of anionic phospholipid membranes at neutral pH, and that in this state it is able to interact with the membranes. When in the intermediate state, holoMb loses its rigid tertiary structure but preserves a pronounced secondary one. The presence of anionic phospholipid membranes substantially accelerates the process of deoxygenation. A possible functional role of the more flexible protein structure acquired in immediate proximity to the membrane surface is discussed.

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.

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

We analyzed the applicability of high-resolution (2)H-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.

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.

Solution structure and dynamics of the chimeric SH3 domains, SHH- and SHA-"Bergeracs".

Two chimeric proteins, SHcapital EN, Cyrillic and SHA of the "SH3-Bergerac" family (where the beta-turn N47D48 in spectrin SH3 domain was substituted for KITVNGKTYE or KATANGKTYE sequences, respectively), were analyzed by high-resolution NMR to resolve their spatial structures and to analyze their dynamics. Although the presence of a stable beta-hairpin in the region of the insertion was confirmed, the introduced extension of the polypeptide chain in SHcapital EN, Cyrillic (approximately 17%) practically did not affect the total molecule topology. Interestingly, the introduced beta-hairpin had higher mobility in comparison with other protein regions. Finally, we performed a disorder prediction with the PONDR VSL2 algorithm and discovered that the inserted beta-hairpin in both SHH and SHA proteins exhibited significant propensity for intrinsic disorder and therefore for high mobility. In agreement with the experimental data, the predisposition for the increased intramolecular mobility was noticeably higher in SHA.

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.