Development of biochemistry in the Biology Department of Kharkhov State University. Formation of a new direction--physiology and biochemistry of aging

The article presents some data on teaching Biochemistry at the Nature Department of Kharkiv Imperial University Physico-Mathematical Faculty, as well as restoration of biochemical education and scientific researches as a result of the University reorganization in the Soviet period and foundation of the Biochemistry Chair at the Biological Faculty in the renovated Kharkiv State University. The analysis of scientific biochemical investigations conducted in the Kharkiv University till now is revealed. The special attention is paid to development of such a scientific trend as age physiology and biochemistry. The article deals with the comprehensive information on scientific and pedagogical activity of the outstanding scientists such as O. V. Nagorny and I. M. Bulankin as founders of the Kharkiv school of age physiology and biochemistry. The work has utilized some archive data.     

V.N. Karazin Kharkov National University as the foundation of biochemistry in Ukraine

The paper deals with the data on foundation and development of physiological chemistry (biochemistry) as independent science and education subject in the V. N. Karazin Kharkov National University before and after the organization of the Department of Physiological Chemistry. Studying the chemistry of natural compounds, their qualitative and quantitative content and transformations in living organisms both by foreign and home researchers made the basis for the appearance of physiological chemistry as static biochemistry. The improvement of the investigation methods and further discoveries caused the appearing of new branches--dynamic and functional biochemistry. The attention is paid to the fact that biochemistry arised at the Kharkov University as the education subject (A. I. Khodnev) and then developed as independent science due to efforts of A. Ya. Danilevskiy as well as biochemical school created by him. The Kazan' and Kharkov periods of scientific activity of A.Ya. Danilevskiy are described. The leading role of A. Ya. Danilevskiy in development of the home school of biochemistry is considered. Important role of A. V. Palladin in the foundation of Kharkov biochemists' school and organization of the Scientific-research Institute of Biochemistry in Kharkov is considered as well. It is stated that the Institute of Biochemistry after its arrival to Kiev and joining the Academy of Sciences became the center of Ukrainian biochemistry. The role of A. V. Nagorny and I. N. Bulankin in further development of biochemistry and foundation of a new scientific branch--age-related physiology and biochemistry--at the Kharkov University after its re-organization is discussed.     

Foundation and development of biochemistry at the Imperial Kharkov University

The article summarizes the biochemical researches carried out at Kharkiv Imperial University from the middle of XIX century up to the cessation of its existence in 1920 as a result of transformation into the Kharkiv Institute of National Education. Scientific activity at the Chair of Medical Chemistry at Medical Department is described in details. Information on professors who led the chair and their researches are represented. Among them a great attention is spared to the Kharkiv works of such famous scientists as A. Danilevsky and V. Gulevich, who made a great contribution to the development of Russian and world biochemistry. There are also some resordes about researches of biological and physiological chemistry carried out at other chairs of Medical Department and Department of Physics and Mathematics of the Kharkiv University. In particular, the article presents the works of well-known plant physiologists and biochemists prof. V. Palladin and V. Zalessky, and the endocrinological researches led by prof. A. Reprev.     

Lessons from nature--protein fibers.

Silks are protein fibers with remarkable mechanical properties. The discovery of the structural features that govern these properties is a challenge for biochemistry and structural biology. This review summarizes the results of the biochemistry of silk proteins as well as the knowledge of the molecular biology of the respective genes. In addition, an overview is presented on the efforts to produce recombinant silk proteins by biotechnological techniques.     

Effect of acrylamide on aldolase structure. I. Induction of intermediate states.

Acrylamide is a fluorescence quencher frequently applied for analysis of protein fluorophores exposure with the silent assumption that it does not affect the native structure of protein. In this report, it is shown that quenching of tryptophan residues in aldolase is a time-dependent process. The Stern-Volmer constant increases from 1.32 to 2.01 M-1 during the first 100 s of incubation of aldolase with acrylamide. Two tryptophan residues/subunit are accessible to quenching after 100 s of aldolase interaction with acrylamide. Up to about 1.2 M acrylamide concentration enzyme inactivation is reversible. Independent analyses of the changes of enzyme activity, 1ANS fluorescence during its displacement from aldolase active-site, UV-difference spectra and near-UV CD spectra were carried out to monitor the transition of aldolase structure. From these measurements a stepwise transformation of aldolase molecules from native state (N) through intermediates: I1, T, I2, to denatured (D) state is concluded. The maxima of I1, T, I2 and D states populations occur at 0.2, 1.0, 2.0 and above 3.0 M of acrylamide concentration, respectively. Above 3.5 M, acrylamide aldolase molecules become irreversibly inactivated.     

Pressure-induced local unfolding of the Ras binding domain of RalGDS

The reliable prediction of the precise three-dimensional structure of proteins from their amino acid sequence is a major, still unresolved problem in biochemistry. Pressure is a parameter that controls folding/unfolding transitions of proteins through the volume change DeltaV of the protein-solvent system. By varying the pressure from 30 to 2,000 bar we detected using 15N/ 1H 2D NMR spectroscopy a unique equilibrium unfolding intermediate I in the Ras binding domain of the Ral guanine nucleotide dissociation stimulator (Ral GDS). It is characterized by a local melting of specific structural elements near hydrophobic cavities while the overall folded structure is maintained.     

N.m.r. and biochemistry

The role and historical progress of n.m.r. applications in biochemistry are briefly outlined. Technical advances over the years have made n.m.r., at last, a technique which can give valuable information about a wide range of biochemical topics, from enzyme kinetics in vivo to the structure of protein-DNA complexes. Emphasis here is placed on studies of proteins, especially those made up from mosaics of modules. It is shown that n.m.r. can readily give detailed structural information about individual protein modules and that valuable information about the structure and function of the intact mosaic protein can be inferred.     

Opportunities and challenges for time-resolved studies of protein structural dynamics at X-ray free-electron lasers

X-ray free-electron lasers (XFELs) are revolutionary X-ray sources. Their time structure, providing X-ray pulses of a few tens of femtoseconds in duration; and their extreme peak brilliance, delivering approximately 10¹² X-ray photons per pulse and facilitating sub-micrometre focusing, distinguish XFEL sources from synchrotron radiation. In this opinion piece, I argue that these properties of XFEL radiation will facilitate new discoveries in life science. I reason that time-resolved serial femtosecond crystallography and time-resolved wide angle X-ray scattering are promising areas of scientific investigation that will be advanced by XFEL capabilities, allowing new scientific questions to be addressed that are not accessible using established methods at storage ring facilities. These questions include visualizing ultrafast protein structural dynamics on the femtosecond to picosecond time-scale, as well as time-resolved diffraction studies of non-cyclic reactions. I argue that these emerging opportunities will stimulate a renaissance of interest in time-resolved structural biochemistry.     

EF手图像超家族成员——肌钙蛋白C的研究进展

EF手图像超家族蛋白泛指一类含有由螺旋区-泡区-螺旋区构成的EF手图像模体的蛋白。这类蛋白通常都具有金属离子结合能力或者形成二聚体的能力。肌钙蛋白C是一种EF手图像蛋白,它具有钙离子结合能力,可以与肌钙蛋白I、肌钙蛋白T形成复合物调节肌肉收缩。目前,国内外对肌钙蛋白C的研究多数集中在脊椎动物上,而对无脊椎动物的研究较少。主要从EF手图像超家族的特性及其家族成员——肌钙蛋白C的分类、结构及功能等方面进行了阐述,并结合笔者自身的研究方向,简要介绍了家蚕肌钙蛋白C的研究情况及前景。     

The Cys3-Cys4 loop of the hydrophobin EAS is not required for rodlet formation and surface activity

Class I hydrophobins are fungal proteins that self-assemble into robust amphipathic rodlet monolayers on the surface of aerial structures such as spores and fruiting bodies. These layers share many structural characteristics with amyloid fibrils and belong to the growing family of functional amyloid-like materials produced by microorganisms. Although the three-dimensional structure of the soluble monomeric form of a class I hydrophobin has been determined, little is known about the molecular structure of the rodlets or their assembly mechanism. Several models have been proposed, some of which suggest that the Cys3-Cys4 loop has a critical role in the initiation of assembly or in the polymeric structure. In order to provide insight into the relationship between hydrophobin sequence and rodlet assembly, we investigated the role of the Cys3-Cys4 loop in EAS, a class I hydrophobin from Neurospora crassa. Remarkably, deletion of up to 15 residues from this 25-residue loop does not impair rodlet formation or reduce the surface activity of the protein, and the physicochemical properties of rodlets formed by this mutant are indistinguishable from those of its full-length counterpart. In addition, the core structure of the truncation mutant is essentially unchanged. Molecular dynamics simulations carried out on the full-length protein and this truncation mutant binding to an air-water interface show that, although it is hydrophobic, the loop does not play a role in positioning the protein at the surface. These results demonstrate that the Cys3-Cys4 loop does not have an integral role in the formation or structure of the rodlets and that the major determinant of the unique properties of these proteins is the amphipathic core structure, which is likely to be preserved in all hydrophobins despite the high degree of sequence variation across the family.     

Conservativeness of residues in nonpolar nuclei of microbial ribonucleases]

We present the application of a new method for analysis of nonpolar structure of proteins. A detailed analysis of the composition and properties of nonpolar nuclei and microclusters of microbial ribonucleases with known sequence have been carried out on the basis of 3D-structure of RNase Pbi and that of RNase Ti. It has been shown that all residues in nonpolar nuclei have high homology, about 95% for proteins with an identical scheme of S-S bridges and about 75% for nonidentical scheme of S-S bridges. The stability of nonpolar nuclei, conservation of their composition and their position in the protein globule allows one to assume that they play an important functional role in protein structure and possibly can be considered as independent structural elements of 3D-structure of a protein.     

On Simplified Global Nonlinear Function for Fitness Landscape: A Case Study of Inverse Protein Folding

The construction of fitness landscape has broad implication in understanding molecular evolution, cellular epigenetic state, and protein structures. We studied the problem of constructing fitness landscape of inverse protein folding or protein design, with the aim to generate amino acid sequences that would fold into an a priori determined structural fold which would enable engineering novel or enhanced biochemistry. For this task, an effective fitness function should allow identification of correct sequences that would fold into the desired structure. In this study, we showed that nonlinear fitness function for protein design can be constructed using a rectangular kernel with a basis set of proteins and decoys chosen a priori. The full landscape for a large number of protein folds can be captured using only 480 native proteins and 3,200 non-protein decoys via a finite Newton method. A blind test of a simplified version of fitness function for sequence design was carried out to discriminate simultaneously 428 native sequences not homologous to any training proteins from 11 million challenging protein-like decoys. This simplified function correctly classified 408 native sequences (20 misclassifications, 95% correct rate), which outperforms several other statistical linear scoring function and optimized linear function. Our results further suggested that for the task of global sequence design of 428 selected proteins, the search space of protein shape and sequence can be effectively parametrized with just about 3,680 carefully chosen basis set of proteins and decoys, and we showed in addition that the overall landscape is not overly sensitive to the specific choice of this set. Our results can be generalized to construct other types of fitness landscape.     

Prediction of the changes in thermodynamic stability of proteins caused by single amino acid substitutions

Prediction of the effect of amino acid substitutions on the thermodynamic stability of proteins is of great importance for studies into the molecular mechanisms underlying the abnormal function of mutant proteins, interpretation of genotyping results, and purposeful design of modified proteins with improved biomedical and biotechnological properties. A set of methods was developed for predicting the changes in free energy (ΔΔG) of mutant proteins containing single substitutions using the information only about protein primary structure or also about the spatial structure. A modified KRAB algorithm was used; its higher accuracy in predicting the changes in the thermodynamic stability of mutant proteins compared with the other known methods designed for solving this problem is demonstrated. Distribution of the positions in the sequence of Malayan pit viper venom protein (kistrin) where the substitutions decrease or increase kistrin stability is analyzed. The substitutions at most positions conserved in the disintegrin family decrease the stability of this protein, except for several positions whose conservation can be determined by functional significance.     

Structure characterization of the central repetitive domain of high molecular weight gluten proteins. II. Characterization in solution and in the dry state.

The structure of the central repetitive domain of high molecular weight HMW) wheat gluten proteins was characterized in solution and in the dry state using HMW proteins Bx6 and Bx7 and a subcloned, bacterially expressed part of the repetitive domain of HMW Dx5. Model studies of the HMW consensus peptides PGQGQQ and GYYPTSPQQ formed the basis for the data analysis (van Dijk AA et al., 1997, Protein Sci 6:637-648). In solution, the repetitive domain contained a continuous nonoverlapping series of both type I and type II II beta-turns at positions predicted from the model studies; type II beta-turns occurred at QPGQ and QQGY sequences and type I beta-turns at YPTS and SPQQ. The subcloned part of the HMW Dx5 repetitive domain sometimes migrated as two bands on SDS-PAGE; we present evidence that this may be caused by a single amino acid insertion that disturbs the regular structure of beta-turns. The type I beta-turns are lost when the protein is dried on a solid surface, probably by conversion to type II beta-turns. The homogeneous type II beta-turn distribution is compatible with the formation of a beta-spiral structure, which provides the protein with elastic properties. The beta-turns and thus the beta-spiral are stabilized by hydrogen bonds within and between turns. Reformation of this hydrogen bonding network after, e.g., mechanical disruption may be important for the elastic properties of gluten proteins.     

基于蛋白质受体的药物分子计算机辅助设计策略

药物分子计算机辅助设计是一种在计算机或者理论上通过构建具有一定潜在药理活性的新化学实体的分子模拟方法。近十几年来,高通量组学技术的快速发展为生物和化学药物分子设计提供了良好的数据支撑和研究契机。另外,现代社会对生物制药合理性以及作用机理理解的要求越来越高,行业普遍要求药物需要有高效、无毒或者低毒以及靶向性强等特点。随着越来越多与药物靶点相关的蛋白质结构通过实验方法解析出来,基于蛋白质受体的药物分子设计方法可行性进一步提高,其方法也变得越来越重要。基于蛋白质受体的药物分子设计方法,一般是以蛋白质以及配体的三维结构出发进行分析,这让药物分子先导物的发现更加理性化。随着相关实验数据的积累以及深度学习等算法的发展,从而可以进行更加科学的药物分子设计,这在一定程度上加快了新药研发的进程,并更有利于探索相应的分子机理。本文对基于蛋白质受体的药物分子设计方法的常用策略进行系统的回顾、总结和展望。     

Solubilization of coat protein from Bacillus thiaminolyticus spores.

Solubilization of spore coat protein of Bacillus thiaminolyticus was investigated using various reagents, and partial characterization of solubilized protein was carried out. Five per cent of the sodium dodecyl sulphate (SDS) treatment was the most effective for solubilization of coat protein, and 5% SDS + 8 M urea and 0.06 N NaOH were also useful. Acrylamide gel disc electrophoresis indicated that the SDS-soluble fraction mainly consists of a single band of protein and its molecular weight was estimated at about 15,000. The SDS+ urea-soluble fraction comprised two proteins with a molecular weight of 14,500 and 32,000, and an alkali-soluble fraction of 12,000 and 25,000 respectively.     

Human serum albumin binding ibuprofen: A 3D description of the unfolding pathway in urea

Small angle X-ray scattering (SAXS) technique, supported by light scattering measurements and spectroscopic data (circular dichroism and fluorescence) allowed us to restore the 3D structure at low resolution of defatted human serum albumin (HSA) in interaction with ibuprofen. The data were carried out on a set of HSA solutions with urea concentrations between 0.00 and 9.00 M. The Singular Value Decomposition method, applied to the complete SAXS data set allowed us to distinguish three different states in solution. In particular a native conformation N (at 0.00 M urea), an intermediate I1 (at 6.05 M urea) and an unfolded structure U (at 9.00 M urea) were recognized. The low-resolution structures of these states were obtained by exploiting both ab initio and rigid body fitting methods. In particular, for the protein without denaturant, a conformation recently described (Leggio et al., PCCP, 2008, 10, 6741–6750), very similar to the crystallographic heart shape, with only a slight reciprocal movement of the three domains, was confirmed. The I1 structure was instead characterized by only a closed domain (domain III) and finally, the recovered structure of the U state revealed the characteristic feature of a completely open state. A direct comparison with the free HSA pointed out that the presence of the ibuprofen provokes a shift of the equilibrium towards higher urea concentrations without changing the unfolding sequence. The work represents a type of analysis which could be exploited in future investigations on proteins in solution, in the binding of drugs or endogenous compounds and in the pharmacokinetic properties as well as in the study of allosteric effects, cooperation or anticooperation mechanisms.     

Ranking valid topologies of the secondary structure elements using a constraint graph

Electron cryo-microscopy is a fast advancing biophysical technique to derive three-dimensional structures of large protein complexes. Using this technique, many density maps have been generated at intermediate resolution such as 6-10 ? resolution. Although it is challenging to derive the backbone of the protein directly from such density maps, secondary structure elements such as helices and β-sheets can be computationally detected. Our work in this paper provides an approach to enumerate the top-ranked possible topologies instead of enumerating the entire population of the topologies. This approach is particularly practical for large proteins. We developed a directed weighted graph, the topology graph, to represent the secondary structure assignment problem. We prove that the problem of finding the valid topology with the minimum cost is NP hard. We developed an O(N(2)2(N)) dynamic programming algorithm to identify the topology with the minimum cost. The test of 15 proteins suggests that our dynamic programming approach is feasible to work with proteins of much larger size than we could before. The largest protein in the test contains 18 helical sticks detected from the density map out of 33 helices in the protein.     

Structure and functional features of enzymes from the Ca2+-phospholipid-dependent protein kinase family

Present day scientific data about the Ca(2+)-phospholipid-dependent protein kinases structure and molecular mechanisms of their activity are summarized and analyzed in this review. Ca(2+)-phospholipid-dependent protein kinases family is well known to include a whole series of enzymes which are homologous by their structure. They play an important role in cell differentiation, growth and proliferation as well as signal transduction through the cytoplasmic membrane. They also take part in cell response realization by phosphorylation of target proteins. Now application of modern biochemical and biophysical methods provided for possibility of the clarification of these enzymes structure features. The great lot of experimental data about the molecular mechanisms of Ca(2+)-phospholipid-dependent protein kinases activity regulation by phosphatydyle serine, phorbol ethers, saturated and unsaturated fatty acids, calcium iones, autophosphorylation and holoferment phosphorylation by other kinases was obtained. As a model of Ca(2+)-phospholipide-dependent protein kinases regulation was their development on the base of the scientific data about this problem.     

Quantitative proteomic analysis of ibuprofen-degrading Patulibacter sp. strain I11

Ibuprofen is the third most consumed pharmaceutical drug in the world. Several isolates have been shown to degrade ibuprofen, but very little is known about the biochemistry of this process. This study investigates the degradation of ibuprofen by Patulibacter sp. strain I11 by quantitative proteomics using a metabolic labelling strategy. The whole-genome of Patulibacter sp. strain I11 was sequenced to provide a species-specific protein platform for optimal protein identification. The bacterial proteomes of actively ibuprofen-degrading cells and cells grown in the absence of ibuprofen was identified and quantified by gel based shotgun-proteomics. In total 251 unique proteins were quantitated using this approach. Biological process and pathway analysis indicated a number of proteins that were up-regulated in response to active degradation of ibuprofen, some of them are known to be involved in the degradation of aromatic compounds. Data analysis revealed that several of these proteins are likely involved in ibuprofen degradation by Patulibacter sp. strain I11.