Alexey V. Finkelstein

De novo design, synthesis and study of albebetin, a polypeptide with a predetermined three-dimensional structure. Probing the structure at the nanogram level.

The de novo polypeptide named albebetin was designed to form the tertiary fold that has not yet been observed in natural proteins. The design was based on the molecular theory of protein structures. The gene coding for this polypeptide was chemically synthesized. For the initial characterization of a protein structure, a new approach has been developed that uses only nanogram amounts of a polypeptide without its previous purification. This approach includes the biosynthesis of radiolabeled protein in a cell-free translation system with subsequent analysis of its compactness and structure by size-exclusion chromatography, urea-gradient electrophoresis and limited proteolysis. According to all tests used, albebetin has a compact stable structure.

How to determine the size of folding nuclei of protofibrils from the concentration dependence of the rate and lag-time of aggregation. I. Modeling the amyloid protofibril formation

The question about the size of nuclei of formation of protofibrils (which constitute mature amyloid fibrils) formed by different proteins and peptides is yet open and debatable because of the absence of solid knowledge of underlying mechanisms of amyloid formation. In this work, a kinetic model of the process of formation of amyloid protofibrils is suggested, which allows calculation of the size of the nuclei using only kinetic data. In addition to the stage of primary nucleation, which is believed to be present in many protein aggregation processes, the given model includes both linear growth of protofibrils (proceeding only at the cost of attaching of monomers to the ends) and exponential growth of protofibrils at the cost of growth from the surface, branching, and fragmentation with the secondary nuclei. Theoretically, only the exponential growth is compatible with the existence of a pronounced lag-period (which can take much more time then the growth of aggregates themselves). The obtained analytical solution allows us to determine the size of the primary and secondary nuclei from the experimentally obtained concentration dependences of the time of growth and the new parameter-the ratio Lrel of the lag-time duration to the time of growth of amyloid protofibrils.

cI and lexA repressors consist of three cro-like domains.

The switching of phages X and 434 from the lysogenic state to the lytic growth or vice versa depends mainly on 2 repressors: cro and cI. This switching can be induced by external agents such as UV light. On the other hand, various DNAdamaging agents (including UV light) induce the ‘SOS-system’ of Escherichia coli [l] which is normally inhibited by IexA repressor. Among the stages of the ‘SOS-system’ work one includes recA protein activation which cleaves proteolytically IexA repressor [2,3]; the same protein cleaves also c1 repressor 1451. binding of c1 to ~2 (which is cooperative with binding to 0~1) turns on (and, moreover, stimulates) the transcription of the c1 gene from PRM promoter. At the same time cro repressor does not bind cooperatively with the sites 0~1, 0~2, OR3 and its binding to OR3 turns off the transcription of c1 gene from mM promoter [lO,l 11. IexA protein binds with 2 DNA regions (each with an approximate 2-fold internal symmetry) before the 1exA gene and with one DNA region before the recA gene turning off the transcription of those genes [ 14,151.

S6 permutein shows that the unusual target topology is not responsible for the absence of rigid tertiary structure in de novo protein albebetin

Ribosomal protein S6 from Thermus thermophilus was modified to form the unusual unique topology designed earlier for a de novo protein albebetin. The S6 gene was cloned, sequenced and circularly permutated by means of genetic engineering methods. The permutated gene was expressed in Escherichia coli and the permutein was isolated and investigated by means of circular dichroism, fluorescence spectroscopy and scanning microcalorimetry. The permutated protein revealed a pronounced secondary structure close to that of the wild type S6 protein and a rigid tertiary structure possessing cooperative temperature melting. It means that the unusual new topology of albebetin is compatible with a rigid tertiary structure, it may be realized in natural proteins and it is not responsible for the absence of rigid structure in albebetin.

Cunning Simplicity of a Stoichiometry Driven Protein Folding Thesis

. present two statements, which, as they say, ‘reveal a surprisingly sim-ple unifying principle of backbone organization in protein folding’ (1). Namely, they assert (i) ‘that protein folding is a direct consequence of a narrow band of stoichiometric occurrences of amino-acids in primary sequences’, and (ii) ‘that “preferential interactions” between amino-acids do not drive protein folding, contrary to all prevalent views’. However, the first of these statements, presented quite ambiguously by Mittal

Prediction of Secondary Structure, Spatial Organization and Distribution of Antigenic Determinants for Hepatitis A Virus Proteins

On the basis of the secondary structure calculations from the known amino acid sequence we came to the conclusion that hepatitis A virus capsid proteins have the typical antiparallel beta-sheet bilayer structure. The predicted secondary structure of the HAV proteins can be well aligned with those of the poliovirus (type 1 Mahoney) and human rhinovirus (type 14). It enabled us to use the X-ray structure of the PV-1M and HRV-14 proteins as a template and then, firstly, to localize the positions of alpha and beta regions in the architecture of the HAV protein molecules and, secondly, to discover the amino acid homologies of the secondary structure regions aligned. The obtained model of the three-dimensional structure for HAV proteins helped us to indicate the exposed regions of the polypeptide chains and to pinpoint the potential neutralizing antigenic sites.

Determination of the Size of the Primary and Secondary Folding Nuclei of Protofibrils from the Concentration Dependence of the Rate and the Lag-Time of Their Formation

A kinetic model of the process of formation of amyloid protofibrils is proposed, allowing calculation of the nucleus size solely from kinetic data. This model includes the primary nucleation, the linear growth of protofibrils binding monomers to their ends, and a possible exponential growth of protofibrils at the cost of either their fragmentation, or branching with the secondary nuclei, or growth from the surface. Theoretically, only the exponential growth is compatible with the existence of a pronounced lag-period (that can take much more time than the following protofibril growth itself). The obtained analytical solution allows us to determine the size of primary and secondary nuclei from experimentally obtained concentration dependences of the growth time of amyloid protofibrils and their lag-time/growth-time ratio. These theoretical results were used to analyze experimental data taken from the literature and our own experimental data on amyloid formation.

Evaluation of the Accuracy of Calculation of the Standard Binding Entropy of Molecules from their Average Mobility in Molecular Crystals

One of the main problems in attempts to predict the binding constants of molecules (or free energies of their binding) is the correct evaluation of configurational binding entropy. This evaluation is possible by methods of molecular dynamics simulation, but these simulations require a lot of computational time. Earlier, we have developed an alternative approach which allows the fast calculation of the binding entropy from summarizing the available data on sublimation of crystals. Our method is based on evaluating the mean amplitude of the movements that are restricted in the bound molecule, e.g., in a crystal, but are not restricted in the free state, e.g., in vapor. In this work, it is shown that the standard entropy of binding of molecules by crystals under standard conditions (1 atm, 25°C) can be assessed rather accurately from geometric and physical parameters of the molecule and the average amplitude of the molecule motions in crystals estimated in our previous work.

Anomalous Kinetics of Amyloidogenesis Suggest a Competition between Oligomers and Fibrils

Meisl et al. have recently observed an anomalous dependence of the amyloid formation rate on the protein concentration. A novel mechanism of fibril growth has been proposed by Meisl et al. to explain the abnormality; it consists in the fibril-catalyzed initiation of fibril formation with saturation of catalytic sites at high concentrations of substrates. Our article describes an alternative explanation of the anomalous kinetics, assuming that the formation of metastable oligomers competes with fibril formation by decreasing the concentration of free monomers. Oligomers are indeed observed in the course of amyloid formation, but are usually considered as seeds of amyloid fibrils rather as their competitors. However, the oligomers visually detectable by electron microscopy were shown to be close in size to those that can be derived from the anomalous dependence of the amyloid growth rate on the protein concentration, given that the anomaly results from competition between oligomer formation and amyloidogenesis.

171 Determination of the size of folding nuclei of protofibrils from the concentration dependence of the rate and lag-time of their formation

repeats are present in proteins with a high number of interactions, we hypothesize that a strong positive selection acts in their evolution. Is there a link between diseases and occurrence of specific homo-repeats? Considering MIM database of human disease, we found that the homo-repeats with length larger than four for such amino acids as Leucine, Serine, Alanine, Glycine, and Proline have a larger propensity to be coupled with disease. Indeed, it has been found that developmental diseases are associated with homo-repeat expansions such as poly-A (alanine): synpolydactyly type II (HOXD13), blepharophimosis (FOXL2), oculopharyngeal muscular dystrophy (PABPN1), infantile spasm syndrome (ARX), and holoprosencephaly (ZIC2). Also, expansion of poly-Q is implicated in several neurodegenerative diseases, including Huntington’s disease and several spinocerebellar ataxia’s. The length of homo-repeats which can affect on aggregation properties has been found for each amino acid and compared with random proteomes. It has been found that the longer homorepeats occur in a protein the stronger aggregation ability we observe for protein sequence. The ability to regulate aggregation of proteins can be one of the general tools for the drug development.