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E I Tiktopulo

Publications and source records attributed to E I Tiktopulo.

At least 19 recordsLinked to original sources
PubMed2002

Extended conformation of mammalian translation elongation factor 1A in solution.

The conformation of mammalian elongation factor eEF1A in solution was examined by the small angle neutron scattering and scanning microcalorimetry. We have found that in contrast to the bacterial analogue the eEF1A molecule has no fixed rigid structure in solution. The radius of gyration of the eEF1A molecule (5.2 nm) is much greater than that of prokaryotic EF1A. The specific heat of denaturation is considerably lower for eEF1A than for EF1A, suggesting that the eEF1A conformation is significantly more disordered. Despite its flexible conformation, eEF1A is found to be highly active in different functional tests. According to the neutron scattering data, eEF1A becomes much more compact in the complex with uncharged tRNA. The absence of a rigid structure and the possibility of large conformational change upon interaction with a partner molecule could be important for eEF1A functioning in channeled protein synthesis and/or for the well-known capability of the protein to interact with different ligands besides the translational components.

Animals↗
PubMed2001

Structure of Cry3A delta-endotoxin within phospholipid membranes.

Interaction of delta-endotoxin and its proteolytic fragments with phospholipid vesicles was studied using electron microscopy, scanning microcalorimetry, and limited proteolysis. It was shown that native protein destroys liposomes. The removal of 4 N-terminal alpha-helices and the extreme 56 C-terminal amino acid residues did not affect this ability. The results obtained by limited proteolysis of delta-endotoxin bound to lipid vesicles show essential conformational changes in three or four N-terminal helices and in the C-terminal region. The calorimetric method used in this study provides a unique possibility for the validation of existing models of protein binding and for a more accurate determination of the regions where conformational changes take place. It was found that the binding of the protein to model liposomes does not alter its structure in the regions starting with the fourth alpha-helix of domain I. This can be concluded from the fact that the activation energy of denaturation of the protein remains unchanged upon its binding to the phospholipid membranes. A new structural model has been proposed which agrees with the data obtained.

Animals↗
PubMed2001

Folding properties of functional domains of tropomodulin.

Tropomodulin (Tmod) stabilizes the actin-tropomyosin filament by capping the slow-growing end (P-end). The N- and C-terminal halves play distinct roles; the N-terminal half interacts with the N-terminal region of tropomyosin, whereas the C-terminal half interacts with actin. Our previous study (A. Kostyukova, K. Maeda, E. Yamauchi, I. Krieger, and Y. Maéda Y., 2000, Eur. J. Biochem. 267:6470-6475) suggested that the two halves are also structurally distinct from each other. We have now studied the folding properties of the two halves, by circular dichroism spectroscopy and by differential scanning calorimetry of the expressed chicken E-type tropomodulin and its large fragments. The results showed that the C-terminal half represents a single, independently folded unit that melts cooperatively through a two-state transition. In contrast, the N-terminal half lacks a definite tertiary structure in solution. The binding of N11, a fragment that corresponds to the first 91 amino acids of the tropomodulin, to tropomyosin substantially stabilized the tropomyosin. This may indicate that the flexible structure of the N-terminal half of tropomodulin in solution is required for binding to tropomyosin and that the N-terminal half acquires its tertiary structure upon binding to tropomyosin.

Animals↗
PubMed1999

Transition state of the rate-limiting step of heat denaturation of Cry3A delta-endotoxin.

Heat denaturation of Cry3A delta-endotoxin from Bacillus thuringiensis var. tenebrionis and its 55 kDa fragment was studied by differential scanning microcalorimetry at low pH. Analysis of the calorimetric data has shown that denaturation of Cry3A delta-endotoxin is a nonequilibrium process at heating rates from 0. 125 to 2 K/min. This means that the stability of delta-endotoxin (the apparent temperature of denaturation Tm) under these conditions is under kinetic control rather than under thermodynamic control. It has been shown that heat denaturation of this protein is a one-step kinetic process. The enthalpy of the process and its activation energy were measured as functions of temperature. The data obtained allow confirmation of the fact that the conformation of delta-endotoxin at the transition state only slightly differs from its native conformation with respect to compactness and extent of hydration. The comparison of the activation energy for intact delta-endotoxin and the 55 kDa fragment showed that the transition of the molecule to a transition state does not cause any changes in the conformation of three N-terminal alpha-helices. Complete removal of the N-terminal domain of delta-endotoxin and 40 amino acids from the C-terminus beta-sheet domain III causes an irreversible loss of the tertiary structure. Thus, during protein folding the nucleation core determining protein stability does not involve its three initial alpha-helices but may include the remaining alpha-helices of the N-terminal domain. The functional significance of peculiarities of structure arrangement of the delta-endotoxin molecule is discussed.

Bacillus thuringiensis↗
PubMed1998

Denaturation of type I collagen fibrils is an endothermic process accompanied by a noticeable change in the partial heat capacity.

Thermal transitions of type I collagen fibrils were investigated by differential scanning calorimetry and spectrophotometry of turbidity within a wide range of external conditions. The advanced microcalorimeter allowed us to carry out the measurements at low concentrations of collagen (0.15-0.3 mg/mL). At these concentrations of collagen and under fibril-forming conditions, the melting curves display two pronounced heat adsorption peaks (at 40 and 55 degreesC). The low-temperature peak was assigned to the melting of monomeric collagen, while the high-temperature peak was assigned to the denaturation of collagen fibrils. It was shown that the denaturation of fibrils, in contrast to the monomeric collagen, is accompanied by a noticeable change in the partial specific heat capacity. Surprisingly, comparison of the collagen calorimetric curves in the fibril-forming and nonforming conditions revealed that DeltaCp of fibril denaturation is caused by a decrease in the Cp of collagen at premelting temperatures. This suggests the existence of an intermediate structural state of collagen in a transparent solution preceding fibril formation. Our study also shows that collagen fibrils formed prior to heating have thermodynamic parameters different from those of fibrils formed and denatured during heating in the calorimeter. Analysis of the data allowed us to determine the denaturation enthalpy of the mature fibrils and to conclude that the enthalpy plays a more important role in fibril stabilization than was previously assumed. The observed large DeltaCp value of fibril denaturation as well as the difference between thermodynamic parameters of the mature and newly formed fibrils is readily explained by the presence of water molecules in the fibril structure.

Absorption↗
PubMed1996

Molten globule-like state of cytochrome c under conditions simulating those near the membrane surface.

Methanol-induced conformational transitions in cytochrome c have been studied by near- and far-UV circular dichroism, Trp fluorescence, microcalorimetry, and diffusion measurements. The existence of at least two cooperative stages of transition has been shown. At the first stage, the native protein is transformed into an intermediate which has only traces of tertiary structure, but has a native-like secondary structure content and is relatively compact; i.e., it has properties of the molten globule state. On the second stage, the alcohol-induced molten globule is transformed into a more helical state, typical of proteins at high alcohol concentrations. The conditions at which the alcohol-induced molten globule exists (moderately low pH and moderately low dielectric constant) could be similar to those existing near negatively charged membrane surfaces. Consequently, these results might explain how the molten globule state can be achieved under physiological conditions.

Animals↗
PubMed1995

Unfolding of tertiary structure of Halobacterium halobium flagellins does not result in flagella destruction.

The structure of Halobacterium halobium R1M1 flagella is investigated by the methods of scanning microcalorimetry, circular dichroism, and electron microscopy. It is shown that melting curves of flagella in solutions with a different concentration of NaCl display only one peak of heat capacity that corresponds to one cooperatively melting domain. It is found that flagella do not dissociate after melting. The possible structural organization of archaebacterial flagella is discussed.

Amino Acid Sequence↗
PubMed1989

Heat capacity and conformation of proteins in the denatured state.

Heat capacity, intrinsic viscosity and ellipticity of a number of globular proteins (pancreatic ribonuclease A, staphylococcal nuclease, hen egg-white lysozyme, myoglobin and cytochrome c) and a fibrillar protein (collagen) in various states (native, denatured, with and without disulfide crosslinks or a heme) have been studied experimentally over a broad range of temperatures. It is shown that the partial heat capacity of denatured protein significantly exceeds the heat capacity of native protein, especially in the case of globular proteins, and is close to the value calculated for an extended polypeptide chain from the known heat capacities of individual amino acid residues. The significant residual structure that appears at room temperature in the denatured states of some globular proteins (e.g. myoglobin and lysozyme) at neutral pH results in a slight decrease of the heat capacity, probably due to partial screening of the protein non-polar groups from water. The heat capacity of the unfolded state increases asymptotically, approaching a constant value at about 100 degrees C. The temperature dependence of the heat capacity of the native state, which can be determined over a much shorter range of temperature than that of the denatured state and, correspondingly, is less certain, appears to be linear up to 80 degrees C. Therefore, the denaturational heat capacity increment seems to be temperature-dependent and is likely to decrease to zero at about 140 degrees C.

Animals↗
PubMed1988

Temperature-induced phase transitions in proteins and lipids. Volume and heat capacity effects.

The partial specific heat capacity and volume of globular proteins and dispersions of phosphatidylcholines in aqueous solutions have been determined over a broad temperature range using a precise scanning microcalorimeter and a vibrational densimeter. It is shown that the temperature-induced, gel-to-liquid crystalline phase transition in phosphatidylcholines proceeds without a noticeable change in heat capacity but with a significant increase in the specific volume, whereas heat denaturation in proteins takes place without a noticeable change in the volume but with a significant increase in heat capacity. This principal difference between temperature-induced conformational phase transitions in proteins and lipids demonstrates clearly that heat denaturation of proteins, in contrast to the gel-to-liquid crystalline phase transition in lipids, cannot be regarded as a process similar to melting. Consequently, the 'molten globule' does not appear to be a suitable model for a heat-denatured protein.

Calorimetry, Differential Scanning↗
PubMed1985

Compact state of a protein molecule with pronounced small-scale mobility: bovine alpha-lactalbumin.

We describe a novel physical state of a protein molecule which is nearly as compact as the native state and has pronounced secondary structure, but differs from the native state by the large increase of thermal fluctuations (in particular, by the large mobility of side groups). This state has been characterized in detail for the acid form of bovine alpha-lactalbumin as a result of the study of physical properties of this state by a large variety of different methods (hydrodynamics, diffuse X-ray scattering, circular dichroism and infrared spectra, polarization of the luminescence, proton magnetic resonance, deuterium exchange and microcalorimetry). It has been shown that bovine alpha-lactalbumin can be transformed into a similar state by thermal denaturation. This process is thermodynamically two state (i.e. all-or-none transition), which means that this state differs from the native one by a phase transition of the first order.

Animals↗
PubMed1982

The central tryptic fragment of histones H1 and H5 is a fully compacted domain and is the only folded region in the polypeptide chain. A thermodynamic study.

Scanning microcalorimetry has been used to show that the globular structure in histones H1 and H5 melts reversibly over a relatively narrow temperature range as a single domain with enthalpy of melting very close to the van't Hoff enthalpy. These proteins therefore exhibit macroscopic cooperativity like that of many well-studied small proteins. For histone H5 the observed melting temperature and melting enthalpy coincide with those previously obtained [Eur. J. Biochem. 67, 379-388 (1976)] using intrinsic Cotton effects in the circular dichroism spectrum, but not with those from extrinsic Cotton effects or NMR chemical shifts. Calorimetric measurements on a central tryptic fragment from histones H1 and H5 show these fragments to have the same molar melting enthalpy as the parent histones. The folded structures of H1 and H5 are therefore located only within these fragments. For histone H1 this conclusion is supported by the finding that the C-terminal fragment (residues 122-213) shows no peak in the specific heat curve.

Calorimetry, Differential Scanning↗