Cholesterol induces uneven curvature of asymmetric lipid bilayers.

A remarkable flexibility is observed in biological membranes, which allows them to form the structures of different curvatures. We addressed the question of intrinsic ability of phospholipid membranes to form highly curved structures and the role of cholesterol in this process. The distribution of cholesterol in the highly curved asymmetric DOPC/DOPS lipid bilayer was investigated by the coarse-grained molecular dynamics simulations in the membrane patches with large aspect ratio. It is shown that cholesterol induces uneven membrane curvature promoting the formation of extended flattened regions of the membrane interleaved by sharp bends. It is shown that the affinity of cholesterol to anionic DOPS or neutral DOPC lipids is curvature dependent. The cholesterol prefers DOPS to DOPC in either planar or highly curved parts of the membrane. In contrast, in the narrow interval of moderate membrane curvatures this preference is inverted. Our data suggest that there is a complex self-consistent interplay between the membrane curvature and cholesterol distribution in the asymmetric lipid bilayers. The suggested new function of cholesterol may have a biological relevance.

[Physical-chemical properties of the mutant (protein) form of D-glucose/D-galactose-binding protein GGBP/H152C with an attached fluorescent dye BADAN].

The influence of various factors on the physico-chemical characteristics and complexation of glucose with a mutant form of D-glucose/D-galactose-binding protein which can be regarded as a sensor of the glucometer, namely the protein GGBP/H152C with solvatochromic dye BADAN attached to the cysteine residue Cys 152, has been investigated. The point mutation His 152Cys and attaching BADAN reduced the affinity of the mutant form GGBP/H152C to glucose more than 8-fold compared to the wild type protein. This allows using this mutant for the determination of sugar content in biological fluids extracted by transdermal technologies. Sufficiently rapid complexation of GGBP/H152C with glucose (the time of protein-glucose complex formation is not more than three seconds even in solutions with a viscosity of 4 cP) provides timely monitoring changes in the concentration of sugar. The changes of ionic strength and pH within the physiological range of values of these variables do not have significant influence on fluorescent characteristics of GGBP/H152C-BADAN. At acidic pH, (see symbol) some of the molecules GGBP/H152C is in the unfolded state. It has been shown that mutant form GGBP/H152C has relatively low resistance to guanidine hydrochloride denaturing effects. This result indicates the need for more stable proteins to create a sensor for glucose biosensor system.

The change of cellular membranes on apoptosis: fluorescence detection.

The strong plasma membrane asymmetry existing in living cells is lost on apoptosis, and it is commonly detected with the probes interacting strongly and specifically with phosphatidylserine (PS). This phospholipid becomes exposed to the cell surface, and the labeled annexin V is used for its detection. The requirement for early and Ca(2+)-independent detection of apoptosis in the formats of spectroscopy of cell suspensions, flow cytometry, microarray technology and confocal or two-photon microscopy stimulated efforts for the development of new methods. Since the PS exposure must produce integrated changes of electrostatic potential and hydration in the outer leaflet of cell membrane, its detection can be provided by direct response of smart fluorescence probes. This review is focused on basic mechanisms underlying the loss of membrane asymmetry during apoptosis and the principles lying in the background of new methods that demonstrate essential advantages over the annexin V-binding assay. The convenient wavelength-ratiometric technique based on fluorescent probe F2N12S is described in detail. It incorporates spontaneously into outer leaflet of cell membrane and the color change of its fluorescent emission associated with apoptosis can be easily detected. This article is part of a Special Issue entitled "Apoptosis: Four Decades Later".

Hierarchical clustering of the correlation patterns: new method of domain identification in proteins.

New method of identification of dynamical domains in proteins - Hierarchical Clustering of the Correlation Patterns (HCCP) is proposed. HCCP allows to identify the domains using single three-dimensional structure of the studied proteins and does not require any adjustable parameters that can influence the results. The method is based on hierarchical clustering performed on the matrices of correlation patterns, which are obtained by the transformation of ordinary pairwise correlation matrices. This approach allows to extract additional information from the correlation matrices, which increases reliability of domain identification. It is shown that HCCP is insensitive to small variations of the pairwise correlation matrices. Particularly it produces identical results if the data obtained for the same protein crystallized with different spatial positions of domains are used for analysis. HCCP can utilize correlation matrices obtained by any method such as normal mode or essential dynamics analysis, Gaussian network or anisotropic network models, etc. These features make HCCP an attractive method for domain identification in proteins.

Heat perturbation of bovine eye lens alpha-crystallin probed by covalently attached ratiometric fluorescent dye 4'-diethylamino-3-hydroxyflavone.

Bovine eye lens alpha-crystallin was covalently labeled with 6-bromomethyl-4'-diethylamino-3-hydroxyflavone and studied under native-like conditions and at the elevated temperature (60 degrees C) that is known to facilitate alpha-crystallin chaperone-like activity. This novel SH-reactive two-band ratiometric fluorescent probe is characterized by two highly emissive N*- and T*-bands; the latter appears due to excited state intramolecular proton transfer reaction. The positions of these bands and the ratio of their intensities for the alpha-crystallin-dye conjugate are the sensitive indicators of polarity of the dye environment and its participation in intermolecular hydrogen bonding. Although we found that the dye labels both the SH and the NH2 groups in alpha-crystallin, a recently developed procedure allowed us to distinguish between the heat-induced spectral changes of the dye molecules attached to SH and NH2 groups. We observed that at elevated temperature the environment of the SH-attached dye becomes more polar and flexible. The number of H-bond acceptor groups in the vicinity of the dye decreases. Since alpha-crystallin contains a single Cys residue within the C-terminal domain of its (alpha)A subunit (the (alpha)B subunit contains none), we can attribute the observed effects to temperature-induced changes in the C-terminal domain of this protein.

Towards realistic description of collective motions in the lattice protein folding models.

Collective motions and the formation of clusters of residues play an important role in the folding of real proteins. However, existing Monte Carlo (MC) techniques of the protein folding simulations based on highly popular lattice models provide only a schematic representation of collective motions, which is rather far from physical reality. The Clustering Monte Carlo (CMC) algorithm was developed with particular aim to provide a realistic description of collective motions on the lattice. CMC allows modeling the cluster dynamics and the effects of the solvent viscosity, which is impossible in conventional algorithms. In this study two 2D lattice peptides, with the ground states of hierarchical and non-hierarchical design, were investigated comparatively using three methods: Metropolis MC with the local move set, Metropolis MC with unspecific rigid rotations and the CMC algorithm. We present evidence that the folding pathways and kinetics of hierarchically folding clustered sequence are not adequately described in conventional MC simulations, and the account for cluster dynamics provided by CMC allows to capture essential features of the folding process. Our data suggest that the methods, which enable specific cluster motions, such as CMC, should be used for a more realistic description of protein folding.

On the excited-state energy transfer between tryptophan residues in proteins: the case of penicillin acylase.

The problem, whether excited-state energy transfer occurs between Trp residues in a multi-tryptophan proteins and if it does, what kind of changes it induces in different parameters of protein fluorescence, is currently under active investigation. In our previous paper [Biophys. Chem. 72 (1998) 265], the energy transfer was found and studied in detail for Na,K-ATPase. It was shown that this transfer influences all parameters of fluorescence emission, which is detected at site-selective conditions (red-edge of excitation, blue and red edges of emission). Present experiments were performed on unusually tryptophan-rich protein, bacterial penicillin acylase (28 Trp per dimer of 82 kDa) and were aimed to extend these observations. They demonstrate substantial heterogeneity in the environments of tryptophan residues within the protein structure. This suggests, that in the present case, if the energy transfer exists, it should be directed from short-wavelength-emitting to long-wavelength-emitting tryptophan residues and thus could be easily observed by a number of time-resolved and steady-state fluorescence techniques. Unexpectedly, no signature of inter-tryptophan energy transfer was found.

Recognition between flexible protein molecules: induced and assisted folding.

This review focuses on a very important but little understood type of molecular recognition--the recognition between highly flexible molecular structures. The formation of a specific complex in this case is a dynamic process that can occur through sequential steps of mutual conformational adaptation. This allows modulation of specificity and affinity of interaction in extremely broad ranges. The interacting partners can interact together to form a complex with entirely new properties and produce conformational signal transduction at substantial distance. We show that this type of recognition is frequent in formation of different protein-protein and protein-nucleic acid complexes. It is also characteristic for self-assembly of protein molecules from their unfolded fragments as well as for interaction of molecular chaperones with their substrates and it can be the origin of 'protein misfolding' diseases. Thermodynamic and kinetic features of this type of dynamic recognition and the principles underlying their modeling and analysis are discussed.

Concepts and misconcepts in the analysis of simple kinetics of protein folding.

Unusually simple two-state kinetics characterizes the folding of a number of small proteins possessing a variety secondary structures. This limits dramatically the number of experimentally resolvable parameters that may characterize this process and also suggests the possibility to describe it based on simple theories borrowed from the field of ordinary chemical reactions. An attempt is made to critically evaluate the basic concepts, which are in the background of this approach. We demonstrate their limitations, which may cast doubt on the interpretation of experimental data. It is shown also that, in contrast to provisions of transition state theory, the simple kinetics of protein folding does not correlate with folded state stability or with the size of the folding unit. Moreover, the folding kinetics exhibits anomalous dependence on temperature and pressure and surprisingly strong dependence on solvent viscosity. The possible role in folding of fluctuations, relaxations and gradient dynamics is discussed. Being overlooked or underestimated, these mechanisms may determine the rate and specificity of the process.

Solid-state conjugation of proteins with hydrophobic compounds in non-denaturing conditions. I. Acylation of proteins by dansyl proline using a polymeric N-hydroxysuccinimide ester.

A method of conjugating protein molecules under native conditions with water-insoluble hydrophobic compounds is developed. It permits very water-insoluble acids to be gently coupled to the primary amines on proteins or other biopolymers. For this purpose we synthesized a polymer (co-polymer of N-hydroxymaleimide and N-vinylpyrrolidone cross-linked by benzidine) which swells equally well in water and in organic solvents. Hydrophobic substances are first activated by esterification to this polymer in organic solvent and then conjugated to protein by acylation in aqueous medium at pH 8.0-8.5. Thus, the contact of native protein with organic co-solvent may be completely avoided. The application of this approach is demonstrated by labeling trypsinogen and plasminogen with dansyl proline.

[Protein folding with molecular chaperones:stochastic process under control]. / Folding belka s molekuliarnymi shaperonami:stakhasticheskii protsess pod kontrolem.

Protein folding in a living cell occurs with the participation of specialized proteins, molecular shaperons. The functional role and molecular mechanism of action of shaperons are discussed. It is shown that shaperons can be considered as proteins that, upon interaction with the folding peptide chain, transform the spontaneous folding to a process controlled and regulated by cellular factors. Models describing these controlled phenomena are discussed.

Fluorescence heterogeneity of tryptophans in Na,K-ATPase: evidences for temperature-dependent energy transfer.

The intrinsic fluorescence emission kinetics of Na,K-ATPase, a large membrane protein containing 16 tryptophan residues, was studied by time-resolved techniques. The lifetime distributions recovered by the Maximum Entropy Method exhibit a strong dependence on the emission wavelength at temperatures between 37 degrees C and -70 degrees C. From the 'blue' edge of the fluorescence emission spectrum up to the maximum of emission, the lifetime distribution at room temperature is the result of four broad peaks which cover the time range 0.3-7 ns. With increasing emission wavelength, these peaks move to longer lifetimes and the peak at shorter times are suppressed at the red edge, while the longest component (6-7 ns) becomes dominant. With decreasing temperature, the number of lifetime components is reduced for the benefit of the long one. At cryogenic temperatures, the emission decay in the red-edge of the fluorescence spectrum consists of one major slow component (6-7 ns) and a fast one (0.5 ns) associated with a negative pre-exponential term. This is a characteristic feature of an excited-state reaction. The temperature dependence of this fast component and the fluorescence anisotropy decay at low temperature in the red-edge, indicate that this excited state reaction may be accounted for a unidirectional inter-tryptophan fluorescence energy transfer from 'blue' populations of donors to 'red' populations of acceptors. This is also illustrated by the time-resolved emission spectra. In the blue edge of the fluorescence emission spectrum, moreover, the time course of the anisotropy decay suggests the existence of homo-transfer of excitation energy involving 'blue' tryptophan residues. The steady-state anisotropy excitation spectrum in vitrified solvent agrees with this suggestion. These different energy transfer mechanisms may be used as structural probes to detect more accurately conformational changes of the protein elicited by effectors and ion binding or release.

Protein fluorescence, dynamics and function: exploration of analogy between electronically excited and biocatalytic transition states.

With the advent and development of time-resolved spectroscopic techniques and substantial progress in understanding of photophysical and photochemical phenomena, a new goal may be achieved: modeling of biochemical reaction or its elementary step by a photochemical event occurring within the probe, bound to a protein molecule. The probe may be located in a well-determined site of the protein matrix and report on the modulation of the reaction rate by the matrix and by the surrounding solvent, or by interactions in multiprotein complexes and in biomembranes. The advantages of this approach are obvious: in contrast to ordinary biochemical reaction, the excited-state reaction may be started by a short light pulse, and its kinetics may be observed directly with high resolution in time. In addition, if the reaction rate is influenced by the dynamics of the protein matrix, these dynamics may be studied simultaneously with the reaction, by using the same or a similar probe and within the same time range. In this review, the prospects for application of probes exhibiting electron transfer, proton transfer, molecular rotations and isomerizations are presented and discussed. The general problem of photochemical modeling of biochemical reactions is discussed. This modeling may result in deeper understanding of enzyme catalyzed reaction mechanisms.

Fluorescence spectroscopic studies on equilibrium dipole-relaxational dynamics of Na,K-ATPase.

Intramolecular dynamics in Na,K-ATPase molecules have been studied by ultraviolet fluorescence spectroscopic methods: determination of temperature-dependent shifts in steady-state spectra, site-selective red-edge effects and their temperature dependence, and time-resolved emission decay as a function of excitation and emission wavelengths. The combination of these methods allows the characterization of the dipolar-relaxational mobility in the environment of the tryptophan residues. Our results show that the mean dipolar-relaxational time is of the order of one nanosecond at room temperature. This is much faster than what is usually observed in globular proteins. The fast dynamics of the protein dipoles are rapid enough so that the dipoles are in dielectric equilibrium during the slower ion transfer processes; this may have important functional consequences.

Intramolecular dynamics in the environment of the single tryptophan residue in staphylococcal nuclease.

The dipole relaxational dynamics in the environment of a single tryptophan residue Trp-140 in staphylococcal nuclease was studied by time-resolved (multi-frequency phase-modulation) spectroscopy and selective red-edge excitation. The long-wavelength position of the fluorescence spectrum (at 343 nm) and the absence of red-edge excitation effects at 0 and 20 degrees C indicate that this residue is surrounded by very mobile protein groups which relax on the subnanosecond time scale. For these temperatures (0-20 degrees C) the steady-state emission spectra did not show the excitation-wavelength dependent shifts (red-edge effects) for excitation wavelengths from 295 to 308 nm; however, the anisotropy decay rate is slow (tens of nanoseconds). This suggests that the spectral relaxation is due to mobility of the surrounding groups rather than the motion of the tryptophan itself. The motions of the tryptophan surrounding are substantially retarded at reduced temperatures in viscous solvent (60% glycerol). The temperature dependence of the difference in position of fluorescence spectra at excitation wavelengths 295 and 305 nm demonstrate the existence of red-edge effect at sub-zero temperatures, reaching a maximum value at -50 degrees C, where the steady-state emission spectrum is shifted to 332 nm. The excitation and emission wavelength dependence of multi-frequency phase-modulation data at the half-transition point (-40 degrees C) demonstrates the existence of the nanosecond dipolar relaxations. At -40 degrees C the time-dependent spectral shift is close to monoexponential with the relaxation time of 1.4 ns.

Does biocatalysis involve inhomogeneous kinetics?

Biocatalytic reactions can occur according to two very different mechanisms: homogeneous, which is described by standard transition state theory (TST) and its modifications, and inhomogeneous (polychromatic), which is characteristic for some of the charge-transfer reactions in liquids and amorphous solids. While most data published on enzyme reactions are interpreted on the basis of homogeneous kinetics, the important recent findings suggest the involvement of inhomogeneous kinetics mechanism.

Selective laser spectroscopy of 1-phenylnaphthylamine in phospholipid membranes.

Time-resolved and steady-state spectra and kinetics of anisotropy of 1-phenylnaphthylamine (1-AN) fluorescent probe in phosphatidylcholine bilayer membranes have been examined using a nanosecond laser spectrofluorimeter. In this system we consider two kinds of inhomogeneous broadening of spectra, the first of which is due to different probe locations in membrane, while the second one is due to the statistical distribution of interaction energy within a given location. This broadening causes the red shift of the fluorescence spectrum at red-edge excitation, the specific dependences of instantaneous fluorescence and fluorescence anisotropy spectra on the wavelength of excitation. A field diagram is presented which, by describing the free energy levels of the polar fluorescent probe in membranes, makes it possible to unambiguously interpret the whole set of experimental data. It is suggested that the release of potential energy of intermolecular interactions which occurs in the process of relaxation, results in accelerated (light-induced) rotation of the probe inside the membrane.

Solvent reorganizational red-edge effect in intramolecular electron transfer.

Polar solvents are characterized by statistical distributions of solute-solvent interaction energies that result in inhomogeneous broadening of the solute electronic spectra. This allows photoselection of the high interaction energy part of the distribution by excitation at the red (long-wavelength) edge of the absorption bands. We observe that intramolecular electron transfer in the bianthryl molecule from the locally excited (LE) to the charge-transfer (CT) state, which requires solvent relaxation and does not occur in vitrified polar solutions, is dramatically facilitated in low-temperature propylene glycol glass by the red-edge excitation. This allows one to obtain spectroscopically the pure CT form and observe its dependence upon the relaxational properties of the solvent. A qualitative potential model of this effect is presented.

Spectroscopic evidence for NADH-induced conformational changes in rabbit muscle aldolase.

The intrinsic fluorescence (steady-state spectra, anisotropy and nanosecond decay) in combination with phosphorescence at room temperature were used to detect and characterize conformational changes in rabbit muscle aldolase accompanying the NADH-binding process. Ligand binding has entailed a decrease in aldolase fluorescence intensity, a blue shift in its maximum and a polarization increase in a long wavelength part of the emission spectrum. The NADH binding induces the changes in room temperature phosphorescence - higher intensity and longer lifetime. The excited state energy transfer from tryptophans to NADH is not observed, and the character of spectroscopic changes on NADH binding allows us to reveal the spectroscopic heterogeneity among the tryptophan residues. The character, location of protein conformational changes associated with the binding of NADH and their relation to the tryptophans' microenvironment in aldolase are discussed.

The solvent effects on the kinetics of bacterial formate dehydrogenase reaction.

The increase in concentration of organic cosolvents results in a 2-2.5-fold increase of the maximal reaction rate and a decrease of Michaelis constant for formate of NAD(+)-dependent formate dehydrogenase from methylotrophic bacteria Pseudomonas sp. 101. These parameters, however, are not affected with the increase of ionic strength. For the logarithm of both Vmax and Km a linear function of the reciprocal of solvent dielectric permittivity was found. The decrease of Km is possibly due to the dielectric screening effect on the substrate binding energy. The increase in Vmax is explained by a model based on a solvent-dependent electrostatic image force, acting on the charges moved in the course of the catalytic step of the enzyme reaction.