A view of the three dimensional structure of globular proteins

A view of the three dimensional structure of globular proteins based on continuous networks of hydrogen bonds is proposed. Active sites of enzymes and ion sites are prominent and, within the networks, there are islands of hydrophobic regions giving an overall piebald effect to the appearance of the molecule. This point of view was originally suggested by the results of quantum mechanical computations on the coupling between hydrogen bonds. A formalism for the total energy of a globular protein in water is also suggested.The study of five lines of experimental evidence supports this suggestion. The analysis of the experimental X-ray data for ten globular proteins, using the NETWORK program, revealed the existence of these hydrogen bond networks; X-ray data showed that water molecules tend to occupy fixed positions relative to the protein molecule; a survey has shown that water molecules tend to occupy specific positions relative to the hydrogen bonding side chains; experimental evidence on the bulk properties of lysozyme showed that there exist tightly bound water molecules; graphics studies of the ribonucleaseA molecule demonstrated the networks and the piebald effect. This point of view is pictorially simple and, to illustrate the use of such networks, we discuss the simple ion pairs which occur as substructures within the networks.     

Optical Kerr effect in biopolymer solutions

The electric field in a single mode, YAG laser beam has been used to induce orientational birefringence in solutions of tobacco rattle virus, DNA, heparin, and hyaluronic acid. Using this laser in its “fixed-Q” mode, laser pulses were generated which persisted for up to 200 μsec in which the effective electric field vector rose to 5 kV cm^?1. The birefringence amplitudes so produced had a quadratic dependence on the effective field strength and thus obeyed Kerr's law. From the birefringence decay rates, relaxation times were determined which, by comparison with the birefringence induced by pulsed static electric fields revealed the biopolymer orientational origins of the effects. This indicated how these experiments can lead to the evaluation of particle geometry, the electronic contribution to electrical polarizabilities, and the optical polarizability of biopolymers in solution.     

Calorimetric study of the formation and melting of thermotropic gel in solutions of globular proteins

Scanning calorimetry has been used for studying lysozyme water solutions of different buffer molarity (mu = 0.5 divided by 1.0) and concentrations (c = 1.5 divided by 25%) at pH 2.0. It is shown that an additional high temperature maximum (HTM) can be observed on the heating curves for lysozyme solutions during irreversible denaturation. Calorimetric and rheological studies under identical heating conditions have shown that aggregation of protein during denaturation leads to the formation of the thermotropic gel. Further increase of temperature brings up the melting of this gel which results in the appearance of HTM on thermograms. Slow cooling of lysozyme gel melt leads to its reconstruction which results in the appearance of exothermic maximum on the corresponding thermograms.     

The three states of globular proteins: acid denaturation.

We describe statistical mechanical theory that aims to predict protein stabilities as a function of temperature, pH, and salt concentration, from the physical properties of the constituent amino acids: (1) the number of nonpolar groups, (2) the chain length, (3) the temperature-dependent free energy of transfer, (4) the pKa's (including those in the native state) and their temperature dependencies. We calculate here the phase diagrams for apomyoglobin and hypothetical variant proteins. The theory captures essential features of protein stability including myoglobin's Tm vs pH as measured by P. L. Privalov [(1979) Advances in Protein Chemistry, Vol. 33, pp. 167-241] and its ionic strength vs pH phase diagram as measured by Y. Goto and A. L. Fink [(1990) Journal of Molecular Biology, Vol. 214, pp. 803-805]. The main predictions here are the following: (1) There are three stable states, corresponding to native (N), compact denatured (C), and highly unfolded (U), with transitions between them. (2) In agreement with experiments, the compact denatured state is predicted to have enthalpy closer to U than N because even though there is considerable hydrophobic "clustering" in C, this nevertheless represents a major loss of hydrophobic contacts relative to configurations (N) that have a hydrophobic "core." (3) C becomes more prominent in the phase diagram with increasing nonpolar content or decreasing chain length, perhaps thus accounting for (a) why lysozyme and alpha-lactalbumin differ in their denatured states, and (b) why shortened Staph nuclease molecules are compact. (4) Of major importance for protein calorimetry is Privalov's observation that the enthalpy of folding, delta H (T, pH) is independent of pH. The theory accounts for this through the prediction that the main electrostatic contribution to stability is not enthalpic; the main contribution is the entropy, mainly due to the different distributions of protons and small ions in the native and denatured states.     

Generalized concentration dependence of globular protein self-diffusion coefficients in aqueous solutions.

The self-diffusion coefficients of globular proteins (myoglobin, bovine serum albumin, barstar, lysozyme) in aqueous solutions at different temperatures and pH values are obtained by pulsed-gradient spin-echo NMR, and their concentration dependence is analyzed. The generalized concentration dependence of globular protein self-diffusion coefficients is empirically established, and compared to the concentration dependence of diffusion coefficients of flexible polymers and rigid Brownian particles.     

Study of the conformational mobility of globular proteins in aqueous solutions according to their proton relaxation in a rotating system of coordinates

Measurements of the nuclear magnetization decay in the rotating frame for protons of SA and RNAase proteins in aqueous solutions indicate the dispersion of the relaxation rate for SA protons within the region of correlation frequencies of 10(5)--10(6) s-1. These frequencies are much lower than frequencies of rotational diffusion movements of the SA macromolecules in aqueous solutions. These must be ascribed to internal movement within protein globules due to which the distances between interacting magnetic moments are changed. This conclusion gives direct evidence in favour of existence of conformational mobility in most of the protein globule volume.     

Determination of the axial ratio of globular proteins in aqueous solution using viscometric measurements

The paper presents the results of viscosity determinations on aqueous solutions of several globular proteins in a wide range of concentrations. On the basis of these measurements a general formula connecting the relative viscosity with concentration and axial ratio of the dissolved proteins was established. By applying the formula the axial ratios of bovine γ-globulin and horse albumin molecules were calculated.     

Association of gold nanorods in water solutions: Influence of globular proteins

By the absorption spectroscopy method, optical properties of gold nanorods (10 × 38 nm) and their interaction with globular protein bovine hemoglobin and bovine serum albumin were investigated. Nanorod behavior was studied in water solution and in solution of 97 mM NaCl under ultrasound action during 90 min and results were then compared. In water solutions, nanorod coagulation (aggregation) was observed with reduced optical density of the longitudinal plasmon band widening at λ > 800 nm. In NaCl solution, absorption spectra evolution had a complex character and was in some degree analogous to the result that was obtained for two-dimensional grids of gold nanoparticles when changing the distance between them. By interacting with serum albumin, stabilization of colloid solution and dissociation of nanorod aggregates were observed.     

Association of gold nanorods in water solutions: influence of globular proteins

By absorption spectroscopy method optical properties of gold nanorods (10x38 nm) and their interaction with globular protein bovine hemoglobin and bovine serum albumin were investigated. Nanorods behavior was studied in water solution and in solution of 97 mM NaCl under ultrasound action during 90 min and results were then compared. In water solutions nanorods coagulation (aggregation) was observed with reduced optical density of the longitudinal plasmon band widening at lamda>800 nm. In NaCI solution absorption spectra evolution had complex character and was in some degree analogous to the result that was obtained for two-dimensional grids of gold nanoparticles when changing the distance between them. By interacting with serum albumin stabilization of colloid solution and dissociation of nanorods aggregates were observed.     

Topology of globular proteins

This paper inquires whether it is reasonable to expect the native structure of proteins to be “knotted”. To this end, some topological properties of polypeptides containing disulfide bridges are discussed using notions from mathematical knot theory and graph theory. The probability of occurrence of knots in random cyclic polymers is calculated as a function of chain length by elementary Monte Carlo methods. The implications of this for protein renaturation and for determining the tertiary structure of proteins are discussed.     

Thermodynamics of globular proteins

The analysis of temperature-induced unfolding of proteins in aqueous solutions was performed. Based on the data of thermodynamic parameters of protein unfolding and using the method of semi-empirical calculations of hydration parameters at reference temperature 298 K, we obtained numerical values of enthalpy, free energy, and entropy which characterize the unfolding of proteins in the ‘gas phase’. It was shown that specific values of the energy of weak intramolecular bonds (?H^int), conformational free energy (?G^conf) and entropy (?S^conf) are the same for proteins with molecular weight 7–25 kDa. Using the energy value (?H^int) and the proposed approach for estimation of the conformational entropy of native protein (S^NC), numerical values of the absolute free energy (G^NC) were obtained.     

On the effect of hydrostatic pressure on the conformational stability of globular proteins

The model developed for cold denaturation (Graziano, PCCP 2010, 12, 14245‐14252) is extended to rationalize the dependence of protein conformational stability upon hydrostatic pressure, at room temperature. A pressure− volume work is associated with the process of cavity creation for the need to enlarge the liquid volume against hydrostatic pressure. This contribution destabilizes the native state that has a molecular volume slightly larger than the denatured state due to voids existing in the protein core. Therefore, there is a hydrostatic pressure value at which the pressure−volume contribution plus the conformational entropy loss of the polypeptide chain are able to overwhelm the stabilizing gain in translational entropy of water molecules, due to the decrease in water accessible surface area upon folding, causing denaturation. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 711–718, 2015.     

A continuum model to study the internal fluctuations in globular proteins

The salient features of the differential equation model to study protein dynamics are presented with results for 19 proteins.     

The effect of autoclaving on the dispersibility and stability of three neutral polysaccharides in dilute aqueous solutions

The dispersibility of three neutral polysaccharides, oat β-glucan, detarium xyloglucan and dextran in a dilute water–cadoxen mixture was studied by viscosity measurement. It was found that intrinsic viscosity measurement, with water–cadoxen mixtures as solvents, is a useful tool to distinguish polymer degradation from disruption of supramolecular aggregates. This approach, in conjunction with size exclusion chromatography, was used to study the effects of heat and pressure treatment on the dispersibility and stability of three polysaccharides in aqueous solutions. Autoclaving treatment at 121°C for 15 min may reduce the degree of aggregation. Following autoclaving in aqueous solution, the Huggins constants decreased from 0.66 to 0.42 for oat β-glucan and from 0.63 to 0.56 for detarium xyloglucan. It remains the same for dextran, indicating good solubility of this polymer in water. The current treatment did not cause evident changes in molecular weight and structures to detarium xyloglucan and dextran. However, degradation occurred with oat β-glucan. The Burchard–Stockmayer–Fixman approach was applied to estimate the unperturbed dimension of oat β-glucan and detarium xyloglucan on samples after autoclaving. The characteristic ratio C_∞ was found to be 7.3 for detarium xyloglucan and 4.7 for oat β-glucan, corresponding to the Kratky–Porod persistence lengths of 2.0 and 1.2 nm, respectively.     

A study of the occurrence of regulator secondary structures in globular proteins

The distribution of regular secondary structures, viz. α-helices and β-strands, along the length of over 70 properties whose secondary structural details have been reported, has been analysed. The occurrence of these regular structures tends to be a maximum at the N- and C-termini. Our analysis suggests that both these free ends could possibly serve as nucleating centers for secondary structures and could play an important role in the folding of proteins.