Dynamics of polyglutamic acids in α-helical and coil states. Comparison with dynamics of some globular proteins. Rayleigh scattering of Mössbauer radiation (RSMR) data

Summary The classical model system poly-L-glutamic acid (poly-Glu), was investigated in a disordered coil state (atpH=7.0) and in helix state (atpH=2.0) by the RSMR technique. By considering that the coil state of poly-Glu models unfolded (random coil) state and α-helix state models the fluctuating secondary structure (during consequent folding of protein), a comparative analysis of the dynamical properties of poly-Glu in different states with the dynamical properties of different proteins in the native state (α-helical myoglobin and HSA, partially β-sheet lysozyme) and in intermediate (molten globule) state (α-lactalbumin) was performed. This comparison brings some unpredicted results: native α-helical proteins behave close to random coil, native partially β-sheet proteins behave close to fluctuating secondary structure (α-helix) and the dynamic behaviour of molten-globule state (partially β-sheet α-lactalbumin) is not different from the behaviour of lysozyme and much more rigid than that of native α-helical proteins. Paper presented at ICAME-95, Rimini, 10–16 September 1995.     

Investigation of different protein states by Rayleigh Scattering of Mössbauer Radiation (RSMR)

A comparative study of a native and molten globule state of α-lactalbumin and polyglutamic acid in helical and coiled states were performed by RSMR. Molten globule state possesses the same dynamical features registrated by RSMR as the native one. Large-scale low-frequency motions were registrated in the coiled molecule without any elements of secondary structure.     

朗之万方程及其在蛋白质折叠动力学中的应用

研究了朗之万方程的动力学性质,并用它模拟了蛋白质分子的折叠过程.首先在相空间中对朗之万方程做连续映射,发现做布朗运动的粒子在位置坐标上存在明显的概率分布,这表明蛋白质折叠过程中分子空间构型是非遍历的.此外,本文还通过数值模拟得到了去折叠态蛋白质的紧密度指标,并验证了它与实验结果以及其他理论方法的一致性.本文还提出了一种利用重整化方法研究熔球体状态蛋白质的理论模型,并提供了考虑疏水基影响的蛋白质折叠过程的模拟思路. 关键词： 朗之万方程 蛋白质折叠非遍历性 紧密度指标 重整化     

Sensing Tryptophan Microenvironment of Amyloid Protein Utilizing Wavelength-Selective Fluorescence Approach

Structural transition among various forms of proteins involves subtle interplay between structure and dynamics and is crucial in human diseases. Red edge excitation shift (REES) represents a suitable approach to explore the environmental organization and dynamics surrounding tryptophan residues in proteins. Although REES from tryptophan residues has been reported for native, molten globule and denatured states of proteins, such data on the amyloid form of proteins is lacking. κ-casein is one of the most important constituents of casein micelles in milk and has a tendency to form amyloid fibril. We report here REES of the sole tryptophan residue for native, acid-denatured and urea-denatured forms of κ-casein. More importantly, we show that the amyloid form of κ-casein displays REES of 4 nm. We analyze these results in terms of tryptophan microenvironment in various forms of κ-casein, particularly the amyloid form. We conclude that REES is a sensitive tool to monitor structural plasticity in proteins.     

Multicanonical chain-growth algorithm

We present a temperature-independent Monte Carlo method for the determination of the density of states of lattice proteins that combines the fast ground-state search strategy of the new pruned-enriched Rosenbluth chain-growth method and multicanonical reweighting for sampling the complete energy space. Since the density of states contains all energetic information of a statistical system, we can directly calculate the mean energy, specific heat, Helmholtz free energy, and entropy for all temperatures. We apply this method to lattice proteins consisting of hydrophobic and polar monomers, and for the examples of sequences considered, we identify the transitions between native, globule, and random coil states. Since no special properties of heteropolymers are involved in this algorithm, the method applies to polymer models as well.     

拉曼光谱法测定天然脂肪球脂质成分

天然脂肪球主要由甘油三酯构成,以不同大小的球状形式分泌而得。不同大小的脂肪球的球体和膜组成成分不同,从而影响了脂肪在乳中的存在形式和最终的乳品功能特性。然而,不同大小的脂肪球成分的差异尚未完全阐明。利用拉曼光谱测定特定大小脂肪球及膜的脂质和脂肪酸组成。拉曼光谱能够从单个脂肪球获得特定拉曼信号,并且在不破坏天然脂肪球构型的情况下进行测定。结果显示,小脂肪球在2 885/2 850 cm-1处条带信号较高,表明小脂肪球趋于形成结晶态的脂肪球膜包裹流动态的甘油三酯内核的结构。此外,小脂肪球与大脂肪球相比,1 655/1 443 cm-1的条带信号较低,表明小脂肪球的脂肪酸不饱和程度较高。总之,从本实验结果可以推断,用特定的小脂肪球分离而得的稀奶油在熔化时较大脂肪球难熔化,搅拌耗时更多,但能形成更柔软的黄油。     

Qualitative and quantitative characterization of the arsenic-binding behaviour of sulfur-containing peptides and proteins by the coupling of reversed phase liquid chromatography to electrospray ionization mass spectrometry

Phenylarsenic-substituted cysteine-containing peptides and proteins were completely differentiated from their unbound original forms by the coupling of reversed phase liquid chromatography with electrospray ionization mass spectrometry. The analysis of biomolecules possessing structure-stabilizing disulfide bridges after reduction provides new insights into requirements concerning the accessibility of cysteine residues for reducing agents as well as for arsenic compounds in a spatial protein structure. Complementary binding studies performed using direct ESI-MS without chromatographic coupling in different solvent systems demonstrated that more than one binding site were activated for aprotinin and lysozyme in denaturing solvents because of a stronger defolding. From the intensities of the different charge states occurring in the mass spectra as well as from the LC elution behaviour, it can be deduced that the folding state of the arsenic-bound protein species resembles the native, oxidized conformation. In contrast, although the milk protein α-lactalbumin has several disulfide bridges, only one phenylarsenic moiety was bound under strongly denaturing conditions. Because of the charge state distribution in the ESI mass spectra, a conformational change to a molten globule structure is assumed. For the second considered milk protein ?-lactoglobulin, a noncovalent interaction with phenylarsine oxide was detected. In general, smaller apparent binding constants for the condensation reactions of the biomolecules with phenylarsine oxide leading to covalent arsenic-sulfur bindings were determined from direct injection ESI-MS measurements than from LC-ESI-MS coupling. The following order of binding affinities for one phenylarsenic group can be assumed from both ESI-MS and LC-ESI-MS: nonapeptide vasopressin?>?nonapeptide vasotocin?>?lysozyme?>?aprotinin?>?α-lactalbumin?>?thioredoxin. Kinetic investigations by LC-ESI-MS yielded a partial reaction order of 2 for vasopressin, Lys and α-lactalbumin and corresponding half-lives of 0.93, 2.56 and 123.5?min, respectively. Copyright ? 2012 John Wiley & Sons, Ltd.     

Observation of persistent α‐helical content and discrete types of backbone disorder during a molten globule to ordered peptide transition via deep‐UV resonance Raman spectroscopy

The molten globule (MG) state can aid in the folding of a protein to a functional structure and is loosely defined as an increase in structural disorder with conservation of the ensemble secondary structure content. Simultaneous observation of persistent secondary structure content with increased disorder has remained experimentally problematic. As a consequence, modeling how the MG state remains stable and how it facilitates proper folding remains difficult due to a lack of amenable spectroscopic techniques to characterize this class of partially unfolded proteins. Previously, deep‐UV resonance Raman (dUVRR) spectroscopy has proven useful in the resolution of global and local structural fluctuations in the secondary structure of proteins. In this work, dUVRR was employed to study the MG to ordered transition of a model four‐helix bundle protein, HP7. Both the average ensemble secondary structure and types of local disorder were monitored, without perturbation of the solvent, pH, or temperature. The MG to ordered transition is induced by stepwise coordination of two heme molecules. Persistent dUVRR spectral features in the amide III region at 1295–1301 and 1335–1338 cm⁻¹ confirm previous observations that HP7 remains predominantly helical in the MG versus the fully ordered state. Additionally, these spectra represent the first demonstration of conserved helical content in a MG protein. With successive heme binding, significant losses are observed in the spectral intensity of the amide III₃ and S regions (1230–1260 and 1390 cm⁻¹, respectively), which are known to be sensitive to local disorder. These observations indicate that there is a decrease in the structural populations able to explore various extended conformations with successive heme binding events. DUVRR spectra indicate that the first heme coordination between two helical segments diminishes exploration of more elongated backbone structural conformations in the inter‐helical regions. A second heme coordination by the remaining two helices further restricts protein motion. Copyright © 2013 John Wiley & Sons, Ltd.     

Fluorescence study on Interactions of α–Crystallin with the Molten Globule State of 1, 4–β–D–Glucan Glucanohydrolase from Thermomonospora sp. induced by guanidine hydrochloride

In this paper, the interaction between α- crystallin and molten globule structure of 1,4–β–D–Glucan Glucohydrolase (TSC) from an alkalothermophilic Thermomonospora sp. was investigated mainly by fluorescence quenching spectra, circular dichroism and three dimensional fluorescence spectra under simulative physiological conditions. Denaturation studies using GdnCl indicated that TSC folds through a partially folded state that resembles molten globule at 1.8 M GdnCl. The chaperone activity of α- crystallin was employed to study refolding of TSC. Here we studied the refolding of GdnCl denatured TSC from its molten globule state (TSC-m complex) in the presence and absence of α-crystallin to elucidate the molecular mechanism of chaperone-mediated in vitro folding. Our results, based on intrinsic tryptophan fluorescence and ANS binding studies, suggest that α-crystallin formed a complex with a putative intermediate molten globule – like intermediate in the refolding pathway of TSC. Reconstitution of the active TSC was observed on cooling the α-crystallin • TSC -m complex to 4°C. Addition of α-crystallin to the molten globule – like intermediate of TSC (TSC-m complex) complex initiated the refolding of TSC with 69 % recovery of the biological activity of the enzyme.     

The role of hydrophobic interaction in phase transition and structure formation of lipid membranes and proteins

The hydrophobic interaction arises from the ordered structure of water around nonpolar groups of molecules in an aqueous solvent. Because biological systems are made of various macromolecules and amphiphiles which are suspended in aqueous solution, the hydrophobic interaction plays a very important role in the formation of higher-order structure and phase transitions in biological systems. Considering the hydrophobic interaction, the van der Waals interaction and the entropic effect, an equation of state of a lipid membrane was obtained which was analogous to the van der Waals equation. The characteristics of the lipid bilayer phase transition as well as the phase behaviors of a lipid monolayer were explained by this equation of state. Experimental evidence was obtained from ultrasonic measurements which indicated that its phase transition accompanys significant critical phenomena. Analysis of the hydrophobicity of amino acid sequences revealed that the morphology of the proteins was determined by the hydrophobicity alone. The essential role of the hydrophobic interaction in the morphogenesis of proteins could be confirmed by a denaturation experiment on a soluble protein, carbonic anhydrase B. Fluorescence measurements showed that an intermediate state, the so-called molten globule state, had a quite hydrophobic core, indicating that the globule shape of this protein is stabilized by the hydrophobic interaction.     

Folding kinetics of two-state proteins based on the model of general random walk in native contact number space

We investigated the folding kinetics of a series of two-state proteins by using the model of general random walk in native contact number space, and derive the observed linear relationship between the logarithms of the folding rate constants and the numbers of native contacts from “kinetic viewpoint”. The protein folding speed limit and stability in this model are consistent with experimental observations.     

Existence of Different Structural Intermediates and Aggregates on the Folding Pathway of Ovalbumin

Structural modifications of ovalbumin in presence of different concentration of guanidine hydrochloride (Gdn HCl) and glucose were investigated by using intrinsic fluorescence, Fourier transform infra-red spectroscopy, circular dichroism and 8-anilino-1-naphthalene-sulphonic acid, to confirm that partially folded intermediates of ovalbumin lead to aggregation. The two partially folded intermediates of ovalbumin were observed one at 1 M Gdn HCl and another in the presence of 20 mM glucose at 3 M Gdn HCl. Both intermediates exist as compact states with altered intrinsic fluorescence, prominent β-sheet secondary structure and enhanced ANS binding. Ovalbumin in the presence of glucose required more concentration of Gdn HCl (3 M) to exist as an intermediate state than control (1 M). Such alpha-helix/beta-sheet transition of proteins is a crucial step in amyloidogenic diseases and represents an internal rearrangement of local contacts in an already folded protein. Further, incubation for 24 h resulted in the formation of aggregates as detected by thioflavin T-assay. On further increasing the concentration of glucose to 50 mM and incubation time for various days resulted in the formation of molten globule state of ovalbumin at 6th day. Later on, at 10th day advanced glycated end products were observed.     

Protein Sequence and Structure: Is One More Fundamental than the Other?

We argue that protein native state structures reside in a novel “phase” of matter which confers on proteins their many amazing characteristics. This phase arises from the common features of all globular proteins and is characterized by a sequence-independent free energy landscape with relatively few low energy minima with funnel-like character. The choice of a sequence that fits well into one of these predetermined structures facilitates rapid and cooperative folding. Our model calculations show that this novel phase facilitates the formation of an efficient route for sequence design starting from random peptides.     

Effect of unfolding on the thickness of the hydration layer of a protein

Properties of water in the hydration layer around a protein is intimately correlated with its function. A knowledge of the thickness of the hydration layer is important to understand its role in guiding the folding-unfolding of the protein. We have performed atomistic molecular dynamics simulations of the folded native and a partially unfolded molten globule structure of the villin headpiece subdomain or HP-36 in aqueous solution to estimate the effect of unfolding on the thickness of hydration layer around different segments of the protein. In particular, several dynamic properties of water around different segments of the folded native and the unfolded structure have been calculated by varying the thickness of the hydration layers. It is found that unfolding of a segment of the protein is correlated with the dynamics of water around it, i.e., within the first hydration layer. The effect of unfolding on water properties has been found to diminish when water molecules present beyond the first hydration layer were included in the calculations.      

Simple Model Study of Phase Transition Properties of Isolated and Aggregated Protein

We investigate the phase transition properties of isolated and aggregated protein by exhaustive numerical study in the confined conformation space with maximally compact lattice model. The study within the confined conformation space shows some general folding properties. Various sequences show different folding properties: two-state folding, three-state folding and prion-like folding behavior. We find that the aggregated protein holds a more evident transition than isolated one and the transition temperature is generally lower than that in isolated case.     

Folding/unfolding kinetics of lattice proteins studied using a simple statistical mechanical model for protein folding, I: Dependence on native structures and amino acid sequences

The folding/unfolding kinetics of a three-dimensional lattice protein was studied using a simple statistical mechanical model for protein folding that we developed earlier. We calculated a characteristic relaxation rate for the free energy profile starting from a completely unfolded structure (or native structure) that is assumed to be associated with a folding rate (or an unfolding rate). The chevron plot of these rates as a function of the inverse temperature was obtained for four lattice proteins, namely, proteins a1, a2, b1, and b2, in order to investigate the dependency of the folding and unfolding rates on their native structures and amino acid sequences. Proteins a1 and a2 fold to the same native conformation, but their amino acid sequences differ. The same is the case for proteins b1 and b2, but their native conformation is different from that of proteins a1 and a2. However, the chevron plots of proteins a1 and a2 are very similar to each other, and those of proteins b1 and b2 differ considerably. Since the contact orders of proteins b1 and b2 are identical, the differences in their kinetics should be attributed to the amino acid sequences and consequently to the interactions between the amino acid residues. A detailed analysis revealed that long-range interactions play an important role in causing the difference in the folding rates. The chevron plots for the four proteins exhibit a chevron rollover under both strongly folding and strongly unfolding conditions. The slower relaxation time on the broad and flat free energy surfaces of the unfolding conformations is considered to be the main origin of the chevron rollover, although the free energy surfaces have features that are rather complicated to be described in detail here. Finally, in order to concretely examine the relationship between changes in the free energy profiles and the chevron plots, we illustrate some examples of single amino acid substitutions that increase the folding rate.     

On hydrodynamic interpretation of folding of an α-helical protein

Using the method of molecular dynamics, the simulation of folding of an α-helical protein from the unfolded to compact and functional (native) state is performed. The protein folding is interpreted as a stationary motion of a compressible “folding fluid”. It is shown that the densities of folding fluxes obey the same similarity relations as the velocities of an incompressible fluid in the Kolmogorov’s turbulence theory, except that instead of the rate of change of kinetic energy per mass unit, the rate of change of flux variance per volume unit plays the role of the key parameter.     

Globules of annealed amphiphilic copolymers: Surface structure and interactions

A mean-field theory of globules of random amphiphilic copolymers in selective solvents is developed for the case of an annealed copolymer sequence: each unit can be in one of two states, H (insoluble) or P (soluble or less insoluble). The study is focussed on the regime when H and P units tend to form long blocks, and when P units dominate in the dilute phase, but are rare in the globule core. A first-order coil-to-globule transition is predicted at some T = T _cg. The globule core density at the transition point increases as the affinity of P units to the solvent, ˜, is increased. Two collapse transitions, coil → “loose” globule and “loose” globule → “dense” globule, are predicted if ˜ is high enough and P units are marginally soluble or weakly insoluble. H and P concentration profiles near the globule surface are obtained and analyzed in detail. It is shown that the surface excess of P units rises as ˜ is increased. The surface tension decreases in parallel. Considering the interaction between close enough surfaces of two globules, we show that they always attract each other at a complete equilibrium. It is pointed out, however, that such equilibrium may be difficult to reach, so that partially equilibrium structures (defined by the condition that a chain forming one globule does not penetrate into the core of the other globule) are relevant. It is shown that at such partial equilibrium the interaction is repulsive, so the globules may be stabilized from aggregation. The strongest repulsion is predicted at the coil-to-globule transition point T _cg: the repulsion force decreases with the distance between the surfaces according to a power law. In the general case (apart from T _cg) the force vs. distance decay becomes exponential; the decay length ξ diverges as T → T _cg. The developed theory explains certain anomalous properties observed for globules of amphiphilic homopolymers.     

There and back again: Two views on the protein folding puzzle

The ability of protein chains to spontaneously form their spatial structures is a long-standing puzzle in molecular biology. Experimentally measured folding times of single-domain globular proteins range from microseconds to hours: the difference (10–11 orders of magnitude) is the same as that between the life span of a mosquito and the age of the universe. This review describes physical theories of rates of overcoming the free-energy barrier separating the natively folded (N) and unfolded (U) states of protein chains in both directions: “U-to-N” and “N-to-U”. In the theory of protein folding rates a special role is played by the point of thermodynamic (and kinetic) equilibrium between the native and unfolded state of the chain; here, the theory obtains the simplest form. Paradoxically, a theoretical estimate of the folding time is easier to get from consideration of protein unfolding (the “N-to-U” transition) rather than folding, because it is easier to outline a good unfolding pathway of any structure than a good folding pathway that leads to the stable fold, which is yet unknown to the folding protein chain. And since the rates of direct and reverse reactions are equal at the equilibrium point (as follows from the physical “detailed balance” principle), the estimated folding time can be derived from the estimated unfolding time. Theoretical analysis of the “N-to-U” transition outlines the range of protein folding rates in a good agreement with experiment. Theoretical analysis of folding (the “U-to-N” transition), performed at the level of formation and assembly of protein secondary structures, outlines the upper limit of protein folding times (i.e., of the time of search for the most stable fold). Both theories come to essentially the same results; this is not a surprise, because they describe overcoming one and the same free-energy barrier, although the way to the top of this barrier from the side of the unfolded state is very different from the way from the side of the native state; and both theories agree with experiment. In addition, they predict the maximal size of protein domains that fold under solely thermodynamic (rather than kinetic) control and explain the observed maximal size of the “foldable” protein domains.