Solvation dynamics of a protein in the pre molten globule state

The nature of solvent molecules around proteins in native and different non-native states is crucial for understanding the protein folding problem. We have characterized two compact denatured states of glutaminyl-tRNA synthetase (GlnRS) under equilibrium conditions in the presence of a naturally occurring osmolyte, l-glutamate. The solvation dynamics of the compact denatured states and the fully unfolded state has been studied using a covalently attached probe, acrylodan, near the active site. The solvation dynamics progressively becomes faster as the protein goes from the native to the molten globule to the pre molten globule to the fully unfolded state. Anisotropy decay measurements suggest that the pre-molten-globule intermediate is more flexible than the molten globule although the secondary structure is largely similar. Dynamic light scattering studies reveal that both the compact denatured states are aggregated under the measurement conditions. The implications of solvation dynamics in aggregated compact denatured states have been discussed.     

Cold Denaturation of α‐Synuclein Amyloid Fibrils

Although amyloid fibrils are associated with numerous pathologies, their conformational stability remains largely unclear. Herein, we probe the thermal stability of various amyloid fibrils. α‐Synuclein fibrils cold‐denatured to monomers at 0–20 °C and heat‐denatured at 60–110 °C. Meanwhile, the fibrils of β2‐microglobulin, Alzheimer’s Aβ1‐40/Aβ1‐42 peptides, and insulin exhibited only heat denaturation, although they showed a decrease in stability at low temperature. A comparison of structural parameters with positive enthalpy and heat capacity changes which showed opposite signs to protein folding suggested that the burial of charged residues in fibril cores contributed to the cold denaturation of α‐synuclein fibrils. We propose that although cold‐denaturation is common to both native proteins and misfolded fibrillar states, the main‐chain dominated amyloid structures may explain amyloid‐specific cold denaturation arising from the unfavorable burial of charged side‐chains in fibril cores.     

Cold denaturation of yeast frataxin offers the clue to understand the effect of alcohols on protein stability

Although alcohols are well-known to be protein denaturants when present at high concentrations, their effect on proteins at low concentrations is much less well characterized. In this paper, we present a study of the effects of alcohols on protein stability using Yfh1, the yeast ortholog of the human protein frataxin. Exploiting the unusual property of this protein of undergoing cold denaturation around 0 degrees C without any ad hoc destabilization, we determined the stability curve on the basis of both high and low temperature unfolding in the presence of three commonly used alcohols: trifluoroethanol, ethanol, and methanol. In all cases, we observed an extended temperature range of protein stability as determined by a modest increase of the high temperature of unfolding but an appreciable decrease in the low temperature of unfolding. On the basis of simple thermodynamic considerations, we are able to interpret the literature on the effects of alcohols on proteins and to generalize our findings. We suggest that alcohols, at low concentration and physiological pH, stabilize proteins by greatly widening the range of temperatures over which the protein is stable. Our results also clarify the molecular mechanism of the interaction and validate the current theoretical interpretation of the mechanism of cold denaturation.     

A molecular dynamics study of the correlations between solvent-accessible surface, molecular volume, and folding state

We analyzed the correlations between molecular volume, solvent-accessible surface, and folding state (secondary structure content) for unfolded conformers of alpha (holo- and apomyoglobin) and beta (retinal-binding protein) proteins and a small water-soluble alanine-rich alpha-helical peptide. Conformers with different degrees of folding were obtained using molecular dynamics at constant temperature and pressure with implicit solvent (dielectric constant adjustment) for all four systems and with explicit solvent for the single helix peptide. Our results support the view that unfolded conformations are not necessary extended, that volume variation is not a good indication of folding state and that the simple model of water penetrating the interior of the protein does not explain the increase in volume upon unfolding.     

Characterization of folding intermediates of a domain-swapped protein by solid-state NMR spectroscopy

We have employed two-dimensional solid-state NMR to study structure and dynamics of insoluble folding states of the domain-swapped protein Crh. Starting from the protein precipitated at its pI, conformational changes due to a modest temperature increase were investigated at the level of individual residues and in real-time. As compared to the crystalline state, Crh pI-precipitates exhibited a higher degree of molecular mobility for several regions of the protein. A rigidly intact center was observed including a subset of residues of the hydrophobic core. Raising the temperature by 13 K to 282 K created a partially unfolded intermediate state that was converted into beta-sheet-rich aggregates that are mostly of spherical character according to electron microscopy. Residue-by-residue analysis indicated that two out of three alpha-helices in aggregated Crh underwent major structural rearrangements while the third helix was preserved. Residues in the hinge region exhibited major chemical-shift changes, indicating that the domain swap was not conserved in the aggregated form. Our study provides direct evidence that protein aggregates of a domain-swapped protein retain a significant fraction of native secondary structure and demonstrates that solid-state NMR can be used to directly monitor slow molecular folding events.     

Residue-specific real-time NMR diffusion experiments define the association states of proteins during folding

Characterizing the association states of proteins during folding is critical for understanding the nature of protein-folding intermediates and protein-folding pathways, protein aggregation, and disease-related aggregation. To study the association states of unfolded, folded, and intermediate species during protein folding, we have introduced a novel residue-specific real-time NMR diffusion experiment. This experiment, a combination of NMR real-time folding experiments and 3D heteronuclear pulsed field gradient NMR diffusion experiments (LED-HSQC), measures hydrodynamic properties, or molecular sizes, of kinetic species directly during the folding process. Application of the residue-specific real-time NMR diffusion experiments to characterize the folding of the collagen triple helix motif shows that this experiment can be used to determine the association states of unfolded, folded, and kinetic intermediates with transient lifetimes simultaneously. The ratio of the apparent translational diffusion coefficients of the unfolded to the folded form of the triple helix is 0.59, which correlates very well with a theoretical ratio for monomer to linear trimer. The apparent diffusion coefficients of the kinetic intermediates formed during triple helix folding indicate the formation of trimer-like associates which is consistent with previously published kinetic and relaxation data. The residue-specific time dependence of apparent diffusion coefficients of monomer and trimer peaks also illustrates the ability to use diffusion data to probe the directionality of triple helix formation. NMR diffusion experiments provide a new strategy for the investigation of protein-folding mechanisms, both to understand the role of kinetic intermediates and to determine the time-dependent aggregation processes in human diseases.     

Modifying the charge state distribution of proteins in electrospray ionization mass spectrometry by chemical derivatization

Electrospray ionization (ESI) of denatured proteins produces a broad distribution of multiply-charged ions leading to multiple peaks in the mass spectrum. We investigated changes in the positive-mode ESI charge state distribution produced by several chemical modifications of denatured proteins. Capping carboxylic acid groups with neutral functional groups yields little change in charge state distribution compared with unmodified proteins. The results indicate that carboxyl groups do not play a significant role in the positive charging of denatured proteins in ESI. The modification of proteins with additional basic sites or fixed positive charges generates substantially higher charge states, providing evidence that the number of ionizable sites, rather than molecular size and shape, determines ESI charging for denatured proteins. Fixed charge modification also significantly reduces the number of protons acquired by a protein, in that the charge state envelope is not increased by the full number of fixed charges appended. This result demonstrates that Coulombic repulsion between positive charges plays a significant role in determining charge state distribution by affecting the gas-phase basicity of ionizable sites. Addition of fixed-charge moieties to a protein is a useful approach for shifting protein charge state distributions to higher charge states, and with further work, it may help limit the distribution of protein ions to fewer charge states.     

The pentapeptide GGAGG has PII conformation

Most of what we know about proteins reflects their native folded structure. Much less is understood about the structure of unfolded proteins, which tends to be referred to as "random coil", lacking extended alpha-helix or beta-strand structure. Recent work suggests that unfolded proteins might adopt significant population of PII structure, an extended left-handed helix found in collagen and proline-rich peptides. A series of short peptides AcGGXGGNH2 has been adopted as a model for studying unfolded protein structure because of the minimal steric effect imposed by flanking glycines. Peptide AcGGAGGNH2 makes possible a host-guest conformation analysis of the middle residue alanine. NMR experiments reveal that the Phi and Psi dihedral angles of the central alanine are -73 degrees and 125 degrees , respectively, placing the alanine in the PII region of the Ramachandran plot. Circular dichroism shows a typical PII spectrum with a strong negative absorbance at 190 nm. Temperature experiments show the alanine structure shifts to increasing beta-strand at high temperature. Because the alanine side chain most closely represents unsubstituted peptide backbone, these results have significant implications for the conformational entropy of unfolded polypeptide chains.     

Raman spectroscopic characterization of secondary structure in natively unfolded proteins: alpha-synuclein

The application of Raman spectroscopy to characterize natively unfolded proteins has been underdeveloped, even though it has significant technical advantages. We propose that a simple three-component band fitting of the amide I region can assist in the conformational characterization of the ensemble of structures present in natively unfolded proteins. The Raman spectra of alpha-synuclein, a prototypical natively unfolded protein, were obtained in the presence and absence of methanol, sodium dodecyl sulfate (SDS), and hexafluoro-2-propanol (HFIP). Consistent with previous CD studies, the secondary structure becomes largely alpha-helical in HFIP and SDS and predominantly beta-sheet in 25% methanol in water. In SDS, an increase in alpha-helical conformation is indicated by the predominant Raman amide I marker band at 1654 cm(-1) and the typical double minimum in the CD spectrum. In 25% HFIP the amide I Raman marker band appears at 1653 cm(-1) with a peak width at half-height of approximately 33 cm(-1), and in 25% methanol the amide I Raman band shifts to 1667 cm(-1) with a peak width at half-height of approximately 26 cm(-1). These well-characterized structural states provide the unequivocal assignment of amide I marker bands in the Raman spectrum of alpha-synuclein and by extrapolation to other natively unfolded proteins. The Raman spectrum of monomeric alpha-synuclein in aqueous solution suggests that the peptide bonds are distributed in both the alpha-helical and extended beta-regions of Ramachandran space. A higher frequency feature of the alpha-synuclein Raman amide I band resembles the Raman amide I band of ionized polyglutamate and polylysine, peptides which adopt a polyproline II helical conformation. Thus, a three-component band fitting is used to characterize the Raman amide I band of alpha-synuclein, phosvitin, alpha-casein, beta-casein, and the non-A beta component (NAC) of Alzheimer's plaque. These analyses demonstrate the ability of Raman spectroscopy to characterize the ensemble of secondary structures present in natively unfolded proteins.     

Plant proteins and their colloidal state

Plant proteins are characterized by a complex colloidal state in their physiological environment. The main reasons are related to the multiple functions of plant proteins as well as the different architectures encountered in the plant cells from various sources. During extraction process to produce ingredients, plant proteins reorganize in several native or denatured colloidal states depending on the energy and the physico–chemical changes applied to the system. In most cases, an equilibrium between the native (soluble monomers or oligomers) and denatured (mostly insoluble) oligomeric/aggregated states is reached. Further, processing of the plant protein ingredients during food production, introducing new hydrophobic phases (e.g., gas, oil), energy (pressure, temperature, shear), and physico–chemical conditions (pH, ionic salts) will lead to a final colloidal state, specific to the structural features of the considered final food product.     

Vapor Treatment of Electrospray Droplets: Evidence for the Folding of Initially Denatured Proteins on the Sub-Millisecond Time-Scale

The exposure of electrospray droplets generated from either highly acidic or highly basic solutions to basic or acidic vapors, respectively, admitted into the counter-current drying gas, has been shown to lead to significant changes in the observed charge state distributions of proteins. In both cases, distributions of charge states changed from relatively high charge states, indicative of largely denatured proteins, to lower charge state distributions that are more consistent with native protein conformations. Ubiquitin, cytochrome c, myoglobin, and carbonic anhydrase were used as model systems. In some cases, bimodal distributions were observed that are not noted under any solution pH conditions. The extent to which changes in charge state distributions occur depends upon the initial solution pH and the pK_a or pK_b of the acidic or basic reagent, respectively. The evolution of charged droplets in the sampling region of the mass spectrometer inlet aperture, where the vapor exposure takes place, occurs within roughly 1 ms. The observed changes in the spectra, therefore, are a function of the magnitude of the pH change as well as the rates at which the proteins can respond to this change. The exposure of electrospray droplets in this fashion may provide means for accessing transient folding states for further characterization by mass spectrometry.     

Single‐Molecule Detection Reveals Knot Sliding in TrmD Denaturation

An increasing number of proteins are found to contain a knot in their polypeptide chain. Although some studies have looked into the folding mechanism of knotted proteins, why and how these complex topologies form are still far from being fully answered. Moreover, no experimental information about how the knot moves during the protein‐folding process is available. Herein, by combining single‐molecule fluorescence resonance energy transfer (smFRET) experiments with molecular dynamics (MD) simulations, we performed a detailed study to characterize the knot in the denatured state of TrmD, a knotted tRNA (guanosine‐1) methyltransferase from Escherichia coli, as a model system. We found that the knot still existed in the unfolded state of TrmD, consistent with the results for two other knotted proteins, YibK and YbeA. More interestingly, both smFRET experiments and MD simulations revealed that the knot slid towards the C‐terminal during the unfolding process, which could be explained by the relatively strong interactions between the β‐sheet core at the N terminal of the native knot region. The size of the knot in the unfolded state is not larger than that in the native state. In addition, the knot slid in a “downhill” mode with simultaneous chain collapse in the denatured state.     

The Pentablock Amphiphilic Copolymer T1107 Prevents Aggregation of Denatured and Reduced Lysozyme

Aggregation of denatured or unfolded proteins establishes a large energy barrier to spontaneous recovery of protein form and function following traumatic injury, tissue cryopreservation, and biopharmaceutical storage. Some tissues utilize small heat shock proteins (sHSPs) to prevent irreversible aggregation, which allows more complex processes to refold or remove the unfolded proteins. It is postulated that large, amphiphilic, and biocompatible block copolymers can mimic sHSP function. Reduced and denatured hen egg white lysozyme (HEWL) is used as a model aggregating protein. The poloxamine T1107 prevents aggregation of HEWL at 37 °C by three complimentary measures. Structural analysis of denatured HEWL reveals a partially folded conformation with preserved or promoted beta‐sheet structures only in the presence of T1107. The physical association of T1107 with denatured HEWL, and the ability to prevent aggregation, is linked to the critical micelle temperature of the polymer. The results suggest that T1107, or a similar amphiphilic block copolymer, can find use as a synthetic chaperone to augment the innate molecular repair mechanisms of natural cells.

     

Forced folding and structural analysis of metastable proteins

A significant fraction of the proteins encoded by the human and other genomes appears to be significantly unfolded in vitro. This will undoubtedly hamper attempts to characterize their structure by classical crystallographic or solution NMR methods. Here we show that encapsulation of a metastable protein within the restricted volume a reverse micelle can be used to force fold the protein and allow its characterization by modern methods of NMR spectroscopy. This may have significant utility in the context of structural proteomics. In addition, variation of the inner volume of the reverse micelle can be used to probe the character of the manifold of unfolded states.     

Mpro-C蛋白三维结构域交换的机理: 来自分子模拟的线索

SARS冠状病毒主蛋白酶(M^pro)在病毒的蛋白酶切过程中发挥着重要作用. M^pro的晶体结构显示它存在两种形式的二聚体: 一种是发生三维结构域交换的形式, 另一种是非交换的形式. M^pro的C端结构域(M^pro-C)单独表达时也能形成与全长M^pro类似的三维结构域交换二聚体. 三维结构域交换通常发生在蛋白质的表面, 但M^pro-C 的结构域交换却发生在疏水核心. 在本文中, 我们利用分子动力学模拟及三维结构域交换预测算法研究了M^pro-C 中被高度埋藏的核心螺旋片段发生交换的机理. 我们发现基于结构与基于序列的已有算法都不能正确预言出M^pro-C和M^pro中发生结构域交换的铰链区位置. 分子模拟结果表明M^pro-C中的交换片段在天然态下埋藏得很好, 但在变性单体中则会被释放并暴露在外面. 因此, 在完全或部分解折叠状态下交换片段的打开有助于促进单体间的相互作用及结构域交换二聚体的形成.     

Ubiquitin: a small protein folding paradigm

For the past twenty years, the small, 76-residue protein ubiquitin has been used as a model system to study protein structure, stability, folding and dynamics. In this time, ubiquitin has become a paradigm for both the experimental and computational folding communities. The folding energy landscape is now uniquely characterised with a plethora of information available on not only the native and denatured states, but partially structured states, alternatively folded states and locally unfolded states, in addition to the transition state ensemble. This Perspective focuses on the experimental characterisation of ubiquitin using a comprehensive range of biophysical techniques.     

Use of the 2D 1H-13C HSQC NMR Methyl Region to Evaluate the Higher Order Structural Integrity of Biopharmaceuticals

The higher-order structure (HOS) of protein therapeutics is directly related to the function and represents a critical quality attribute. Currently, the HOS of protein therapeutics is characterized by methods with low to medium structural resolution, such as Fourier transform infrared (FTIR), circular dichroism (CD), intrinsic fluorescence spectroscopy (FLD), and differential scanning calorimetry (DSC). High-resolution nuclear magnetic resonance (NMR) methods have now been introduced, representing powerful approaches for HOS characterization (HOS by NMR). NMR is a multi-attribute method with unique abilities to give information on all structural levels of proteins in solution. In this study, we have compared 2D ¹H-¹³C HSQC NMR with two established biophysical methods, i.e., near-ultraviolet circular dichroism (NUV-CD) and intrinsic fluorescence spectroscopy, for the HOS assessments for the folded and unfolded states of two monoclonal antibodies belonging to the subclasses IgG1 and IgG2. The study shows that the methyl region of the ¹H-¹³C HSQC NMR spectrum is sensitive to both the secondary and tertiary structure of proteins and therefore represents a powerful tool in assessing the overall higher-order structural integrity of biopharmaceutical molecules.     

The Thermodynamics of Folding of a β Hairpin Peptide Probed Through Replica Exchange Molecular Dynamics Simulations

Peptides that possess a well defined native state are ideal model systems to study the folding of proteins. They possess many of the complexities of larger proteins, yet their small size renders their study computationally tractable. Recent advances in sampling techniques, including replica exchange molecular dynamics, now permit a full characterization of the thermodynamics of folding of small peptides. These simulations not only yield insight into the folding of larger proteins, but equally importantly, they allow, through comparison with experiment, an objective test of the accuracy of force fields, water models and of different numerical schemes for dealing with electrostatic interactions. In this account, we present a molecular dynamics simulation of a small β-hairpin peptide using the replica exchange algorithm and illustrate how this enhanced sampling scheme enables a thorough characterization of the native and unfolded states, and sheds new light into its folding mechanism.