Dynamics of water in the hydration layer of a partially unfolded structure of the protein HP-36

Atomistic molecular dynamics simulations of the folded native structure and a partially unfolded molten globule structure of the protein villin headpiece subdomain or HP-36 have been carried out with explicit solvent to explore the effects of unfolding on the dynamical behavior of water present in the hydration layers of different segments (three alpha-helices) of the protein. The calculations revealed that the unfolding of helix-2 influences the translational and rotational motions of water present in the hydration layers of the three helices in a heterogeneous manner. It is observed that a correlation exists between the unfolding of helix-2 and the microscopic kinetics of protein-water hydrogen bonds formed by its residues. This in turn has an influence on the rigidity of the hydration layers of the helices in the unfolded structure versus that in the folded native structure. These results should provide a microscopic explanation to recent solvation dynamics experiments on folded native and unfolded structures of proteins.     

Coupling between hydration layer dynamics and unfolding kinetics of HP-36

We have performed atomistic molecular dynamics simulations of aqueous solutions of HP-36 at 300 K in its native state, as well as at high temperatures to explore the unfolding dynamics of the protein and its correlation with the motion of water around it. On increasing the temperature a partially unfolded molten globule state is formed where the smallest alpha helix (helix 2) unfolds into a coil. It is observed that the unfolding is initiated around the residue Phe-18 which shows a sharp displacement during unfolding. We have noticed that the unfolding of the protein affects the density of water near the protein surface. Besides, the dynamics of water in the protein hydration layer has been found to be strongly correlated with the time evolution of the unfolding process. We have introduced and calculated a displacement time correlation function to monitor the change in water motion relative to the protein backbone during unfolding. We find that the unfolding of helix 2 is associated with an increase in mobility of water around it as compared to water around the other two helices. We have also explored the microscopic aspects of secondary structure specific and site specific solvation dynamics of the protein. The calculations reveal that unfolding influences the solvation dynamics of the protein molecule in a heterogeneous manner depending on the location of the polar probe residues. This seems to be in agreement with recent experimental findings.     

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.     

Protonation/deprotonation effects on the stability of the Trp-cage miniprotein

The Trp-cage miniprotein is a 20 amino acid peptide that exhibits many of the properties of globular proteins. In this protein, the hydrophobic core is formed by a buried Trp side chain. The folded state is stabilized by an ion pair between aspartic acid and an arginine side chain. The effect of protonating the aspartic acid on the Trp-cage miniprotein folding/unfolding equilibrium is studied by explicit solvent molecular dynamics simulations of the protein in the charged and protonated Asp9 states. Unbiased Replica Exchange Molecular Dynamics (REMD) simulations, spanning a wide temperature range, are carried out to the microsecond time scale, using the AMBER99SB forcefield in explicit TIP3P water. The protein structural ensembles are studied in terms of various order parameters that differentiate the folded and unfolded states. We observe that in the folded state the root mean square distance (rmsd) from the backbone of the NMR structure shows two highly populated basins close to the native state with peaks at 0.06 nm and 0.16 nm, which are consistent with previous simulations using the same forcefield. The fraction of folded replicas shows a drastic decrease because of the absence of the salt bridge. However, significant populations of conformations with the arginine side chain exposed to the solvent, but within the folded basin, are found. This shows the possibility to reach the folded state without formation of the ion pair. We also characterize changes in the unfolded state. The equilibrium populations of the folded and unfolded states are used to characterize the thermodynamics of the system. We find that the change in free energy difference due to the protonation of the Asp amino acid is 3 kJ mol(-1) at 297 K, favoring the charged state, and resulting in ΔpK(1) = 0.5 units for Asp9. We also study the differences in the unfolded state ensembles for the two charge states and find significant changes at low temperature, where the protonated Asp side chain makes multiple hydrogen bonds to the protein backbone.     

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.     

Zinc-porphyrin Solvation in folded and unfolded states of Zn-cytochrome c

After a brief review of the use of photochemical triggers and heme metal substitution to probe the folding dynamics of cytochrome c, we present new results on the photophysics and photochemistry of folded and unfolded states of the zinc-substituted protein (Zn-cyt c). Our measurements of Zn-cyt c triplet state decay kinetics reveal a systematic isotope effect on lifetimes: the decay in the folded protein (tau(H)2(O) approximately 10 ms) is only modestly affected by isotopically substituted buffers (k(H)2(O)/k(D)2(O) = 1.2), whereas a reduced triplet lifetime (approximately 1.3 ms) and greater isotope effect (1.4) were found for the chemically denatured, fully unfolded protein. The shortest lifetime (0.1-0.4 ms) and greatest isotope effect (1.5) were found for a fully exposed model compound, zinc-substituted N-acetyl-microperoxidase-8 (ZnAcMP8), implying that the unfolded protein provides some protection to the Zn-porphyrin group even under fully denaturing conditions. Further evidence for partial structure in unfolded Zn-cyt c comes from bimolecular quenching experiments using Ru(NH(3))(6)(3+) as an external Zn-porphyrin triplet state quencher. In the presence of quencher, partially unfolded protein at midpoint guanidinium chloride (GdmCl) and urea concentrations exhibits biphasic triplet decay kinetics, a fast component corresponding to an extended, solvent-exposed state (6.6 x 10(8) M(-1) s(-1) in GdmCl, 6.3 x 10(8) M(-1) s(-1) in urea) and a slow component attributable to a compact, relatively solvent-inaccessible, state (5.9 x 10(7) M(-1) s(-1) in GdmCl, 8.6 x 10(6) M(-1) s(-1) in urea). The variation in Zn-porphyrin solvation for the compact states in the two denaturants reveals that the cofactor in the partially unfolded protein is better protected in urea solutions.     

Towards a Quantitative Understanding of Protein Hydration and Volumetric Properties

Herein, we probe by pressure perturbation calorimetry (PPC) the coefficient of thermal expansion, the volumetric and the hydration properties of variants of a hyperstable variant of staphylococcal nuclease (SNase), Δ+PHS. The temperature‐dependent volumetric properties of the folded and unfolded states of the wild‐type protein are calculated with previously published data. The present PPC results are used to interpret the volume diagram and expansivity at a molecular level. We conclude that the expansivity of the unfolded state is, to a first approximation, temperature independent, while that of the folded state decreases with increasing temperature. Our data suggest that at low temperature the defining contribution to ΔV comes mainly from excluded volume differences and ΔV for unfolding is negative. In contrast, at high temperatures, differential solvation due to the increased exposed surface area of the unfolded state and, in particular, its larger thermal volume linked to the increased conformational dynamics of the unfolded state ensemble takes over and ΔV for unfolding eventually becomes positive.     

Sensitivity of polar solvation dynamics to the secondary structures of aqueous proteins and the role of surface exposure of the probe

The structure and dynamics of water around a protein is expected to be sensitive to the details of the adjacent secondary structure of the protein. In this article, we explore this sensitivity by calculating both the orientational dynamics of the surface water molecules and the equilibrium solvation time correlation function of the polar amino acid residues in each of the three helical segments of the protein HP-36, using atomistic molecular dynamics simulations. The solvation dynamics of polar amino acid residues in helix-2 is found to be faster than that of the other two helices (the average time constant is smaller by a factor of 2), although the interfacial water molecules around helix-2 exhibit much slower orientational dynamics than that around the other two helices. A careful analysis shows that the origin of such a counterintuitive behavior lies in the dependence of the solvation time correlation function on the surface exposure of the probe-the more exposed is the probe, the faster the solvation dynamics. We discuss that these results are useful in explaining recent solvation dynamics experiments.     

Implications of the unfolded state in the folding energetics of heterogeneous-backbone protein mimetics

Sequence-encoded folding is the foundation of protein structure and is also possible in synthetic chains of artificial chemical composition. In natural proteins, the characteristics of the unfolded state are as important as those of the folded state in determining folding energetics. While much is known about folded structures adopted by artificial protein-like chains, corresponding information about the unfolded states of these molecules is lacking. Here, we report the consequences of altered backbone composition on the structure, stability, and dynamics of the folded and unfolded states of a compact helix-rich protein. Characterization through a combination of biophysical experiments and atomistic simulation reveals effects of backbone modification that depend on both the type of artificial monomers employed and where they are applied in sequence. In general, introducing artificial connectivity in a way that reinforces characteristics of the unfolded state ensemble of the prototype natural protein minimizes the impact of chemical changes on folded stability. These findings have implications in the design of protein mimetics and provide an atomically detailed picture of the unfolded state of a natural protein and artificial analogues under non-denaturing conditions.

Biophysical experiments and atomistic simulation reveal impacts of protein backbone alteration on the ensemble that defines the unfolded state. These effects have implications on folded stability of protein mimetics.     

Intramolecular vibrational excitation of unfolding reactions in ZnII-substituted and metal-free cytochromes c: activation enthalpies from integrated fluorescence stokes shift and line shape excitation profiles

We have employed continuous-wave fluorescence spectroscopy to observe the light-induced formation of partially unfolded states of Zn(II)-substituted and metal-free (or free-base) cytochrome c (ZnCytc and fbCytc, respectively). In these experiments, the intrinsic porphyrin chromophore provides a vibrational excitation to the protein structure via intramolecular vibrational redistribution of the excess vibronic energy above the first excited singlet state. As the excitation light source is tuned, the fluorescence spectrum of both systems exhibits steplike transitions of the integrated Stokes shift, vibronic structure, and line width that mark apparent activation enthalpy barriers for structural transitions of the protein from the native state to a set of at least three partially unfolded states. The vibronic structure of the ZnCytc spectrum reports the exchange of the Zn(II) ion's native H18 and M80 axial ligands with non-native ligands as the excitation wavenumber is scanned through the three barriers. The metal ion's axial ligands contribute substantially to the stability of ZnCytc; the activation enthalpies for the corresponding transitions in fbCytc are one-third of those in ZnCytc. A comparison of the present results from ZnCytc with those obtained previously with picosecond time-resolved methods [Lampa-Pastirk and Beck, J. Phys. Chem. B 2006, 110, 22971-22974] indicates that the vibrationally excited protein structure propagates along an unfolding pathway from the native state that specifically populates the three states in order of their activation enthalpies. The excitation-wavenumber profile of the fluorescence line width is markedly inconsistent with a Maxwell-Boltzmann distribution over the three states. These results contrast with the general expectation of the protein-folding funnel hypothesis that a distribution of intermediate structures should result from the diffusive propagation of a nonequilibrium protein structure.     

Contribution of charged groups to the enthalpic stabilization of the folded states of globular proteins

In this paper we use the results from all-atom molecular dynamics (MD) simulations of proteins and peptides to assess the individual contribution of charged atomic groups to the enthalpic stability of the native state of globular proteins and investigate how the distribution of charged atomic groups in terms of solvent accessibility relates to protein enthalpic stability. The contributions of charged groups is calculated using a comparison of nonbonded interaction energy terms from equilibrium simulations of charged amino acid dipeptides in water (the "unfolded state") and charged amino acids in globular proteins (the "folded state"). Contrary to expectation, the analysis shows that many buried, charged atomic groups contribute favorably to protein enthalpic stability. The strongest enthalpic contributions favoring the folded state come from the carboxylate (COO(-)) groups of either Glu or Asp. The contributions from Arg guanidinium groups are generally somewhat stabilizing, while N(+)(3) groups from Lys contribute little toward stabilizing the folded state. The average enthalpic gain due to the transfer of a methyl group in an apolar amino acid from solution to the protein interior is described for comparison. Notably, charged groups that are less exposed to solvent contribute more favorably to protein native-state enthalpic stability than charged groups that are solvent exposed. While solvent reorganization/release has favorable contributions to folding for all charged atomic groups, the variation in folded state stability among proteins comes mainly from the change in the nonbonded interaction energy of charged groups between the unfolded and folded states. A key outcome is that the calculated enthalpic stabilization is found to be inversely proportional to the excess charge density on the surface, in support of an hypothesis proposed previously.     

A solution study on the local and global structure changes of cytochrome c: an unfolding process induced by urea

The local and global structural changes of cytochrome c induced by urea in aqueous solution have been studied using X-ray absorption spectroscopy (XAS) and small-angle X-ray scattering (SAXS). According to the XAS result, both the native (folded) protein and the unfolded protein exhibit the same preedge features taken at Fe K-edge, indicating that the Fe(III) in the heme group of the protein maintains a six-coordinated local structure in both the folded and unfolded states. Furthermore, the discernible differences in the X-ray absorption near-edge structure (XANES) of these two states are attributed to a possible spin transition of the Fe(III) from a low-spin state to a high-spin state during the unfolding process. The perseverance of six-coordination and the spin transition of the iron are reconciled by a proposed ligand exchange, with urea and water molecules replacing the methionine-80 and histidine-18 axial ligands, respectively. The SAXS result reveals a significant morphology change of cytochrome c from a globular shape of a radius of gyration R(g) = 12.8 A of the native protein to an elongated ellipsoid shape of R(g) = 29.7 A for the unfolded protein in the presence of concentrated urea. The extended X-ray absorption fine structure (EXAFS) data unveil the coordination geometries of Fe(III) in both the folded and unfolded state of cytochrome c. An initial spin transition of Fe(III) followed by an axial ligand exchange, accompanied by the change in the global envelope, is proposed for what happened in the protein unfolding process of cytochrome c.     

A minimalist model for exploring conformational effects on the electrospray charge state distribution of proteins

The electrospray ionization (ESI) charge state distribution of proteins is highly sensitive to the protein structure in solution. Unfolded conformations generally form higher charge states than tightly folded structures. The current study employs a minimalist molecular dynamics model for simulating the final stages of the ESI process in order to gain insights into the physical reasons underlying this empirical relationship. The protein is described as a string of 27 beads ("residues"), 9 of which are negatively charged and represent possible protonation sites. The unfolded state of this bead string is a random coil, whereas the native conformation adopts a compact fold. The ESI process is simulated by placing the protein inside a solvent droplet with a 2.5 nm radius consisting of 1600 Lennard-Jones particles. In addition, the droplet contains 14 protons which are modeled as highly mobile point charges. Disintegration of the droplet rapidly releases the protein into the gas phase, resulting in average charge states of 4.8+ and 7.4+ for the folded and unfolded conformation, respectively. The protonation probabilities of individual residues in the folded state reveal a characteristic pattern, with values ranging from 0.2 to 0.8. In contrast, the protonation probabilities of the unfolded protein are more uniform and cover the range from 0.8 to 1.0. The origin of these differences can be traced back to a combination of steric and electrostatic effects. Residues exhibiting a small accessible surface area are less likely to capture a proton, an effect that is exacerbated by partial electrostatic shielding from nearby positive residues. Conversely, sites that are sterically exposed are associated with electrostatic funnels that greatly increase the likelihood of protonation. Unfolding enhances the steric and electrostatic exposure of protonation sites, thereby causing the protein to capture a greater number of protons during the droplet disintegration process.     

Site-specific hydration dynamics in the nonpolar core of a molten globule by dynamic nuclear polarization of water

Water-protein interactions play a direct role in protein folding. The chain collapse that accompanies protein folding involves extrusion of water from the nonpolar core. For many proteins, including apomyoglobin (apoMb), hydrophobic interactions drive an initial collapse to an intermediate state before folding to the final structure. However, the debate continues as to whether the core of the collapsed intermediate state is hydrated and, if so, what the dynamic nature of this water is. A key challenge is that protein hydration dynamics is significantly heterogeneous, yet suitable experimental techniques for measuring hydration dynamics with site-specificity are lacking. Here, we introduce Overhauser dynamic nuclear polarization at 0.35 T via site-specific nitroxide spin labels as a unique tool to probe internal and surface protein hydration dynamics with site-specific resolution in the molten globular, native, and unfolded protein states. The (1)H NMR signal enhancement of water carries information about the local dynamics of the solvent within ～10 ? of a spin label. EPR is used synergistically to gain insights on local polarity and mobility of the spin-labeled protein. Several buried and solvent-exposed sites of apoMb are examined, each bearing a covalently bound nitroxide spin label. We find that the nonpoloar core of the apoMb molten globule is hydrated with water bearing significant translational dynamics, only 4-6-fold slower than that of bulk water. The hydration dynamics of the native state is heterogeneous, while the acid-unfolded state bears fast-diffusing hydration water. This study provides a high-resolution glimpse at the folding-dependent nature of protein hydration dynamics.     

Ultrafast relaxation dynamics of a biologically relevant probe dansyl at the micellar surface

We report picosecond-resolved measurement of the fluorescence of a well-known biologically relevant probe, dansyl chromophore at the surface of a cationic micelle (cetyltrimethylammonium bromide, CTAB). The dansyl chromophore has environmentally sensitive fluorescence quantum yields and emission maxima, along with large Stokes shift. In order to study the solvation dynamics of the micellar environment, we measured the fluorescence of dansyl chromophore attached to the micellar surface. The fluorescence transients were observed to decay (with time constant approximately 350 ps) in the blue end and rise with similar timescale in the red end, indicative of solvation dynamics of the environment. The solvation correlation function is measured to decay with time constant 338 ps, which is much slower than that of ordinary bulk water. Time-resolved anisotropy of the dansyl chromophore shows a bi-exponential decay with time constants 413 ps (23%) and 1.3 ns (77%), which is considerably slower than that in free solvents revealing the rigidity of the dansyl-micelle complex. Time-resolved area-normalized emission spectroscopic (TRANES) analysis of the time dependent emission spectra of the dansyl chromophore in the micellar environment shows an isoemissive point at 21066 cm-1. This indicates the fluorescence of the chromophore contains emission from two kinds of excited states namely locally excited state (prior to charge transfer) and charge transfer state. The nature of the solvation dynamics in the micellar environments is therefore explored from the time-resolved anisotropy measurement coupled with the TRANES analysis of the fluorescence transients. The time scale of the solvation is important for the mechanism of molecular recognition.     

An Off‐Pathway Folding Intermediate of an Acyl Carrier Protein Domain Coexists with the Folded and Unfolded States under Native Conditions

A protein can exist in multiple states under native conditions and those states with low populations are often critical to biological function and self‐assembly. To investigate the role of the minor states of an acyl carrier protein, NMR techniques were applied to determine the number of minor states and characterize their structures and kinetics. The acyl carrier protein from Micromonospora echinospora was found to exist in one major folded state (95.2 %), one unfolded state (4.1 %), and one intermediate state (0.7 %) under native conditions. The three states are in dynamic equilibrium and the intermediate state very likely adopts a native‐like structure and is an off‐pathway folding product. The intermediate state may mediate the formation of oligomers in vitro and play an important role in the recognition of partner enzymes in vivo.     

脲和盐酸胍诱导过氧化氢酶去折叠的研究

用荧光相图法分别研究了脲和盐酸胍诱导牛肝过氧化氢酶去折叠的过程。当脲 浓度从0依次增大至0.50,4.5和8.0 mol/L时,过氧化氢酶从天然四聚体依次转变 为蓬松的四聚体、部分折叠的无活性二聚体和去折叠态,而当盐酸胍浓度从0依次 变化至0.65,2.5和6.0 mol/L时,过氧化氢酶则从天然四聚体集资转变为部分折叠 的激活二聚体、部分折叠的单体和去折叠态,这表明无论是用脲还是用盐酸胍作为 变性剂,该蛋白的变性过程都符合“四态模型”,但这两种变性剂诱导该蛋白去折 叠的途径和机制有较大差异。实验结果表明荧光相图法可以检测蛋白质去折叠的中 间态。用等温滴定量去热法研究了盐酸胍诱导过氧化氢酶去折叠过程的热力学, 25.0 ℃时低浓度盐酸胍诱导该蛋白从天然四聚体转变为部分折叠的激活二聚体的 本征摩尔构象变化焓、Gibbs自由能和熵分别为-69.2 kJ·mol~(-1),6.43 kJ· mol~(-1)和-254 J·K~(-1)·mol~(-1),据此推断盐酸胍通过熵效应和静电效应来 稳定和激活该二聚体。     

Folding kinetics of a lattice protein via a forward flux sampling approach

We implement a forward flux sampling approach [R. J. Allen et al., J. Chem. Phys. 124, 194111 (2006)] for calculating transition rate constants and for sampling paths of protein folding events. The algorithm generates trajectories for the transition between the unfolded and folded states as chains of partially connected paths, which can be used to obtain the transition-state ensemble and the properties that characterize these intermediates. We apply this approach to Monte Carlo simulations of a model lattice protein in open space and in confined spaces of varying dimensions. We study the effect of confinement on both protein thermodynamic stability and folding kinetics; the former by mapping free-energy landscapes and the latter by the determination of rate constants and mechanistic details of the folding pathway. Our results show that, for the range of temperatures where the native state is stable, confinement of a protein destabilizes the unfolded state by reducing its entropy, resulting in increased thermodynamic stability of the folded state. Relative to the folding in open space, we find that the kinetics can be accelerated at temperatures above the temperature at which the unconfined protein folds fastest and that the rate constant increases with the number of constrained dimensions. By examining the statistical properties of the transition-state ensemble, we detect signs of a classical nucleation folding mechanism for a core of native contacts formed at an early stage of the process. This nucleus acts as folding foci and is composed of those residues that have higher probability to form native contacts in the transition-state intermediates, which can vary depending on the confinement conditions of the system.     

脲和盐酸胍诱导溶菌酶去折叠的荧光相图法研究

用荧光相图法分别研究了脲和盐酸胍诱导卵清溶菌酶去抓叠的过程。当变性体 系中无还原剂2－巯基乙醇存在、脲浓度从0变化至4.0 mol/L（或盐酸胍浓度从0变 化至3.0 mol/L）时,溶菌酶从天然态转变为部分折叠中间态,当脲浓度从4.0 mol/L变化至8.0 mol/L（或盐酸胍浓度从3.0 mol/L变化至6.0 mol/L）时,溶菌 酶从中间态转变为去折叠态,此时该蛋白的变性过程符合“三态模型”。而当变性 体系中有该还原剂存在时,溶菌酶则由天然态直接转变为去折叠态,此时脲诱导该 蛋白去折叠的过程符合曲型的“二态模型”。实难结果表明荧光相图法可以检测蛋 白南去抓叠的中间态。