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.     

Separation and characterisation of beta2-microglobulin folding conformers by ion-exchange liquid chromatography and ion-exchange liquid chromatography–mass spectrometry

In this work we present for the first time the use of ion-exchange liquid chromatography to separate the native form and a partially structured intermediate of the folding of the amyloidogenic protein beta₂-microglobulin. Using a strong anion-exchange column that accounts for the differences in charge exposure of the two conformers, a LC–UV method is initially optimised in terms of mobile phase pH, composition and temperature. The preferred mobile phase conditions that afford useful information were found to be 35 mM ammonium formate, pH 7.4 at 25 °C. The dynamic equilibrium of the two species is demonstrated upon increasing the concentration of acetonitrile in the protein sample. Then, the chromatographic method is transferred to MS detection and the respective charge state distributions of the separated conformers are identified. The LC–MS results demonstrate that one of the conformers is partially unfolded, compared with the native and more compact species. The correspondence with previous results obtained in free solution by capillary electrophoresis suggest that strong ion exchange LC–MS does not alter beta₂-microglobulin conformation and maintains the dynamic equilibrium already observed between the native protein and its folding intermediate.     

Congophilicity (Congo red affinity) of different beta2-microglobulin conformations characterized by dye affinity capillary electrophoresis

The amyloidogenic protein beta-microglobulin was characterized by affinity capillary electrophoresis (CE). CE could separate conformational variants of beta2-microglobulin and with the amyloid-specific dye Congo red as a buffer additive it was possible to measure different Congo red-affinities of native and abnormally folded beta2-microglobulin. We find that native beta2-microglobulin has an intermediate affinity for Congo red at pH 7.3 and that binding involves electrostatic interactions. The conformational variant of beta2-microglobulin that appears in acetonitrile solutions binds Congo red more strongly. Affinity CE using Congo red as a buffer additive is a new, simple, fast, and quantitative micromethod for the characterization of soluble conformational intermediates of amyloidogenic proteins.     

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。     

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。     

Detection of the equilibrium folding intermediate of beta-lactoglobulin in the presence of trifluoroethanol by mass spectrometry

Nano-electrospray ionization mass spectrometry (nano-ESI-MS) was used to monitor the effect of trifluoroethanol (TFE) on the conformational properties of beta-lactoglobulin (BLG). TFE stabilizes protein secondary structure, particularly alpha-helices. However, it also acts as a denaturant above critical concentrations. In the case of BLG, TFE at low concentrations is known to induce formation of an equilibrium intermediate that contains non-native helical structure. Such an intermediate is thought to form also under physiological conditions, playing a role in BLG folding in vivo by preventing aggregation. This well-characterized system was chosen in order to test species distributions obtained by nano-ESI-MS. BLG spectra at increasing concentrations of TFE at pH 2 indicate transient accumulation of a conformer whose charge-state distribution (CSD) falls between that of the native and that of the denatured protein, indicating that the TFE-induced, partially folded form can be selectively monitored by this technique. The condition of its maximum accumulation corresponds to 16% TFE, in excellent agreement with results from solution experiments. In contrast, titrations with methanol or acetonitrile (ACN) reveal apparent two-state transitions from native to fully unfolded BLG. At 10% TFE, the protein appears to be still fully folded at room temperature but, if unfolding is elicited by the combination with other denaturing agents, e.g. heat or low concentrations of ACN, it proceeds via formation of the intermediate. Thus, TFE can also induce formation of the BLG intermediate in synergism with generic denaturing agents. This study indicates good agreement between ESI-MS and other biophysical methods monitoring protein conformational transitions in the presence of TFE.     

Temperature dependent equilibrium native to unfolded protein dynamics and properties observed with IR absorption and 2D IR vibrational echo experiments

Dynamic and structural properties of carbonmonoxy (CO)-coordinated cytochrome c(552) from Hydrogenobacter thermophilus (Ht-M61A) at different temperatures under thermal equilibrium conditions were studied with infrared absorption spectroscopy and ultrafast two-dimensional infrared (2D IR) vibrational echo experiments using the heme-bound CO as the vibrational probe. Depending on the temperature, the stretching mode of CO shows two distinct bands corresponding to the native and unfolded proteins. As the temperature is increased from low temperature, a new absorption band for the unfolded protein grows in and the native band decreases in amplitude. Both the temperature-dependent circular dichroism and the IR absorption area ratio R(A)(T), defined as the ratio of the area under the unfolded band to the sum of the areas of the native and unfolded bands, suggest a two-state transition from the native to the unfolded protein. However, it is found that the absorption spectrum of the unfolded protein increases its inhomogeneous line width and the center frequency shifts as the temperature is increased. The changes in line width and center frequency demonstrate that the unfolding does not follow simple two-state behavior. The temperature-dependent 2D IR vibrational echo experiments show that the fast dynamics of the native protein are virtually temperature independent. In contrast, the fast dynamics of the unfolded protein are slower than those of the native protein, and the unfolded protein fast dynamics and at least a portion of the slower dynamics of the unfolded protein change significantly, becoming faster as the temperature is raised. The temperature dependence of the absorption spectrum and the changes in dynamics measured with the 2D IR experiments confirm that the unfolded ensemble of conformers continuously changes its nature as unfolding proceeds, in contrast to the native state, which displays a temperature-independent distribution of structures.     

荧光相图法研究脲诱导牛血清白蛋白的去折叠过程

作为从分子水平上阐明生命奥秘的中心课题之一,蛋白质的折叠问题一直受到生物化学、生物物理学和结构生物学等领域研究工作者的高度关注。在蛋白质的变性过程中,它们往往达不到完全去折叠,而是会形成不同的部分折叠中间态[1-3],这些部分折叠中间态在蛋白质折叠过程中起着重要作     

The influence of Cu(2+) on the unfolding and refolding of intact and proteolytically processed beta(2)-microglobulin

Human beta(2)-microglobulin (beta(2)m) is an amyloidogenic protein in patients suffering from chronic kidney disease and especially in those patients that need intermittent hemodialysis for longer periods, e.g., when awaiting transplantation. While many in vitro conditions induce beta(2)m-amyloid formation from wild-type (wt) beta(2)m and while a number of structurally altered beta(2)m molecules are known to be conformationally unstable and amyloidogenic on their own, it is not known why beta(2)m-amyloid is generated in some dialysis patients. For many amyloid proteins it is known that divalent metal ions, especially Cu(2+), display strong binding and distinct destabilizing effects on protein conformation. The present study uses CE to assess conformational states of wt and cleaved beta(2)m (dK58-beta(2)m, beta(2)m cleaved at lysine-58, a modification found in the circulation of hemodialysis patients) in the presence of divalent metal ions. The experiments provide both qualitative and quantitative data showing the specific destabilizing effects of Cu(2+)-ions on the folding of wt beta(2)m. Both refolding after acid denaturation and solution structure of beta(2)m under otherwise native conditions are severely influenced by Cu(2+). An increased unfolding, aggregation, and induction of Congo red-reactive molecular species in Cu(2+)-incubated wt-beta(2)m could be demonstrated while the refolding kinetics of dK58-beta(2)m, already slower than the wt molecule, appeared not to be further decreased by Cu(2+). Given the interest in the actions of metal ions in other types of amyloidosis, including, e.g., Alzheimer's disease and the prion encephalopathies, the use of microelectrophoretic methods to monitor unfolding and refolding of biomolecules available in scarce amounts as shown in this study is an attractive option.     

Monitoring folding/unfolding transitions of proteins by capillary zone electrophoresis: measurement of deltaG and its variation along the pH scale.

Free-solution capillary zone electrophoresis (CZE) can be used to monitor folding/unfolding transitions of proteins and to construct the classical sigmoidal transition curve describing this isomerization process. By performing a series of CZE experiments along the pH scale (here between pH 2.5 and 6.0) it is possible to measure the parameter [urea]1/2, which represents the concentration of urea at the midpoint of each transition curve, and its dependence from the local pH value. The [urea]1/2 parameter provides an idea of the stability of the protein at a given pH; in the case of cytochrome c, for example, it shows that at and below pH 2 the protein will spontaneously unfold even in the absence of a denaturant. The equation describing the sigmoidal folding/unfolding transition can be used for deriving the term deltaG degrees, which refers to the intrinsic difference in the Gibb's free energy between the (total or partial) denatured state and the reference state, taken usually as the native configuration of a protein. The variation of deltaG degrees between the two extremes of our measurements (pH 2.5 and 6.0) along the stated pH interval has been measured (and theoretically calculated) to be of the order of 7-10 kcal/mol and is here interpreted by assuming that at pH 2.5 and below there is an additionally stretching of the polypeptide coil due to coulombic repulsion, as the unfolded chain looses its zwitterionic character and assumes a pure (or very nearly so) cationic surface. Given the minute amounts of sample required, the fully automated state of the analysis, the rapidity and ease of operation, it is hoped that the CZE technique will become more and more popular in the years to come for monitoring folding/unfolding transitions 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.     

Exploration of the secondary structure specific differential solvation dynamics between the native and molten globule states of the protein HP-36

Recent experiments have shown that the time dependence of fluorescence Stokes shift of a chromophore is substantially different when the chromophore is located in a molten globule (MG) state and in the native state of the same protein. To understand the origin of this difference, particularly the role of water in the differential solvation of the protein in the native and the MG states, we have carried out fully atomistic molecular dynamics simulations with explicit water of a partially unfolded MG state of the protein HP-36 and compared the results with the solvation dynamics of the protein in the folded native state. It is observed that the polar solvation dynamics of the three helical segments of the protein is influenced in a nonuniform heterogeneous manner in the MG state. While the equilibrium solvation time correlation function for helix-3 has been found to relax faster in the MG state as compared to that in the native state, the decay of the corresponding function for the other two helices slows down in the MG state. A careful analysis shows that the origin of such heterogeneous relative solvation behavior lies in the differential location of the polar probe residues and their exposure to bulk solvent. We find a significant negative cross-correlation between the contribution (to the solvation energy of a tagged amino acid residue) of water and the other groups of the protein, indicating a competing role in solvation. The sensitivity of solvation dynamics to the secondary structure and the immediate environment can be used to discriminate the partially unfolded and folded states. These results therefore should be useful in explaining recent solvation dynamics experiments on native and MG states of proteins.     

Sulfonated molecules that bind a partially structured species of beta2-microglobulin also influence refolding and fibrillogenesis

Human beta2-microglobulin (beta2-m) is a small amyloidogenic protein responsible for dialysis-related amyloidosis, which represents a severe complication of long-term hemodialysis. A therapeutic approach for this amyloidosis could be based on the stabilization of beta2-m through the binding to a small molecule, to possibly inhibit protein misfolding and amyloid fibril formation. The search of a strong ligand of this protein is extremely challenging: by using CE in affinity and refolding experiments we study the effect that previously selected sulfonated molecules have on the equilibrium between the native form and an ensemble of conformers populating the slow phase of beta2-m folding. These data are correlated with the effect that the same molecules exert on in vitro fibrillogenesis experiments.     

Design of a hyperstable protein by rational consideration of unfolded state interactions

Stabilization of proteins is a long-sought objective. Targeting the unfolded state interactions of a protein is not a method used for this purpose, although many proteins are known to contain such interactions. The N-terminal domain of ribosomal protein L9 (NTL9) has a lysine residue at position 12, which makes strong non-native interactions in the unfolded state. Substitution of a d-alanine for G34 in NTL9 is known to stabilize the protein by reducing the entropy of the unfolded state. Here we combine these two mutations to design a hyperstable protein. The structure of the variant is the same as that of wild-type as judged by 2D NMR. The variant is hyperstable as judged by denaturation experiments, where complete thermal unfolding of the protein does not occur in native buffer.     

Protein Unfolding of metMyoglobin Monitored by Spectroscopic Techniques

In this experiment the unfolding of the protein, myoglobin, will be monitored using both fluorescence and UV-vis absorption spectroscopy. Changes in the absorbance at 409.5 nm, the absorption maximum of the native state, will be monitored in order to probe changes in the protein conformation after initiation of unfolding by addition of a chemical denaturant. Protein unfolding will also be monitored after exciting the sample at 280 nm and following protein fluorescence emission at 345 nm, the fluorescence maximum for the unfolded state. The absorption run follows the time-dependent decrease in the concentration of the native-state species, whereas the fluorescence experiment monitors the increase in the concentration of the unfolded state. Kinetic rate constants obtained using the two techniques will be compared.     

Capillary electrophoresis analysis of different variants of the amyloidogenic protein β2‐microglobulin as a simple tool for misfolding and stability studies

Free solution capillary electrophoresis with UV detection is here used to retrieve information on the conformational changes of wild‐type β₂‐microglobulin and a series of naturally and artificially created variants known to have different stability and amyloidogenic potential. Under nondenaturing conditions, the resolution of at least two folding conformers at equilibrium is obtained and a third species is detected for the less stable isoforms. Partial denaturation by using chaotropic agents such as acetonitrile or trifluoroethanol reveals that the separated peaks are at equilibrium, as the presence of less structured species is either enhanced or induced at the expenses of the native form. Reproducible CE data allow to obtain an interesting semiquantitative correlation between the peak areas observed and the protein stability. Thermal unfolding over the range 25–42°C is induced inside the capillary for the two pathogenic proteins (wtβ₂‐microglobulin and D76N variant): the large differences observed upon small temperature variation draw attention on the robustness of analytical methods when dealing with proteins prone to misfolding and aggregation.     

Characterisation of cytochrome c unfolding by nano-electrospray ionization time-of-fight and Fourier transform ion cyclotron resonance mass spectrometry

Protein charge-state distributions (CSDs) in electrospray-ionization mass spectrometry (ESI-MS) represent a sensitive tool to probe different conformational states. We describe here the effect of trifluoroethanol (TFE) on cytochrome c equilibrium unfolding at different pH by nano-ESI-MS. While even low concentrations of TFE destabilize the protein native structure at low pH, a TFE content of 2.5%-5% is found to favor cyt c folding at pH approximately 7. Furthermore, we perform comparison of CSDs obtained by time-of-flight (ToF) and Fourier-transform-ion- cyclotron-resonance (FT-ICR) mass analyzers. To this purpose, we analyze spectra of cyt c in the presence of different kind of denaturants. In particular, experiments with 1-propanol suggest that also by FT-ICR-MS, as previously observed on an ESI-ToF instrument, CSDs do not appear to be controlled by the solvent surface tension as predicted by the Rayleigh-charge model. Moreover, there is general good agreement in conformational effects revealed by the different instruments under several buffer conditions. Nevertheless, the ToF instrument appears to discriminate better between unfolded and partially unfolded forms.     

Spectroscopic study on acid-induced unfolding and refolding of apo-neuroglobin

pH-induced unfolding and refolding of apo-neuroglobin (apo-Ngb) were investigated by UV, fluorescence, circular dichroism (CD) spectra and light scattering measurements. Results revealed that apo-Ngb became partially unfolded at around pH 5.0, with evidences from a red shift in the fluorescence spectra, a decrease in the far-UV CD and a sharp peak in the light scattering intensity. Further lowering of the pH reversed these effects, suggesting that apo-Ngb folds back to a compact state. At pH 2.0, the apo-Ngb forms a folding intermediate known as molten globule (MG), which is possessed of native-like secondary structure and almost complete loss of tertiary structure. Based on these results, the acid-induced denaturation pathway of apo-Ngb can be illustrated from the native state (N), via a partially unfolded state (U_A) to the molten globule state (MG).     

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.     

Chemical Stimulus‐responsive Folding and Unfolding of a Dendrimeric Assembly

A dendrimeric trimer undergoes folding and unfolding in response to a chemical stimulus. The trimer of interest contains a central dendrimer with a butadiyne‐linked zinc porphyrin dimer ((ZnP)₂) core, in addition to two terminal dendrimers with zinc porphyrin (ZnP) cores. The obtained absorption spectra indicate that the unfolded form is the exclusive conformer in chloroform, while the addition of 1,4‐diazabicyclo[2.2.2]octane (DABCO) in chloroform leads to transformation from the unfolded to the folded structure containing two DABCO units per trimer; the folded structure originates from the cross‐linking of (ZnP)₂ and ZnP with DABCO. Moreover, the addition of excess DABCO promotes the generation of the unfolded structure containing four DABCO units.