Nonnative interactions in the FF domain folding pathway from an atomic resolution structure of a sparsely populated intermediate: an NMR relaxation dispersion study

Several all-helical single-domain proteins have been shown to fold rapidly (microsecond time scale) to a compact intermediate state and subsequently rearrange more slowly to the native conformation. An understanding of this process has been hindered by difficulties in experimental studies of intermediates in cases where they are both low-populated and only transiently formed. One such example is provided by the on-pathway folding intermediate of the small four-helix bundle FF domain from HYPA/FBP11 that is populated at several percent with a millisecond lifetime at room temperature. Here we have studied the L24A mutant that has been shown previously to form nonnative interactions in the folding transition state. A suite of Carr-Purcell-Meiboom-Gill relaxation dispersion NMR experiments have been used to measure backbone chemical shifts and amide bond vector orientations of the invisible folding intermediate that form the input restraints in calculations of atomic resolution models of its structure. Despite the fact that the intermediate structure has many features that are similar to that of the native state, a set of nonnative contacts is observed that is even more extensive than noted previously for the wild-type (WT) folding intermediate. Such nonnative interactions, which must be broken prior to adoption of the native conformation, explain why the transition from the intermediate state to the native conformer (millisecond time scale) is significantly slower than from the unfolded ensemble to the intermediate and why the L24A mutant folds more slowly than the WT.     

Effect of urea on the β-hairpin conformational ensemble and protein denaturation mechanism

Despite the daily use of urea to influence protein folding and stability, the molecular mechanism with which urea acts is still not well understood. Here the use of combined parallel tempering and metadynamics simulation allows us to study the free-energy landscape associated with the folding/unfolding of β-hairpin GB1 equilibrium in 8 M urea and pure water. The nature of the unfolded state in both solutions has been analyzed: in urea solution the addition of denaturants acts to expand the denatured state, while in pure water solution the unfolded state is noticeably more compact. For what concerns the mechanism by which urea acts as a denaturant, a preferential direct interaction between urea molecules and protein backbone has been found. However, the bias toward urea solvation is largest at intermediate values of the gyration radius.     

Probing the transition state ensemble of a protein folding reaction by pressure-dependent NMR relaxation dispersion

The F61A/A90G mutant of a redesigned form of apocytochrome b562 folds by an apparent two-state mechanism. We have used the pressure dependence of 15N NMR relaxation dispersion rate profiles to study the changes in volumetric parameters that accompany the folding reaction of this protein at 45 degrees C. The experiments were performed under conditions where the folding/unfolding equilibrium could be studied at each pressure without addition of denaturants. The exquisite sensitivity of the methodology to small changes in folding/unfolding rates facilitated the use of relatively low-pressure values (between 1 and 270 bar) so that pressure-induced changes to the unfolded state ensemble could be minimized. A volume change for unfolding of -81 mL/mol is measured (at 1 bar), a factor of 1.4 larger (in absolute value) than the volume difference between the transition state ensemble (TSE) and the unfolded state. Notably, the changes in the free energy difference between folded and unfolded states and in the activation free energy for folding were not linear with pressure. Thus, the difference in the isothermal compressibility upon unfolding (-0.11 mL mol(-1) bar(-1)) and, for the first time, the compressibility of the TSE relative to the unfolded state (0.15 mL mol(-1) bar(-1)) could be calculated. The results argue for a TSE that is collapsed but loosely packed relative to the folded state and significantly hydrated, suggesting that the release of water occurs after the rate-limiting step in protein folding. The notion of a collapsed and hydrated TSE is consistent with expectations based on earlier temperature-dependent folding studies, showing that the barrier to folding at 45 degrees C is entropic (Choy, W. Y.; Zhou, Z.; Bai, Y.; Kay, L. E. J. Am. Chem. Soc. 2005, 127, 5066-5072).     

Refolding of Cold-Denatured Barstar Induced by Radio-Frequency Heating: A New Method to Study Protein Folding by Real-Time NMR Spectroscopy

The C40A/C82A double mutant of barstar has been shown to undergo cold denaturation above the water freezing point. By rapidly applying radio-frequency power to lossy aqueous samples, refolding of barstar from its cold-denatured state can be followed by real-time NMR spectroscopy. Since temperature-induced unfolding and refolding is reversible for this double mutant, multiple cycling can be utilized to obtain 2D real-time NMR data. Barstar contains two proline residues that adopt a mix of cis and trans conformations in the low-temperature-unfolded state, which can potentially induce multiple folding pathways. The high time resolution real-time 2D-NMR measurements reported here show evidence for multiple folding pathways related to proline isomerization, and stable intermediates are populated. By application of advanced heating cycles and state-correlated spectroscopy, an alternative folding pathway circumventing the rate-limiting cis-trans isomerization could be observed. The kinetic data revealed intermediates on both, the slow and the fast folding pathway.     

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.     

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).     

Surface Arginine Saturation Effect on Unfolding Reaction of Firefly Luciferase: A Thermodynamic and Kinetic Perspective

Replacement of some hydrophobic solvent‐exposed residues in Lampyris turkestanicus luciferase with arginine increases thermostability of this enzyme. Herein, thermodynamic and kinetic of unfolding reactions of wild type (WT), E354R/356R, E354R/356R‐I232R and E354R/356R‐Q35R/L182R/I232R variants, has been investigated. Fluorescence and Far‐UV circular dichroism measurements using urea as a chemical denaturant indicated that the value of for all variants is greater than that of WT enzyme. Analysis of m‐values, as a measure of difference in the solvent accessible surface area between the native and denatured states of protein, revealed that higher stability of mutants is related to their higher degree of compactness in the folded state. Results of unfolding kinetic experiments showed that all variants have three‐exponential behavior in which they unfolded with three rate constants and corresponding amplitudes. Increasing the rate constants of fast unfolding phase in mutants relative to WT protein may be attributed to more compactness and more kinetic sensitivity of their folded state to urea. However, more population of WT protein was unfolded from fast unfolding phase. Results of this investigation highlight kinetic stability of luciferase via a slow rate of unfolding.     

Identification of Folding Intermediates of Streblin,The Most Stable Serine Protease: Biophysical Analysis

Streblin, a serine proteinase from plant Streblus asper, has been used to investigate the conformational changes induced by pH, temperature, and chaotropes. The near/far UV circular dichroism activities under fluorescence emission spectroscopy and 8-aniline-1-naphthalene sulfonate (ANS) binding have been carried out to understand the unfolding of the protein in the presence of denaturants. Spectroscopic studies reveal that streblin belongs to the α+β class of proteins and exhibits stability towards chemical denaturants, guanidine hydrochloride (GuHCl). The pH-induced transition of this protein is noncooperative for transition phases between pH 0.5 and 2.5 (midpoint, 1.5) and pH 2.5 and 10.0 (midpoint, 6.5). At pH 1.0 or lower, the protein unfolds to form acid-unfolded state, and for pH 7.5 and above, protein turns into an alkaline denatured state characterized by the absence of ANS binding. At pH 2.0 (1 M GuHCl), streblin exists in a partially unfolded state with characteristics of a molten globule state. The protein is found to exhibit strong and predominant ANS binding. In total, six different intermediate states has been identified to show protein folding pathways.     

Dehydration in the folding of reduced cytochrome c revealed by the electron-transfer-triggered folding under high pressure

We determined the activation volume associated with protein folding of reduced cytochrome c from the collapsed intermediate to the native state. The folding rate was followed by a change in the absorption (420 nm) at various pressures between 0.1 and 200 MPa and at various concentrations of denaturant (guanidine hydrochloride) between 3.2 and 4.0 M. Dependence of the folding rate on both these factors revealed that the activation volume at ambient pressure in the absence of denaturant is negative (DeltaVf0 = -14 (+/-8) cm3.mol-1). Such a negative activation volume can be accounted for by a decrease in volume resulting from the dehydration of hydrophobic groups, primarily the heme group. Dehydration, which increases the entropy of the protein system, compensates for a decrease in the entropy accompanying the formation of the more compact and ordered transition state. We, therefore, propose that the positive change in the activation entropy for the folding reaction is due to the dehydration of hydrophobic groups. Furthermore, dehydration entropically promotes the protein folding reaction.     

The mechanism of GroEL/GroES folding/refolding of protein substrates revisited

The thermodynamics and kinetics of zinc-cytochrome c (ZnCyt c) interactions with Escherichia coli molecular chaperone GroEL (Chaperonin 60; Cpn60) are described. Zinc(II)-porphyrin represents a flexible fluorescent probe for thermodynamic complex formation between GroEL and ZnCyt c, as well as for stopped-flow fluorescence kinetic experiments. Data suggests that GroEL and GroEL/GroES-assisted refolding of unfolded ZnCyt c takes place by a mechanism that is quite close to the Anfinsen Cage hypothesis for molecular chaperone activity. However, even in the presence of ATP, GroEL/GroES-assisted refolding of ZnCyt c takes place at approximately half the rate of refolding of ZnCyt c alone. On the other hand, there is little evidence for refolding behaviour consistent with the Iterative Annealing hypothesis. This includes a complete lack of GroEL or GroEL/GroES-assisted enhancement of refolding rate constant k(2) associated with the unfolding of a putative misfolded state I (Zn) on the pathway to the native state. Reviewing our data in the light of data from other laboratories, we observe that all forward rate enhancements or reductions could be accounted for in terms of thermodynamic coupling (adjusting positions of refolding equilibria) due to binding interactions between GroEL and unfolded protein substrates, driven by thermodynamic considerations. Therefore, we propose that passive kinetic partitioning should be considered the core mechanism of the GroEL/GroES molecular chaperone machinery, wherein the core function is to bind unfolded protein substrates leading to a blockade of aggregation pathways and to increases in molecular flux through productive folding pathway(s).     

Reversible Unfolding–Refolding of Rubredoxin: A Single‐Molecule Force Spectroscopy Study

In metalloproteins, metal centers serve as active sites for a range of functional purposes and as important structural elements to facilitate protein folding and assembly. It is challenging to observe the reversible unfolding and refolding of metalloproteins because of a loss or decomposition of the metal center. Here, the reversible unfolding–refolding of the iron–sulfur protein rubredoxin was observed directly using single‐molecule force spectroscopy. The results demonstrate that the iron can remain attached to the CXXC motif when rubredoxin is unfolded. Upon relaxation, the unfolded rubredoxin can refold into its native holo state with the reconstituted FeS₄ center. The possible loss of iron from the unfolded protein prevents rubredoxin from refolding into its native holo state. These results demonstrated that unfolding of rubredoxin is reversible, as long as the iron remains attached, and provide experimental evidence for the iron‐priming mechanism for the folding of rubredoxin.     

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.     

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     

Relaxation rate for an ultrafast folding protein is independent of chemical denaturant concentration

The connection between free-energy surfaces and chevron plots has been investigated in a laser temperature jump kinetic study of a small ultrafast folding protein, the 35-residue subdomain from the villin headpiece. Unlike all other proteins that have been studied so far, no measurable dependence of the unfolding/refolding relaxation rate on denaturant concentration was observed over a wide range of guanidinium chloride concentration. Analysis with a simple Ising-like theoretical model shows that this denaturant-invariant relaxation rate can be explained by a large movement of the major free energy barrier, together with a denaturant- and reaction coordinate-dependent diffusion coefficient.     

The fast and the slow: folding and trapping of λ6-85

Molecular dynamics simulations combining many microsecond trajectories have recently predicted that a very fast folding protein like lambda repressor fragment λ(6-85) D14A could have a slow millisecond kinetic phase. We investigated this possibility by detecting temperature-jump relaxation to 5 ms. While λ(6-85) D14A has no significant slow phase, two even more stable mutants do. A slow phase of λ(6-85) D14A does appear in mild denaturant. The experimental data and computational modeling together suggest the following hypothesis: λ(6-85) takes only microseconds to reach its native state from an extensively unfolded state, while the latter takes milliseconds to reach compact β-rich traps. λ(6-85) is not only thermodynamically but also kinetically protected from reaching such "intramolecular amyloids" while folding.     

Capillary electrophoresis investigation of a partially unfolded conformation of beta(2)-microglobulin

Dialysis-related amyloidosis is a disease in which partial unfolding of beta(2)-microglobulin plays a key pathogenetic role in the formation of the amyloid fibrils. We have recently demonstrated that a partially unfolded conformer of beta(2)-microglobulin is involved in fibrillogenesis and that this species is significantly populated under physiological conditions. In this work capillary electrophoresis has been used to measure the equilibrium between the native protein and this conformer in samples known to have a higher or lower amyloidogenic potential, namely full-length beta(2)-microglobulin, two truncated species and a mutant, created by replacing histidine in position 31 with thyrosine. In addition, for all protein species folding stability experiments have been carried out by monitoring the secondary structure by circular dichroism at increasing concentrations of guanidinium chloride. The values of free energy of unfolding in the absence of denaturant, obtained by elaboration of these experiments, were found to be inversely correlated to the area percent of the partially unfolded conformer, as measured by capillary electrophoresis. Affinity capillary electrophoresis experiments have been also carried out under nondenaturing conditions to assess the affinity of copper and suramin to either the native form or the conformational intermediate of full-length beta(2)-microglobulin.     

In Situ Monitored Vortex Fluidic-Mediated Protein Refolding/Unfolding Using an Aggregation-Induced Emission Bioprobe

Protein folding is important for protein homeostasis/proteostasis in the human body. We have established the ability to manipulate protein unfolding/refolding for β-lactoglobulin using the induced mechanical energy in the thin film microfluidic vortex fluidic device (VFD) with monitoring as such using an aggregation-induced emission luminogen (AIEgen), TPE-MI. When denaturant (guanidine hydrochloride) is present with β-lactoglobulin, the VFD accelerates the denaturation reaction in a controlled way. Conversely, rapid renaturation of the unfolded protein occurs in the VFD in the absence of the denaturant. The novel TPE-MI reacts with exposed cysteine thiol when the protein unfolds, as established with an increase in fluorescence intensity. TPE-MI provides an easy and accurate way to monitor the protein folding, with comparable results established using conventional circular dichroism. The controlled VFD-mediated protein folding coupled with in situ bioprobe AIEgen monitoring is a viable methodology for studying the denaturing of proteins.     

Direct Observation of the Reversible Two‐State Unfolding and Refolding of an α/β Protein by Single‐Molecule Atomic Force Microscopy

Directly observing protein folding in real time using atomic force microscopy (AFM) is challenging. Here the use of AFM to directly monitor the folding of an α/β protein, NuG2, by using low‐drift AFM cantilevers is demonstrated. At slow pulling speeds (<50 nm s^?1), the refolding of NuG2 can be clearly observed. Lowering the pulling speed reduces the difference between the unfolding and refolding forces, bringing the non‐equilibrium unfolding–refolding reactions towards equilibrium. At very low pulling speeds (ca. 2 nm s^?1), unfolding and refolding were observed to occur in near equilibrium. Based on the Crooks fluctuation theorem, we then measured the equilibrium free energy change between folded and unfolded states of NuG2. The improved long‐term stability of AFM achieved using gold‐free cantilevers allows folding–unfolding reactions of α/β proteins to be directly monitored near equilibrium, opening the avenue towards probing the folding reactions of other mechanically important α/β and all‐β elastomeric proteins.     

Is there a unique melting temperature for two-state proteins?

Thermal unfolding (or folding) in many proteins occurs in an apparent two-state manner, suggesting that only two states, unfolded and folded, are populated. At the melting temperature, Tm, the two states coexist. Using lattice models with side chains we show that individual residues become structured at temperatures that deviate from Tm, which implies that partially folded conformations make substantial contribution to thermodynamic properties of two-state proteins. We also find that the folding cooperativity for a given residue is linked to its accessible surface area. These results are consistent with the experiments on GCN4-like zipper peptide, which showed that local melting temperatures differ from Tm. Analysis of thermal unfolding of six proteins shows that deltaT/Tm approximately N(-1), where deltaT is the transition width and N is the number of residues. This scaling allows us to conclude that, when corrected for finite size effects, folding cooperativity can be captured using coarse grained models.     

Denaturant sensitive regions in creatine kinase identified by hydrogen/deuterium exchange

The GdmHCl-induced unfolding of creatine kinase (CK) has been studied by hydrogen/deuterium (H/D) exchange combined with mass spectrometry. MM-CK unfolded for various periods in different denaturant concentrations was pulsed-labeled with deuterium to identify different conformational intermediate states. For all denaturation times or GdmHCl concentrations, we observed variable proportions of only two species. The low-mass envelope of isotope peaks corresponds to a species that has gained about 10 deuteriums more than native CK, and the high-mass envelope to a completely deuterated species. To localize precisely the unfolded regions in the states highly populated during denaturation, the protein was digested with two proteases (pepsin and type XIII protease) after H/D exchange and rapid quenching of the reaction. The two sets of fragments obtained were analyzed by liquid chromatography coupled to mass spectrometry to determine the deuterium level in each fragment. Bimodal distributions of deuterium were found for most peptides, indicating that these regions were either folded or unfolded. This behavior is consistent with cooperative, localized unfolding. However, we observed a monomodal distribution of deuterium in two regions (1-12 and 162-186). We conclude that the increment of mass observed in the low-mass species of the intact protein (+10 Da) has its origin in these two segments. These regions, which are very sensitive to low GdmHCl concentrations, are involved in the monomer-monomer interface of CK and their perturbation is likely to weaken the dimeric structure. At higher denaturant concentration, this would induce dissociation of the dimer.