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.     

Electrical conductivity of cytochrome c anhydrous film

Electrical conducitivities of both ferricytochrome c and ferrocytochrome c anhydrous films were measured at physiological temperatures. A film-casting method was applied to prepare the films. Rather low values of resistivities (55°C) of 6.5 × 10¹⁰ Ω cm for ferrocytochrome c in comparison with the previously reported value of 10¹¹ Ω cm (127°C) were obtained. Apparent activation energies for the above conductivities, 0.6 eV, were the same in both samples.     

Viscosity scaling for the glassy phase of protein folding

Although commendable progress has been made in the understanding of the physics of protein folding, a key unresolved issue is whether Kramers' diffusion model of chemical reactions is generally applicable to activated barrier crossing events during folding. To examine the solvent viscosity effect on the folding transition of native-like trapped intermediates, laser flash photolysis has been used to measure the microsecond folding kinetics of a natively folded state of CO-liganded ferrocytochrome c (M-state) in the 1-250 cP range of glycerol viscosity at pH 7.0, 20 degrees C. The single rate coefficient for the folding of the M-state to the native state of the protein (i.e., the M --> N folding process) decreases initially when the solvent viscosity is low (<10 cP), but saturates at higher viscosity, indicating that Kramers model is not general enough for scaling the viscosity dependence of post-transition folding involving glassy dynamics. Analysis based on the Grote-Hynes idea of time dependent friction in conjunction with defect diffusion dynamics can account for the observed non-Kramers scaling.     

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.     

温控分子动力学研究微管蛋白活性肽链的折叠机制

运用温控和常温分子动力学方法, 研究了微管蛋白活性部位Pep1-28肽链的折叠机制, 总模拟时间为380.0 ns. 对于温控分子动力学, 逐渐降温可以清晰显示Pep1-28肽链的折叠途径, 发生明显折叠的温度约为550 K, 其折叠和去折叠可逆机制为U(>1200 K)←→I1(1200-1000 K)←→I2(800 K)←→I3(600 K)←→I4(450 K)←→F1(400 K)←→F2(300 K), 其中U为去折叠态构象, I1、I2、I3和I4是折叠过程中的四个重要的中间态构象, F1和F2是两个结构相近的折叠态构象. 对于常温(300 K)分子动力学, 其构象转变和折叠过程相当迅速, 很难观察到有效、稳定的中间态构象. 尤其引人注意的是, 其折叠态结构陷入了能量局域极小点, 与温控(300 K)的有较大差异, 两者能量差高达297.53 kJ·mol-1. 可见, 温控分子动力学方法不仅清晰地显示多肽和蛋白质折叠过程的重要中间态构象, 为折叠和去折叠机制提供直接、可靠的依据, 而且还有助于跨越较高的构象能垒, 促使多肽和蛋白质折叠以形成全局能量最低的稳定结构.     

Production of O2- in photolyzed water demonstrated through the use of superoxide dismutase

Abstract— The photolysis of water has been studied using ferricytochrome c as the detector of reducing radicals and ferrocytochrome c as the detector of oxidizing radicals. Mannitol was used as a scavenger of hydroxyl radicals and superoxide dismutase was used to expose the specific involvement of superoxide radicals. Aerobic photolysis caused a reduction of ferricytochrome c, which was inhibited by superoxide dismutase and was enhanced by mannitol. Aerobic photolysis also caused the oxidation of ferrocytochrome c, which was inhibited by mannitol and augmented by superoxide dismutase. The presence of superoxide dismutase also eliminated the effects of mannitol on the aerobic oxidation of ferrocytochrome c. Photolysis in the absence of oxygen also caused the reduction of ferricytochrome c and the oxidation of ferrocytochrome c, but under these anaerobic conditions neither mannitol nor superoxide dismutase exerted significant effects. An explanation of these observations is offered in terms of the reactivities of H^., OH^. and O^-2 radicals.     

Ultrafast Heme Dynamics of Ferric Cytochrome c in Different Environments: Electronic,Vibrational, and Conformational Relaxation

The excited‐state dynamics of ferric cytochrome c (Cyt c), an important electron‐transfer heme protein, in acidic to alkaline medium and in its unfolded form are investigated by using femtosecond pump–probe spectroscopy, exciting the heme and Tryptophan (Trp) to understand the electronic, vibrational, and conformational relaxation of the heme. At 390 nm excitation, the electronic relaxation of heme is found to be ≈150 fs at different pH values, increasing to 480 fs in the unfolded form. Multistep vibrational relaxation dynamics of the heme, including fast and slow processes, are observed at pH 7. However, in the unfolded form and at pH 2 and 11, fast phases of vibrational relaxation dominate, revealing the energy dissipation occurring through the covalent bond interaction between the heme and the nearest amino acids. A significant shortening of the excited‐state lifetime of Trp is observed at various pH values at 280 nm excitation due to resonance energy transfer to the heme. The longer time constant (25 ps) observed in the unfolded form is attributed to a complete global conformational relaxation of Cyt c.     

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).     

Fast folding of a four-helical bundle protein

The FK506-FKBP12 binding-domain of the kinase FRAP (FRB) forms a classic up-down four-helical bundle. The folding pathway of this protein has been investigated using a combination of equilibrium and kinetic studies. The native state of the protein is stable with respect to the unfolded state by some 7 kcal mol(-1) at pH 6.0, 10 degrees C. A kinetic analysis of unfolding and refolding rate constants as a function of chemical denaturant concentration suggests that an intermediate state may be populated during folding at low concentrations of denaturant. The presence of this intermediate state is confirmed by refolding experiments performed in the presence of the hydrophobic dye 8-anilinonaphthalene-1 sulfonate (ANS). ANS binds to the partially folded intermediate state populated during the folding of FRB and undergoes a large change in fluorescence that can be detected using stopped-flow techniques. Analysis of the kinetic data suggests that the intermediate state is compact and it may even be a misfolded species that has to partially unfold before it can reach the transition state. Folding and unfolding rate constants in water are approximately 150-200 s(-1) and 0.005-0.06 s(-1), respectively, at neutral pH and 10 degrees C. The folding of FRB is somewhat slower than for other all-helical proteins, probably as a consequence of the formation of a metastable intermediate state. The folding rate constant in the absence of any populated intermediate can be estimated to be 8800 s(-1). Despite the presence of an intermediate state, which effectively slows folding, the protein still folds rapidly with a half-life of 5 ms at 10 degrees C. The dependence of the rate constants on denaturant concentration indicates that the transition state for folding is compact with some 80% of the surface area exposed in the unfolded state buried in the transition state. Data presented for FRB is compared with kinetic data obtained for other all-helical proteins.     

Photochemical reduction of cytochrome c by a 1,4,5,8-naphthalenediimide radical anion

Steady-state UV irradiation of aqueous solutions containing cytochrome c (cyt c) and N,N'-bis(2-phosphonoethyl)-1,4,5,8-naphthalenediimide (BPNDI), a water-soluble aromatic imide, resulted in the reduction of the heme iron from the Fe(III) to the Fe(II) oxidation state. The reaction kinetics were followed by the increase of the ferrocytochrome c absorbance band at 549 nm. The rate of the photochemical reaction was pH dependent, reaching its maximum values over the pH range 4-7. Addition of electrolyte (NaCl) at pH 5 resulted in a decrease in the reaction rate, as expected for reactions between oppositely charged species. Flash photolysis studies revealed that the actual reductant in the reaction was a photogenerated BPNDI radical anion, which transferred an electron to the cyt c heme iron. The participation of imide radicals in the process was confirmed by the ready reduction of cyt c by BPNDI radicals chemically generated with sodium dithionite.     

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).     

Structural and redox properties of mitochondrial cytochrome c co-sorbed with phosphate on hematite (alpha-Fe2O3) surfaces

The interaction of metalloproteins with oxides has implications not only for bioanalytical systems and biosensors but also in the areas of biomimetic photovoltaic devices, bioremediation, and bacterial metal reduction. Here, we investigate mitochondrial ferricytochrome c (Cyt c) co-sorption with 0.01 and 0.1 M phosphate on hematite (alpha-Fe2O3) surfaces as a function of pH (2-11). Although Cyt c sorption to hematite in the presence of phosphate is consistent with electrostatic attraction, other forces act upon Cyt c as well. The occurrence of multilayer adsorption, and our AFM observations, suggest that Cyt c aggregates as the pH approaches the Cyt c isoelectric point. In solution, methionine coordination of heme Fe occurs only between pH 3 and 7, but in the presence of phosphate this coordination is retained up to pH 10. Electrochemical evidence for the presence of native Cyt c occurs down to pH 3 and up to pH 10 in the absence of phosphate, and this range is extended to pH 2 and 11 in the presence of phosphate. Cyt c that initially adsorbs to a hematite surface may undergo conformation change and coat the surface with unfolded protein such that subsequently adsorbing protein is more likely to retain the native conformational state. AFM provides evidence for rapid sorption kinetics for Cyt c co-sorbed with 0.01 or 0.1 M phosphate. Cyt c co-sorbed with 0.01 M phosphate appears to unfold on the surface of hematite while Cyt c co-sorbed with 0.1 M phosphate possibly retains native conformation due to aggregation.     

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.     

Redox-coupled dynamics and folding in cytochrome c

Cytochrome c functions as an electron carrier in the mitochondrial electron-transport chain using the Fe(II)-Fe(III) redox couple of a covalently attached heme prosthetic group, and it has served as a paradigm for both biological redox activity and protein folding. On the basis of a wide variety of biophysical techniques, it has been suggested that the protein is more flexible in the oxidized state than in the reduced state, which has led to speculation that it is the dynamics of the protein that has been evolved to control the cofactor's redox properties. To test this hypothesis, we incorporated carbon-deuterium bonds throughout cytochrome c and characterized their absorption frequencies and line widths using IR spectroscopy. The absorption frequencies of several residues on the proximal side of the heme show redox-dependent changes, but none show changes in line width, implying that the flexibility of the oxidized and reduced proteins is not different. However, the spectra demonstrate that folded protein is in equilibrium with a surprisingly large amount of locally unfolded protein, which increases with oxidation for residues localized to the proximal side of the heme. The data suggest that while the oxidized protein is not more flexible than the reduced protein, it is more locally unfolded. Local unfolding of cytochrome c might be one mechanism whereby the protein evolved to control electron transfer.     

Effects of pH on the kinetic reaction

In most cases, kinetic unfolding reactions of proteins follow a simple one-step mechanism that does not involve any detectable intermediates. One example for a more complicated unfolding reaction is the acid-induced denaturation of holo-myoglobin (hMb). This reaction proceeds through a transient intermediate and can be described by a sequential two-step mechanism (Konermann et al. Biochemistry 1997, 36, 6448-6454). Time-resolved electrospray ionization mass spectrometry (ESI MS) is a new technique for monitoring the kinetics of protein folding and unfolding in solution. Different protein conformations can be distinguished by the different charge state distributions that they generate during ESI. At the same time this technique allows monitoring the loss or binding of noncovalent protein ligands. In this work, time-resolved ESI MS is used to study the dependence of the kinetic unfolding mechanism of hMb on the specific solvent conditions used in the experiment. It is shown that hMb unfolds through a short-lived intermediate only at acidic pH. Under basic conditions no intermediate is observed. These findings are confirmed by the results of optical stopped-flow absorption experiments. This appears to be the first time that a dependence of the kinetic mechanism for protein unfolding on external conditions such as pH has been observed.     

Energetic basis of human telomeric DNA folding into G-quadruplex structures

Recent theoretical studies performed on the folding/unfolding mechanism of the model telomeric human DNA, 5'-AGGGTTAGGGTTAGGGTTAGGG-3' (Tel22), have indicated that in the presence of K(+) ions Tel22 folds into two hybrid G-quadruplex structures characterized by one double and two reversal TTA loops arranged in a different way. They predicted a new unfolding pathway from the initial mixture of hybrid G-quadruplexes via the corresponding intermediate triplex structures into the final, fully unfolded state. Significantly, no experimental evidence supporting the suggested pathway has been reported. In the current work, we performed a comprehensive global thermodynamic analysis of calorimetric (DSC, ITC) and spectroscopic (CD) data obtained on monitoring the folding/unfolding of Tel22 induced by changes of temperature and K(+) concentration. We show that unfolding of Tel22 may be described as a monomolecular equilibrium three-state process that involves thermodynamically distinguishable folded (F), intermediate (I), and unfolded (U) state. Considering that calorimetric methods cannot distinguish between energetically similar G-quadruplex or triplex conformations predicted by the theoretical model one can conclude that our results represent the first experimental support of the suggested unfolding/folding mechanism of Tel22. This conclusion is confirmed by the fact that the estimated number of K(+) ions released upon each unfolding step in our thermodynamic model agrees well with the corresponding values predicted by the theoretical model and that the observed changes in enthalpy, entropy, and heat capacity accompanying the F → I and I → U transitions can be reasonably explained only if the intermediate state I is considered to be a triplex structural conformation.     

Evidence for a Molten Globule State in Cicer α-Galactosidase Induced by pH, Temperature, and Guanidine Hydrochloride

Physiologically as well as industrially, α-galactosidases are very important enzymes, but very little is known about the stability and folding aspect of enzyme. In the present study, we have investigated the temperature, pH, and guanidine hydrochloride (GuHCl) induced unfolding of Cicer α-galactosidase using circular dichroism and fluorescence spectroscopy. Strong negative ellipticities at 208, 215, and 222 nm indicate the presence of both α and β structures in Cicer α-galactosidase and showed that its secondary structure belongs to α?+?β class of proteins with 31 % α-helicity. For Cicer α-galactosidase the emission maximum was found to be 345 nm which suggests that tryptophan residues are less exposed to solvent. However, at pH?2.0, protein showed blue-shift. This state of protein lacked activity but it retained significant secondary structure. Enhanced binding of ANS at pH?2.0 indicated significant unfolding and exposure of hydrophobic regions. The unfolded state of Cicer α-galactosidase showed a red-shift of 15 nm with a concomitant decrease in the fluorescence intensity. The enzyme maintained its native structure and full activity up to 40 °C; however, above this temperature, denaturation was observed.     

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.     

Comparison of continuous and pulsed labeling amide hydrogen exchange/mass spectrometry for studies of protein dynamics

In contrast to the rigid structures portrayed by X-ray diffraction, proteins in solution display constant motion which leads to populations that are momentarily unfolded. To begin to understand protein dynamics, we must have experimental methods for determining rates of folding and unfolding, as well as for identifying structures of folding and unfolding intermediates. Amide hydrogen exchange has become an important tool for such measurements. When urea is used to stabilize unfolded forms of proteins, the refolding rates may become slower than the rates of isotope exchange. In such cases, the intermolecular distribution of deuterium among the entire population of molecules may become bimodal, giving rise to a bimodal distribution of isotope peaks in mass spectra of the protein or its peptic fragments. When the protein is exposed continuously to D2O, the relative intensities of the two envelopes of isotope peaks give an integrated account of populations participating in the folding/unfolding process. However, when the protein is exposed only briefly to D2O, the relative intensities of the two envelopes of isotope peaks give an instantaneous measure of the folded/unfolded populations. Application of these two labeling methods to a large protein, aldolase, is described along with a discussion of specific parameters required to optimize these experiments.