COSMIC analysis of the major alpha-helix of barnase during folding

The structures of transition states and intermediates in protein folding may be analysed by protein engineering methods that remove simple interactions that stabilize the folded state. We have now extended the range and reliability of the procedure by using the COSMIC (Combination of Sequential Mutant Interaction Cycles) technique, in which a series of double-mutant cycles is constructed. In each cycle, the side-chains of two amino acid residues that interact in the folded state are mutated separately and together. Kinetic and equilibrium measurements on folding for each cycle show unambiguously whether or not two residues interact during protein folding. A series of such cycles has been constructed to leapfrog along the major alpha-helix of barnase, comprising residues 6 to 18. The helix is found to be intact from its C terminus to residue 12 but begins to unwind towards the N terminus in both the transition state for unfolding and in a folding intermediate.     

Temperature-dependent downhill unfolding of ubiquitin. I. Nanosecond-to-millisecond resolved nonlinear infrared spectroscopy

Transient thermal unfolding of ubiquitin is investigated using nonlinear infrared spectroscopy after a nanosecond laser temperature jump (T-jump). The abrupt change in the unfolding free energy surface and the ns time resolution allow us to observe a fast response on ns to micros time-scales, which we attribute to downhill unfolding, before a cross-over to ms kinetics. The downhill unfolding by a sub-population of folded proteins is induced through a shift of the barrier toward the native state. By adjusting the T-jump width, the effect of the initial (T(i)) and final (T(f)) temperature on the unfolding dynamics can be separated. From the amplitude of the fast downhill unfolding, the fractional population prepared at the unfolding transition state is obtained. This population increases with both T(i) and with T(f). A two-state kinetic analysis of the ms refolding provides thermodynamic information about the barrier height. By a combination of the fast and slow unfolding and folding parameters, a quasi-two-state kinetic analysis is performed to calculate the time-dependent population changes of the folded state. This calculation coincides with the experimentally obtained population changes at low temperature but deviations are found in the T-jump from 67 to 78 degrees C. Using temperature-dependent barrier height changes, a temperature Phi value analysis is performed. The result shows a decreasing trend of Phi(T) with temperature, which indicates an increase of the heterogeneity of the transition state. We conclude that ubiquitin unfolds along a well-defined pathway at low temperature which expands with increasing temperature to include multiple routes.     

Early events in apomyoglobin unfolding probed by laser T-jump/UV resonance Raman spectroscopy

Early events in the unfolding of apomyoglobin are studied with time-resolved ultraviolet resonance Raman (UVRR) spectroscopy coupled to a laser-induced temperature jump (T-jump). The UVRR spectra provide simultaneous probes of the aromatic side-chain environment and the amide backbone conformation. The amide bands reveal helix melting, with relaxation times of 70 and 16 micros at pH 5.5 and 4, respectively, in reasonable agreement with previously reported amide I' FTIR/T-jump relaxations (132 and 14 micros at pD 5.5 and 3). The acceleration at pH 4 is consistent with destabilization of the hydrophobic AGH core of the protein via protonation of a pair of buried histidines. The same relaxation times are found for intensity loss by the phenylalanine F12 band, signaling solvent exposure of the phenyl rings. There are seven Phe residues, distributed throughout the protein; they produce a global response, parallel to helix melting. Relaxation of the tryptophan W16 intensity also parallels helix melting at pH 5.5 but is twice as fast, 7 micros, at pH 4. The pH 5.5 signal arises from Trp 7, which is partially solvent-exposed, while the pH 4 signal arises from the buried Trp 14. Thus, Trp 14 is exposed to the solvent prior to helix melting of the AGH core, suggesting initial displacement of the A helix, upon which Trp 14 resides. All of the UVRR signals show a prompt response, within the instrument resolution (approximately 60 ns), which accounts for half of the total relaxation amplitude. This response is attributed to solvent penetration into the protein, possibly convoluted with melting of hydrated helix segments.     

Core formation in apomyoglobin: probing the upper reaches of the folding energy landscape

An acid-destabilized form of apomyoglobin, the so-called E state, consists of a set of heterogeneous structures that are all characterized by a stable hydrophobic core composed of 30-40 residues at the intersection of the A, G, and H helices of the protein, with little other secondary structure and no other tertiary structure. Relaxation kinetics studies were carried out to characterize the dynamics of core melting and formation in this protein. The unfolding and/or refolding response is induced by a laser-induced temperature jump between the folded and unfolded forms of E, and structural changes are monitored using the infrared amide I' absorbance at 1648-1651 cm(-1) that reports on the formation of solvent-protected, native-like helix in the core and by fluorescence emission changes from apomyoglobin's Trp14, a measure of burial of the indole group of this residue. The fluorescence kinetics data are monoexponential with a relaxation time of 14 micros. However, infrared kinetics data are best fit to a biexponential function with relaxation times of 14 and 59 micros. These relaxation times are very fast, close to the limits placed on folding reactions by diffusion. The 14 micros relaxation time is weakly temperature dependent and thus represents a pathway that is energetically downhill. The appearance of this relaxation time in both the fluorescence and infrared measurements indicates that this folding event proceeds by a concomitant formation of compact secondary and tertiary structures. The 59 micros relaxation time is much more strongly temperature dependent and has no fluorescence counterpart, indicating an activated process with a large energy barrier wherein nonspecific hydrophobic interactions between helix A and the G and H helices cause some helix burial but Trp14 remains solvent exposed. These results are best fit by a multiple-pathway kinetic model when U collapses to form the various folded core structures of E. Thus, the results suggest very robust dynamics for core formation involving multiple folding pathways and provide significant insight into the primary processes of protein folding.     

Folding of the multidomain ribosomal protein L9: the two domains fold independently with remarkably different rates

The folding and unfolding behavior of the multidomain ribosomal protein L9 from Bacillus stearothermophilus was studied by a novel combination of stopped-flow fluorescence and nuclear magnetic resonance (NMR) spectroscopy. One-dimensional 1H spectra acquired at various temperatures show that the C-terminal domain unfolds at a lower temperature than the N-terminal domain (Tm = 67 degrees C for the C-terminal domain, 80 degrees C for the N-terminal domain). NMR line-shape analysis was used to determine the folding and unfolding rates for the N-terminal domain. At 72 degrees C, the folding rate constant equals 2980 s-1 and the unfolding rate constant equals 640 s-1. For the C-terminal domain, saturation transfer experiments performed at 69 degrees C were used to determine the folding rate constant, 3.3 s-1, and the unfolding rate constant, 9.0 s-1. Stopped-flow fluorescence experiments detected two resolved phases: a fast phase for the N-terminal domain and a slow phase for the C-terminal domain. The folding and unfolding rate constants determined by stopped-flow fluorescence are 760 s-1 and 0.36 s-1, respectively, for the N-terminal domain at 25 degrees C and 3.0 s-1 and 0.0025 s-1 for the C-terminal domain. The Chevron plots for both domains show a V-shaped curve that is indicative of two-state folding. The measured folding rate constants for the N-terminal domain in the intact protein are very similar to the values determined for the isolated N-terminal domain, demonstrating that the folding kinetics of this domain is not affected by the rest of the protein. The remarkably different rate constants between the N- and C-terminal domains suggest that the two domains can fold and unfold independently. The folding behavior of L9 argues that extremely rapid folding is not necessarily functionally important.     

Experimental tests of villin subdomain folding simulations

We have used laser temperature-jump to investigate the kinetics and mechanism of folding the 35 residue subdomain of the villin headpiece. The relaxation kinetics are biphasic with a sub-microsecond phase corresponding to a helix-coil transition and a slower microsecond phase corresponding to overall unfolding/refolding. At 300 K, the folding time is 4.3(+/-0.6) micros, making it the fastest folding, naturally occurring protein, with a rate close to the theoretical speed limit. This time is in remarkable agreement with the prediction of 5 (+11,-3) micros by Zagrovic et al. from atomistic molecular dynamics simulations using an implicit solvent model. We test their prediction that replacement of the C-terminal phenylalanine residue with alanine will increase the folding rate by removing a transient non-native interaction. We find that the alanine substitution has no effect on the folding rate or on the equilibrium constant. Implications of this result for the validity of the simulated folding mechanism are discussed.     

Ligand exchange during unfolding of cytochrome c.

The productive folding pathway of cytochrome c passes through an obligatory HW intermediate in which the heme is coordinated by a solvent water molecule and a native ligand, His-18, prior to the formation of the folded HM state with both the native His-18 and Met-80 heme coordination. Two off pathway intermediates, a five-coordinated state (5C) and a bis-histidine state (HH), were also identified during the folding reaction. In the present work, the thermodynamics and the kinetics of the unfolding reaction of cytochrome c were investigated with resonance Raman scattering, tryptophan fluorescence spectroscopy, and circular dichroism. The objective of these experiments was to determine if the protein opens up and diverges into the differing heme ligation states through a many pathway mechanism or if it passes through intermediate states analogous to those observed during the folding reaction. Equilibrium unfolding results indicate that, in contrast to 5C, the stability of HH with respect to HW decreases as the concentration of GdnHCl increases. The difference in their response to the denaturant indicates that the polypeptide structure of 5C is relatively loose as compared with HH in which the polypeptide is misfolded. Time-resolved resonance Raman measurements show that strikingly similar ligand exchange reactions occur during unfolding as were observed during folding. Combined with fluorescence data, a kinetic model is proposed in which local structural rearrangements controlled by heme ligand exchange reactions appear prior to the global relaxation of the polypeptide chain.     

Probing the folding and unfolding dynamics of secondary and tertiary structures in a three-helix bundle protein

Fast relaxation kinetics studies of the B-domain of staphylococcal protein A were performed to characterize the folding and unfolding of this small three-helix bundle protein. The relaxation kinetics were initiated using a laser-induced temperature jump and probed using time-resolved infrared spectroscopy. The kinetics monitored within the amide I' absorbance of the polypeptide backbone exhibit two distinct kinetics phases with nanosecond and microsecond relaxation times. The fast kinetics relaxation time is close to the diffusion limits placed on protein folding reactions. The fast kinetics phase is dominated by the relaxation of the solvated helix (nu = 1632 cm(-1)), which reports on the fast relaxation of the individual helices. The slow kinetics phase follows the cooperative relaxation of the native helical bundle core that is monitored by both solvated (nu = 1632 cm(-1)) and buried helical IR bands (nu = 1652 cm(-1)). The folding rates of the slow kinetics phase calculated over an extended temperature range indicate that the core formation of this protein follows a pathway that is energetically downhill. The unfolding rates are much more strongly temperature-dependent indicating an activated process with a large energy barrier. These results provide significant insight into the primary process of protein folding and suggest that fast formation of helices can drive the folding of helical proteins.     

Cytochrome c folds through a smooth funnel

A dominant feature of folding of cytochrome c is the presence of nonnative His-heme kinetic traps, which either pre-exist in the unfolded protein or are formed soon after initiation of folding. The kinetically trapped species can constitute the majority of folding species, and their breakdown limits the rate of folding to the native state. A temperature jump (T-jump) relaxation technique has been used to compare the unfolding/folding kinetics of yeast iso-2 cytochrome c and a genetically engineered double mutant that lacks His-heme kinetic traps, H33N,H39K iso-2. The results show that the thermodynamic properties of the transition states are very similar. A single relaxation time tau(obs) is observed for both proteins by absorbance changes at 287 nm, a measure of solvent exclusion from aromatic residues. At temperatures near Tm, the midpoint of the thermal unfolding transitions, tau(obs) is four to eight times faster for H33N,H39K iso-2 (tau(obs) approximately 4-10 ms) than for iso-2 (tau(obs) approximately 20-30 ms). T-jumps show that there are no kinetically unresolved (tau < 1-3 micros T-jump dead time) "burst" phases for either protein. Using a two-state model, the folding (k(f)) and unfolding (k(u)) rate constants and the thermodynamic activation parameters standard deltaGf, standard deltaGu, standard deltaHf, standard deltaHu, standard deltaSf, standard deltaSu are evaluated by fitting the data to a function describing the temperature dependence of the apparent rate constant k(obs) (= tau(obs)(-1)) = k(f) + k(u). The results show that there is a small activation enthalpy for folding, suggesting that the barrier to folding is largely entropic. In the "new view," a purely entropic kinetic barrier to folding is consistent with a smooth funnel folding landscape.     

Molecular basis of cooperativity in protein folding IV. Core: A general cooperative folding model

The cooperative nature of the protein folding process is independent of the characteristic fold and the specific secondary structure attributes of a globular protein. A general folding/unfolding model should, therefore, be based upon structural features that transcend the peculiarities of α-helices, β-sheets, and other structural motifs found in proteins. The studies presented in this paper suggest that a single structural characteristic common to all globular proteins is essential for cooperative folding. The formation of a partly folded state from the native state results in the exposure to solvent of two distinct regions: (1) the portions of the protein that are unfolded; and (2) the “complementary surfaces,” located in the regions of the protein that remain folded. The cooperative character of the folding/unfolding transition is determined largely by the energetics of exposing complementary surface regions to the solvent. By definition, complementary regions are present only in partly folded states; they are absent from the native and unfolded states. An unfavorable free energy lowers the probability of partly folded states and increases the cooperativity of the transition. In this paper we present a mathematical formulation of this behavior and develop a general cooperative folding/unfolding model, termed the “complementary region” (CORE) model. This model successfully reproduces the main properties of folding/unfolding transitions without limiting the number of partly folded states accessible to the protein, thereby permitting a systematic examination of the structural and solvent conditions under which intermediates become populated. It is shown that the CORE model predicts two-state folding/unfolding behavior, even though the two-state character is not assumed in the model. © 1993 Wiley-Liss, Inc.     

Temperature dependence of the folding and unfolding kinetics of the GCN4 leucine zipper via 13C(alpha)-NMR

Studies by one-dimensional NMR are reported on the interconversion of folded and unfolded forms of the GCN4 leucine zipper in neutral saline buffer. The peptide bears 99% 13C(alpha) labels at three sites: V9, L12, and G31. Time-domain 13C(alpha)-NMR spectra are interpreted by global Bayesian lineshape analysis to extract the rate constants for both unfolding and folding as functions of temperature in the range 47-71 degrees C. The data are well fit by the assumption that the same rate constants apply at each labeled site, confirming that only two conformational states need be considered. Results show that 1) both processes require a free energy of activation; 2) unfolding is kinetically enthalpy-opposed and entropy-driven, while folding is the opposite; and 3) the transition state dimer ensemble averages approximately 40% helical. The activation parameters for unfolding, derived from NMR data at the elevated temperatures where both conformations are populated, lead to estimates of the rate constant at low temperatures (5-15 degrees C) that agree with extant values determined by stopped-flow CD via dilution from denaturing media. However, the corresponding estimated values for the folding rate constant are larger by two to three orders of magnitude than those obtained by stopped flow. We propose that this apparent disagreement is caused by the necessity, in the stopped-flow experiment, for initiation of new helices as the highly denaturant-unfolded molecule adjusts to the newly created benign solvent conditions. This must reduce the success rate of collisions in producing the folded molecule. In the NMR determinations, however, the unfolded chains always have a small, but essential, helix content that makes such initiation unnecessary. Support for this hypothesis is adduced from recent extant experiments on the helix-coil transition in single-chain helical peptides and from demonstration that the folding rate constants for coiled coils, as obtained by stopped flow, are influenced by the nature of the denaturant used.     

Wild type, mutant protein unfolding and phase transition detected by single-nanopore recording

Understanding protein folding remains a challenge. A difficulty is to investigate experimentally all the conformations in the energy landscape. Only single molecule methods, fluorescence and force spectroscopy, allow observing individual molecules along their folding pathway. Here we observe that single-nanopore recording can be used as a new single molecule method to explore the unfolding transition and to examine the conformational space of native or variant proteins. We show that we can distinguish unfolded states from partially folded ones with the aerolysin pore. The unfolding transition curves of the destabilized variant are shifted toward the lower values of the denaturant agent compared to the wild type protein. The dynamics of the partially unfolded wild type protein follows a first-order transition. The denaturation curve obtained with the aerolysin pore is similar to that obtained with the α-hemolysin pore. The nanopore geometry or net charge does not influence the folding transition but changes the dynamics.     

Multifrequency calorimetry of the folding/unfolding transition of cytochrome c.

The folding-unfolding transition of Fe(III) cytochrome c has been studied with the new technique of multifrequency calorimetry. Multifrequency calorimetry is aimed at measuring directly the dynamics of the energetic events that take place during a thermally induced transition by measuring the frequency dispersion of the heat capacity. This is done by modulating the folding/unfolding equilibrium using a variable frequency, small oscillatory temperature perturbation (approximately 0.05-0.1 degrees C) centered at the equilibrium temperature of the system. Fe(III) cytochrome c at pH 4 undergoes a fully reversible folding/unfolding transition centered at 67.7 degrees C and characterized by an enthalpy change of 81 kcal/mol and heat capacity difference between unfolded and folded states of 0.9 kcal/K*mol. By measuring the temperature dependence of the frequency dispersion of the heat capacity in the frequency range of 0.1-1 Hz it has been possible to examine the time regime of the enthalpic events associated with the transition. The multifrequency calorimetry results indicate that approximately 85% of the excess heat capacity associated with the folding/unfolding transition relaxes with a single relaxation time of 326 +/- 68 ms at the midpoint of the transition region. This is the first time that the time regime in which heat is absorbed and released during protein folding/unfolding has been measured.     

Reversible denaturation of the gene V protein of bacteriophage f1

The guanidine hydrochloride (GuHCl)-induced denaturation of the gene V protein of bacteriophage f1 has been studied, using the chemical reactivity of a cysteine residue that is buried in the folded protein and the circular dichroism (CD) at 211 and 229 nm as measures of the fraction of polypeptide chains in the folded form. It is found that this dimeric protein unfolds in a single cooperative transition from a folded dimer to two unfolded monomers. A folded, monomeric form of the gene V protein was not detected at equilibrium. The kinetics of unfolding of the gene V protein in 3 M GuHCl and the refolding in 2 M GuHCl are also consistent with a transition between a folded dimer and two unfolded monomers. The GuHCl concentration dependence of the rates of folding and unfolding suggests that the transition state for folding is near the folded conformation.     

Molecular basis of co-operativity in protein folding.

The folding/unfolding transition of proteins is a highly co-operative process characterized by the presence of very few or no thermodynamically stable partially folded intermediate states. The purpose of this paper is to present a thermodynamic formalism aimed at describing quantitatively the co-operative folding behavior of proteins. In order to account for this behavior, a hierarchical algorithm aimed at evaluating the folding/unfolding partition function has been developed. This formalism defines the partition function in terms of multiple levels of interacting co-operative folding units. A co-operative folding unit is defined as a protein structural element that exhibits two-state folding/unfolding behavior. At the most fundamental level are those structural elements that behave co-operatively as a result of purely local interactions. Higher-order co-operative folding units are formed through interactions between different structural elements. The hierarchical formalism utilizes the crystallographic structure of the protein as a template to generate partially folded conformations defined in terms of co-operative folding units. The Gibbs free energy of those states and their corresponding statistical weights are then computed using experimental energetic parameters determined calorimetrically. This formalism has been applied to the case of myoglobin. It is shown that the hierarchical partition function correctly predicts the presence, energetics and co-operativity of the heat and cold denaturation transitions. The major contribution to the co-operative folding behavior arises from the solvent exposure of non-polar residues located in regions complementary to those that have undergone unfolding. This entropically uncompensated and energetically unfavorable solvent exposure characterizes all partially folded states but not the unfolded state, thus minimizing the population of partially folded intermediates throughout the folding/unfolding transition.     

Insights into conformation and dynamics of protein GB1 during folding and unfolding by NMR

Understanding protein stability requires characterization of structural determinants of the folded and unfolded states. Many proteins are capable of populating partially folded states under specific solution conditions. Occasionally, coexistence of the folded and an unfolded state under non- or mildly denaturing conditions can be observed by NMR, allowing us to structurally probe these states under identical conditions. Here we report on a destabilized mutant of the B1 domain of protein G (GB1) whose equilibrium unfolding was systematically investigated. Backbone amide residual dipolar couplings (RDCs), the tryptophan Nepsilon-H resonance and the amide nitrogen transverse relaxation rates (R2s) for varying pH values and different temperatures were measured. The backbone amide RDCs indicate that prior to complete unfolding, two melting hot spots are formed at the turn around T11, L12 and K13 and the N terminus of the helix at A24 and T25. The RDCs for the low pH, thermally unfolded state of GB1 are very small and do not indicate the presence of any native-like structure. Amide nitrogen transverse relaxation rates for GB1 in the folded state at different temperatures exhibit large contributions from exchange processes and the associated dynamics display considerable heterogeneity. Our data provide clear evidence for intermediate conformations and multi-state equilibrium un/folding for this GB1 variant.     

Molecular dynamics simulations of synthetic peptide folding

Because the time scale of protein folding is much greater than that of the widely used simulations of native structures, a detailed report of molecular dynamics simulations of folding has not been available. In this study, we Included the average solvent effect in the potential functions to simplify the calculation of the solvent effect and carried out long molecular dynamics simulations of the alanine-based synthetic peptides at 274 K. From either an extended or a randomly generated conformation, the simulations approached a helix-coil equilibrium in about 3 ns. The multiple minima problem did not prevent helix folding. The calculated helical ratio of Ac-AAQAAAAQAAAAQAAY-NH₂ was 47%, in good agreement with the circular dichroism measurement (about 50%). A helical segment with frayed ends was the most stable conformation, but the hydrophobic interaction favored the compact, distorted helix-turn-helix conformations. The transition between the two types of conformations occurred in a much larger time scale than helix propagation. The transient hydrogen bonds between the glutamine side chain and the backbone carbonyl group could reduce the free energy barrier of helix folding and unfolding. The substitution of a single alanine residue in the middle of the peptide with valine or glycine decreased the average helical ratio significantly, in agreement with experimental observations. © 1996 Wiley-Liss, Inc.     

Infrared spectroscopic discrimination between the loop and alpha-helices and determination of the loop diffusion kinetics by temperature-jump time-resolved infrared spectroscopy for cytochrome c

The infrared (IR) absorption of the amide I band for the loop structure may overlap with that of the alpha-helices, which can lead to the misassignment of the protein secondary structures. A resolution-enhanced Fourier transform infrared (FTIR) spectroscopic method and temperature-jump (T-jump) time-resolved IR absorbance difference spectra were used to identify one specific loop absorption from the helical IR absorption bands of horse heart cytochrome c in D2O at a pD around 7.0. This small loop consists of residues 70-85 with Met-80 binding to the heme Fe(III). The FTIR spectra in amide I' region indicate that the loop and the helical absorption bands overlap at 1653 cm(-1) at room temperature. Thermal titration of the amide I' intensity at 1653 cm(-1) reveals that a transition in loop structural change occurs at lower temperature (Tm=45 degrees C), well before the global unfolding of the secondary structure (Tm approximately 82 degrees C). This loop structural change is assigned as being triggered by the Met-80 deligation from the heme Fe(III). T-jump time-resolved IR absorbance difference spectra reveal that a T-jump from 25 degrees C to 35 degrees C breaks the Fe-S bond between the Met-80 and the iron reversibly, which leads to a loop (1653 cm(-1), overlap with the helical absorption) to random coil (1645 cm(-1)) transition. The observed unfolding rate constant interpreted as the intrachain diffusion rate for this 16 residue loop was approximately 3.6x10(6) s(-1).