Proline scanning mutagenesis reveals non-native fold in the molten globule state of equine beta-lactoglobulin

The secondary structure in the molten globule state (an equilibrium analogue of a burst-phase folding intermediate) of equine beta-lactoglobulin was investigated by changes in the circular dichroic spectrum induced by a series of site-directed proline substitutions. The results challenge the structural picture obtained from previous hydrogen/deuterium exchange experiments. A stable non-native alpha-helix was found to exist in the region corresponding to the eighth strand (H strand) in the native structure, where the backbone amide protons are the most strongly protected from exchange. Therefore, the backbone topology in the folding core is significantly different from that in the native structure. This indicates that the burst-phase folding intermediate of beta-lactoglobulin is a trapped species because of misfolded backbone topology.     

Early formation of a beta hairpin during folding of staphylococcal nuclease H124L as detected by pulsed hydrogen exchange

Pulsed hydrogen exchange methods were used to follow the formation of structure during the refolding of acid-denatured staphylococcal nuclease containing a stabilizing Leu substitution at position 124 (H124L SNase). The protection of more than 60 backbone amide protons in uniformly (15)N-labeled H124L SNase was monitored as a function of refolding time by heteronuclear two-dimensional NMR spectroscopy. As found in previous studies of staphylococcal nuclease, partial protection was observed for a subset of amide protons even at the earliest folding time point (10 msec). Protection indicative of marginally stable hydrogen-bonded structure in an early folding intermediate was observed at over 30 amide positions located primarily in the beta-barrel and to a lesser degree in the alpha-helical domain of H124L SNase. To further characterize the folding intermediate, protection factors for individual amide sites were measured by varying the pH of the labeling pulse at a fixed refolding time of 16 msec. Protection factors >5.0 were observed only for amide positions in a beta-hairpin formed by strands 2 and 3 of the beta-barrel domain and a single site near the C-terminus. The results indicate that formation of stable hydrogen-bonded structure in a core region of the beta-sheet is among the earliest structural events in the folding of SNase and may serve as a nucleation site for further structure formation.     

Investigation of the four cooperative unfolding units existing in the MICAL-1 CH domain

The solution structure of human MICAL-1 calpolnin homology (CH) domain is composed of six alpha helices and one 3(10) helix. To study the unfolding of this domain, we carry out native-state hydrogen exchange, intrinsic fluorescence and far-UV circular dichroism experiments. The free energy of unfolding, DeltaG(H2O), is calculated to be 7.11+/-0.58 kcal mol(-1) from GuHCl denaturation at pH 6.5. Four cooperative unfolding units are found using native-state hydrogen exchange experiment. Forty-seven slow-exchange residues can be studied by native-state hydrogen exchange experiments. From the concentration dependence of exchange rates, free energy of amide hydrogen with solvent, DeltaG(HX) and m-value (sensitivity of exposure to denaturant) are obtained, which reveal four cooperative unfolding units. The slowest exchanging protons are distributed throughout the whole hydrophobic core of the protein, which might be the folding core. These results will help us understand the structure of MICAL-1 CH domain more deeply.     

Folding of an all-helical Greek-key protein monitored by quenched-flow hydrogen–deuterium exchange and NMR spectroscopy

To advance our understanding of the protein folding process, we use stopped-flow far-ultraviolet (far-UV) circular dichroism and quenched-flow hydrogen–deuterium exchange coupled with nuclear magnetic resonance (NMR) spectroscopy to monitor the formation of hydrogen-bonded secondary structure in the C-terminal domain of the Fas-associated death domain (Fadd-DD). The death domain superfamily fold consists of six α-helices arranged in a Greek-key topology, which is shared by the all-β-sheet immunoglobulin and mixed α/β-plait superfamilies. Fadd-DD is selected as our model death domain protein system because the structure of this protein has been solved by NMR spectroscopy, and both thermodynamic and kinetic analysis indicate it to be a stable, monomeric protein with a rapidly formed hydrophobic core. Stopped-flow far-UV circular dichroism spectroscopy revealed that the folding process was monophasic and the rate is 23.4 s⁻¹. Twenty-two amide hydrogens in the backbone of the helices and two in the backbone of the loops were monitored, and the folding of all six helices was determined to be monophasic with rate constants between 19 and 22 s⁻¹. These results indicate that the formation of secondary structure is largely cooperative and concomitant with the hydrophobic collapse. This study also provides unprecedented insight into the formation of secondary structure within the highly populated Greek-key fold more generally. Additional insights are gained by calculating the exchange rates of 23 residues from equilibrium hydrogen–deuterium exchange experiments. The majority of protected amide protons are found on helices 2, 4, and 5, which make up core structural elements of the Greek-key topology.     

Peptide models of folding initiation sites of bovine beta-lactoglobulin: identification of nativelike hydrophobic interactions involving G and H strands

In an attempt to characterize the early folding events in bovine beta-lactoglobulin (BLG), a set of peptides, covering the flexible N-terminal region and the stable C-terminus beta-core, was synthesized and analyzed by circular dichroism and by nuclear magnetic resonance in water, trifluoroethanol (TFE), and sodium dodecyl sulfate (SDS) below and above the critical micellar concentration. The role of local and long-range hydrophobic interactions in guiding the folding has been investigated. For the peptide fragment covering the more flexible N-terminal region of BLG (beta-strands A, B), where both theoretical predictions and kinetic refolding experiments suggested the formation of non-native alpha-helix, no native long-range contacts were identified, and a helical secondary structure was stabilized only in the presence of 25 mM SDS. At variance, in 50% (v/v) TFE, native, long-range hydrophobic interactions were observed in the peptide covering the core region comprising G and H beta-strands. The side chains involved in these interactions form a nativelike hydrophobic cluster, thus suggesting that the GH region may act as the folding initiation site for BLG. This result is reinforced by the identification, in the urea denaturated BLG, of residual structure located at the level of the GH interface, as evidenced by NMR analysis. These results, in excellent agreement with kinetic, thermodynamic, and cold denaturation folding data, once more underline the utmost importance of the GH region for the stability and folding of BLG. Severe aggregation effects prevented the structural analysis of the peptide covering the EFGH region, indicating that this larger segment does not represent an independent folding domain and that the terminal alpha-helix is necessary for stabilizing the BLG folding core.     

Stable intermediate states and high energy barriers in the unfolding of GFP

We present a study of the denaturation of a truncated, cycle3 variant of green fluorescent protein (GFP). Chemical denaturation is used to unfold the protein, with changes in structure being monitored by the green fluorescence, tyrosine fluorescence and far-UV circular dichroism. The results show that the denaturation behaviour of GFP is complex compared to many small proteins: equilibrium is established only very slowly, over the time course of weeks, suggesting that there are high folding/unfolding energy barriers. Unfolding kinetics confirm that the rates of unfolding at low concentrations of denaturant are very low, consistent with the slow establishment of the equilibrium. In addition, we find that GFP significantly populates an intermediate state under equilibrium conditions, which is compact and stable with respect to the unfolded state (m(IU)=4.6 kcal mol(-1) M(-1) and Delta G(IU)=12.5 kcal mol(-1)). The global and local stability of GFP was probed further by measuring the hydrogen/deuterium (H/D) NMR exchange rates of more than 157 assigned amide protons. Analysis at two different values of pH showed that amide protons within the beta-barrel structure exchange at the EX2 limit, consequently, free energies of exchange could be calculated and compared to those obtained from the denaturation-curve studies providing further support for the three-state model and the existence of a stable intermediate state. Analysis reveals that amide protons in beta-strands 7, 8, 9 and 10 have, on average, higher exchange rates than others in the beta-barrel, suggesting that there is greater flexibility in this region of the protein. Forty or so amide protons were found which do not undergo significant exchange even after several months and these are clustered into a core region encompassing most of the beta-strands, at least at one end of the barrel structure. It is likely that these residues play an important role in stabilizing the structure of the intermediate state. The intermediate state observed in the chemical denaturation studies described here, is similar to that observed at pH 4 in other studies.     

Amide proton exchange used to monitor the formation of a stable alpha-helix by residues 3 to 13 during folding of ribonuclease S

We make use of the known exchange rates of individual amide proton in the S-peptide moiety of ribonuclease S (RNAase S) to determine when during folding the alpha-helix formed by residues 3 to 13 becomes stable. The method is based on pulse-labeling with [3H]H2O during the folding followed by an exchange-out step after folding that removes 3H from all amide protons of the S-peptide except from residues 7 to 14, after which S-peptide is separated rapidly from S-protein by high performance liquid chromatography. The slow-folding species of unfolded RNAase S are studied. Folding takes place in strongly native conditions (pH 6.0, 10 degrees C). The seven H-bonded amide protons of the 3-13 helix become stable to exchange at a late stage in folding at the same time as the tertiary structure of RNAase S is formed, as monitored by tyrosine absorbance. At this stage in folding, the isomerization reaction that creates the major slow-folding species has not yet been reversed. Our result for the 3-13 helix is consistent with the finding of Labhardt (1984), who has studied the kinetics of folding of RNAase S at 32 degrees C by fast circular dichroism. He finds the dichroic change expected for formation of the 3-13 helix occurring when the tertiary structure is formed. Protected amide protons are found in the S-protein moiety earlier in folding. Formation or stabilization of this folding intermediate depends upon S-peptide: the intermediate is not observed when S-protein folds alone, and folding of S-protein is twice as slow in the absence of S-peptide. Although S-peptide combines with S-protein early in folding and is needed to stabilize an S-protein folding intermediate, the S-peptide helix does not itself become stable until the tertiary structure of RNAase S is formed.     

Folding and stability of sweet protein single-chain monellin. An insight to protein engineering

Engineered single-chain monellin (SCM) proteins were constructed by recombinant technology without disrupting the topology and sweet activity of native protein. Data from 8-anilinonaphthalene-1-sulfonic acid fluorescence, size-exclusion chromatography, and heteronuclear NMR strongly suggest the presence of a folding intermediate at 1.5 m GdnHCl for SCM protein. The structural feature of the folding intermediate from NMR data reveals that the secondary structures became mostly unstable, and protein experiences a dynamic equilibrium between native and unfolded state. All backbone amide protons exchange within 10 min, which imply that no stable hydrogen bonds exist in the secondary structural regions in the folding intermediate. From equilibrium unfolding and mutagenesis studies, the unfolding transition midpoints of mutant proteins gradually shifted toward lower denaturant concentration, indicating stability reductions of mutant proteins. Our results suggest that stability and folding pathways of SCM proteins could be regulated by a combined study of spectroscopy and mutagenesis, and these studies will provide useful information for understanding the folding kinetics of novel engineered proteins.     

The folding pathway of T4 lysozyme: an on-pathway hidden folding intermediate

T4 lysozyme has two easily distinguishable but energetically coupled domains: the N and C-terminal domains. In earlier studies, an amide hydrogen/deuterium exchange pulse-labeling experiment detected a stable submillisecond intermediate that accumulates before the rate-limiting transition state. It involves the formation of structures in both the N and C-terminal regions. However, a native-state hydrogen exchange experiment subsequently detected an equilibrium intermediate that only involves the formation of the C-terminal domain. Here, using stopped-flow circular dichroism and fluorescence, amide hydrogen exchange-folding competition, and protein engineering methods, we re-examined the folding pathway of T4-lysozyme. We found no evidence for the existence of a stable folding intermediate before the rate-limiting transition state at neutral pH. In addition, using native-state hydrogen exchange-directed protein engineering, we created a mimic of the equilibrium intermediate. We found that the intermediate mimic folds with the same rate as the wild-type protein, suggesting that the equilibrium intermediate is an on-pathway intermediate that exists after the rate-limiting transition state.     

GroEL accelerates the refolding of hen lysozyme without changing its folding mechanism.

The chaperonin GroEL binds folding intermediates of four-disulfidehen lysozyme transiently within its central cavity. Using stopped flow fluorescence we show that GroEL binds early intermediates in folding and accelerates the slow kinetic phase that reflects the reversal of non-native interactions involving tryptophan residues and the formation of the native state. Pulsed hydrogen exchange monitored by electrospray ionization mass spectrometry demonstrates that GroEL does not alter the folding mechanism, nor are protected species unfolded by the chaperonin. The data suggest a mechanism for GroEL-assisted folding in which the reorganization of non-native tertiary interactions is facilitated but domain folding is unperturbed.     

The apomyoglobin folding pathway revisited: structural heterogeneity in the kinetic burst phase intermediate

Extensive analysis of accurate quench-flow hydrogen exchange results indicates that the burst phase kinetic intermediate in the folding of apomyoglobin (apoMb) from urea is structurally heterogeneous. The structural variability is associated with the partial folding of the E helix during the burst phase (<6.4ms) of the folding process. Analysis of the effects of exchange-out of amide proton labels during the labeling pulse ( approximately pH 10) of the quench-flow process indicates that three of the amide protons in the E helix are in fact largely protected in the burst phase of folding, while the remainder of the E helix has a substantial complement of amide protons that show biphasic kinetics, i.e. are protected partly during the burst phase and partly during the slow phase of folding. The locations of these amide protons can be used to map the sites of structural heterogeneity in the kinetic molten globule. These sites include, besides the E helix, the ends of the A and B helices and part of the C helix. Our results give significant support to the hypothesis that the kinetic molten globule intermediate of apoMb is native-like.     

Study of early events in the protein folding of villin headpiece using molecular dynamics simulation

Protein folding is scientifically and computationally challenging problem. The early phases of protein folding are interesting due to various events like nascent secondary structure formation, hydrophobic collapse leading to formation of non-native or meta-stable conformations. These events occur within a very short time span of 100ns as compared to total folding time of few microseconds. It is highly difficult to observe these events experimentally due to very short lifetime. Molecular dynamics simulation technique can efficiently probe the detailed atomic level understanding about these events. In the present paper, all atom molecular dynamics simulation trajectory of nearly 200ns was carried out for fully solvated villin headpiece with PME treatment using AMBER 7 package. Initial hydrophobic collapse along with secondary structure formation resulted into formation of partially stable non-native conformations. The formation of secondary structural elements and hydrophobic collapse takes place simultaneously in the folding process.     

Strange kinetic phase in the extremely early folding process of beta-lactoglobulin

A continuous-wave probed laser-induced temperature jump system was constructed and applied to monitor the changes in tryptophan fluorescence of the beta-lactoglobulin during its folding; the kinetic phases were traced from 300 ns to 10 ms after a temperature jump. Notably, an early phase with typical squeezed-exponential characteristics, [exp[-(kt)(beta)], beta>1.0], was observed around several tens of microseconds after the temperature jump, which is actually the earliest phase ever observed for beta-lactoglobulin. This process can be explained by conformational shift occurring within the unfolded ensemble (U-->U'), which is followed by the non-native intermediate (I) formation of this protein.     

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.     

Dissection of the pathway of molecular recognition by calmodulin

Amide hydrogen exchange has been used to examine the structural dynamics and energetics of the interaction of a peptide corresponding to the calmodulin-binding domain of smooth muscle myosin light chain kinase (smMLCKp) with calcium-saturated calmodulin. Heteronuclear NMR (15)N-(1)H correlation spectroscopy was used to quantify amide proton exchange rates of the uniformly (15)N-labeled domain bound to calmodulin. A key feature of a proposed model for molecular recognition by calmodulin [Ehrhardt et al. (1995) Biochemistry 34, 2731-2738] is tested by examination of the dependence of amide hydrogen exchange on applied hydrostatic pressure. Hydrogen exchange rates and corresponding protection factors (1/K(op)) for individual amide protons of the bound smMLCKp domain span 5 orders of magnitude at ambient pressure. Individual protection factors decrease significantly in a linear fashion with increasing hydrostatic pressure. A common pressure dependence is revealed by a constant large negative volume change across the residues comprising the core of the bound helical domain. The pattern of protection factors and their response to hydrostatic pressure is consistent with a structural reorganization that results in the concerted disruption of ion pairs between calmodulin and the bound domain. These observations reinforce a model for the molecular recognition pathway where formation of the initial encounter complex is followed by helix-coil transitions in the bound state and subsequent concerted formation of the extensive ion pair network defining the intermolecular contact surface between CaM and the target domain in the final, compact complex structure.     

Apparent local stability of the secondary structure of Azotobacter vinelandii holoflavodoxin II as probed by hydrogen exchange: implications for redox potential regulation and flavodoxin folding.

As a first step to determine the folding pathway of a protein with an alpha/beta doubly wound topology, the 1H, 13C, and 15N backbone chemical shifts of Azotobacter vinelandii holoflavodoxin II (179 residues) have been determined using multidimensional NMR spectroscopy. Its secondary structure is shown to contain a five-stranded parallel beta-sheet (beta2-beta1-beta3-beta4-beta5) and five alpha-helices. Exchange rates for the individual amide protons of holoflavodoxin were determined using the hydrogen exchange method. The amide protons of 65 residues distributed throughout the structure of holoflavodoxin exchange slowly at pH* 6.2 [kex < 10(-5) s(-1)] and can be used as probes in future folding studies. Measured exchange rates relate to apparent local free energies for transient opening. We propose that the amide protons in the core of holoflavodoxin only exchange by global unfolding of the apo state of the protein. The results obtained are discussed with respect to their implications for flavodoxin folding and for modulation of the flavin redox potential by the apoprotein. We do not find any evidence that A. vinelandii holoflavodoxin II is divided into two subdomains based on its amide proton exchange rates, as opposed to what is found for the structurally but not sequentially homologous alpha/beta doubly wound protein Che Y.     

Secondary structures and structural fluctuation in a dimeric protein, Streptomyces subtilisin inhibitor.

Based on the nuclear magnetic resonance assignments of a dimeric protein, Streptomyces subtilisin inhibitor (SSI), microscopic details of secondary structures in solution have been elucidated. The chemical shift index of C(alpha) signals, together with information on the hydrogen exchange rates of the backbone amide protons, were used to identify secondary structures. The locations of these secondary structures were found to be different in some critical points from those determined earlier by X-ray crystallography of the crystal. Notably, the beta3 strand is completely missing and the alpha2 helix is extended toward the C-terminus. Furthermore, hydrogen exchange experiments of individual peptide NH protons under strongly folding conditions revealed mechanisms of global and local structural fluctuation within the dimeric structure. It has been suggested that the global fluctuation of the monomeric unit occurs without affecting the accompanying monomer, in contrast to the equilibrium thermal unfolding, which is cooperative. Higher protection against hydrogen exchange for residues in part of the beta4 strand implies that this region might serve as a folding core.     

Partially unfolded forms and non-two-state folding of a beta-sandwich: FHA domain from Arabidopsis receptor kinase-associated protein phosphatase

FHA domains adopt a beta-sandwich fold with 11 strands. The first evidence of partially unfolded forms of a beta-sandwich is derived from native-state hydrogen exchange (NHX) of the forkhead-associated (FHA) domain from kinase-associated protein phosphatase from Arabidopsis. The folding kinetics of this FHA domain indicate that EX2 behavior prevails at pH 6.3. In the chevron plot, rollover in the folding arm and bends in the unfolding arm suggest folding intermediates. NHX of this FHA domain suggests a core of six most stable beta-strands and two loops, characterized by rare global unfolding events. Flanking this stable core are beta-strands and recognition loops with less stability, termed subglobal motifs. These suggest partially unfolded forms (near-native intermediates) with two levels of stability. The spatial separation of the subglobal motifs on the flanks suggests possible parallelism in their folding as additional beta-strands align with the stable core of six strands. Intermediates may contribute to differences in stabilities and m-values suggested by NHX or kinetics relative to chemical denaturation. Residual structure in the unfolded regime is suggested by superprotection of beta-strand 6 and by GdmCl-dependence of adjustments in amide NMR spectra and residual optical signal. The global folding stability depends strongly on pH, with at least 3 kcal/mol more stability at pH 7.3 than at pH 6.3. This FHA domain is hypothesized to fold progressively with initial hydrophobic collapse of its stable six-stranded core followed by addition of less stable flanking beta-strands and ordering of recognition loops.     

Hydrogen exchange of the tetramerization domain of the human tumour suppressor p53 probed by denaturants and temperature.

We have analysed the hydrogen/deuterium exchange of the tetramerization domain of human tumour suppressor p53 under mild chemical denaturation conditions, and at different temperatures. Exchange behaviour has been measured for 16 amide protons in the chemical-denaturation studies and for seven protons in the temperature-denaturation studies. The exchange rates are in the range observed for other proteins with similar elements of secondary structure. The slowest-exchange core includes contributions from residues in the alpha helix and the beta sheet. However, only some of the slowest-exchanging protons correspond to residues involved in native interactions in the transient intermediate detected during the folding of this domain. The guanidinium-chloride denaturation curves of all residues seem to merge together, although they are well below the main isotherm of global unfolding. Thus, there is no evidence for several subglobal unfolding units. The activation parameters obtained from the temperature-denaturation experiments are similar to those obtained for monomeric proteins, and well below the global unfolding enthalpy obtained by circular dichroism measurements. Thus, the exchange studies at different denaturant concentrations and temperatures indicate that no particular folding intermediate is populated under those conditions.