Residual Structure of Unfolded Ubiquitin as Revealed by Hydrogen/Deuterium-Exchange 2D NMR

The characterization of residual structures persistent in unfolded proteins in concentrated denaturant solution is currently an important issue in studies of protein folding because the residual structure present, if any, in the unfolded state may form a folding initiation site and guide the subsequent folding reactions. Here, we studied the hydrogen/deuterium (H/D)-exchange behavior of unfolded human ubiquitin in 6 M guanidinium chloride. We employed a dimethylsulfoxide (DMSO)-quenched H/D-exchange NMR technique with the use of spin desalting columns, which allowed us to perform a quick medium exchange from 6 M guanidinium chloride to a quenching DMSO solution. Based on the backbone resonance assignment of ubiquitin in the DMSO solution, we successfully investigated the H/D-exchange kinetics of 60 identified peptide amide groups in the ubiquitin sequence. Although a majority of these amide groups were not protected, certain amide groups involved in a middle helix (residues 23–34) and an N-terminal β-hairpin (residues 2–16) were significantly protected with a protection factor of 2.1–4.2, indicating that there were residual structures in unfolded ubiquitin and that these amide groups were more than 52% hydrogen bonded in the residual structures. We show that the hydrogen-bonded residual structures in the α-helix and the β-hairpin are formed even in 6 M guanidinium chloride, suggesting that these residual structures may function as a folding initiation site to guide the subsequent folding reactions of ubiquitin.     

Protection of amide protons in folding intermediates of ribonuclease A measured by pH-pulse exchange curves

pH-pulse exchange curves have been measured for samples taken during the folding of ribonuclease A. The curve gives the number of protected amide protons remaining after a 10-s pulse of exchange at pHs from 6.0 to 9.5, at 10 degrees C. Amide proton exchange is base catalyzed, and the rate of exchange increases 3000-fold between pH 6.0 and pH 9.5. The pH at which exchange occurs depends on the degree of protection against exchange provided by structure. Pulse exchange curves have been measured for samples taken at three times during folding, and these are compared to the pulse exchange curves of N, the native protein, of U, the unfolded protein in 4 M guanidinium chloride, and of IN, the native-like intermediate obtained by the prefolding method of Schmid. The results are used to determine whether folding intermediates are present that can be distinguished from N and U and to measure the average degree of protection of the protected protons in folding intermediates. The amide (peptide NH) protons of unfolded ribonuclease A were prelabeled with 3H by a previous procedure that labels only the slow-folding species. Folding was initiated at pH 4.0, 10 degrees C, where amide proton exchange is slower than the folding of the slow-folding species. Samples were taken at 0-, 10-, and 20-s folding, and their pH-pulse exchange curves were measured.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hydrogen exchange in a large 29 kD protein and characterization of molten globule aggregation by NMR

The nature of denatured ensembles of the enzyme human carbonic anhydrase (HCA) has been extensively studied by various methods in the past. The protein constitutes an interesting model for folding studies that does not unfold by a simple two-state transition, instead a molten globule intermediate is highly populated at 1.5 M GuHCl. In this work, NMR and H/D exchange studies have been conducted on one of the isozymes, HCA I. The H/D exchange studies, which were enabled by the previously obtained resonance assignment of HCA I, have been used to identify unfolded forms that are accessible from the native state. In addition, the GuHCl-induced unfolded states of HCA I have also been characterized by NMR at GuHCl concentrations in the 0-5 M range. The most important findings in this work are as follows: (1) Amide protons located in the center of the beta-sheet require global unfolding events for efficient H/D exchange. (2) The molten globule and the native state give similar protection against H/D exchange for all of the observable amide protons (i.e., water seems not to efficiently penetrate the interior of the molten globule). (3) At high protein concentrations, the molten globule can form large aggregates, which are not detectable by solution-state NMR methods. (4) The unfolded state (U), present at GuHCl concentrations above 2 M, is composed of an ensemble of conformations having residual structures with different stabilities.     

Two-dimensional 1H NMR studies of cytochrome c: hydrogen exchange in the N-terminal helix

The hydrogen exchange behavior of the N-terminal helical segment in horse heart cytochrome c was studied in both the reduced and the oxidized forms by use of two-dimensional nuclear magnetic resonance methods. The amide protons of the first six residues are not H bonded and exchange rapidly with solvent protons. The most N-terminal H-bonded groups--the amide NH of Lys-7 to Phe-10--exhibit a sharp gradient in exchange rate indicative of dynamic fraying behavior, consistent with statistical-mechanical principles. This occurs identically in both reduced and oxidized cytochrome c. In the oxidized form, residues 11-14, which form the last helical turn, all exchange with a similar rate, about one million times slower than the rate characteristic of freely exposed peptide NH, even though some are on the aqueous face of the helix and others are fully buried. These and similar observations in several other proteins appear to document local cooperative unfolding reactions as determinants of protein H exchange reactions. The N-terminal segment of cytochrome c is insensitive to the heme redox state, as in the crystallographic model, except for residues closest to the heme (Cys-14 and Ala-15), which exchange about 15-fold more slowly in the reduced form. The cytochrome c H exchange results can be further considered in terms of the conformation of the native and the transiently unfolded forms and their free energy relationships in both the reduced and the oxidized states.     

The use of spin desalting columns in DMSO‐quenched H/D‐exchange NMR experiments

Dimethylsulfoxide (DMSO)‐quenched hydrogen/deuterium (H/D)‐exchange is a powerful method to characterize the H/D‐exchange behaviors of proteins and protein assemblies, and it is potentially useful for investigating non‐protected fast‐exchanging amide protons in the unfolded state. However, the method has not been used for studies on fully unfolded proteins in a concentrated denaturant or protein solutions at high salt concentrations. In all of the current DMSO‐quenched H/D‐exchange studies of proteins so far reported, lyophilization was used to remove D₂O from the protein solution, and the lyophilized protein was dissolved in the DMSO solution to quench the H/D exchange reactions and to measure the amide proton signals by two‐dimensional nuclear magnetic resonance (2D NMR) spectra. The denaturants or salts remaining after lyophilization thus prevent the measurement of good NMR spectra. In this article, we report that the use of spin desalting columns is a very effective alternative to lyophilization for the medium exchange from the D₂O buffer to the DMSO solution. We show that the medium exchange by a spin desalting column takes only about 10 min in contrast to an overnight length of time required for lyophilization, and that the use of spin desalting columns has made it possible to monitor the H/D‐exchange behavior of a fully unfolded protein in a concentrated denaturant. We report the results of unfolded ubiquitin in 6.0M guanidinium chloride.     

Strategy for trapping intermediates in the folding of ribonuclease and for using 1H-nmr to determine their structures

The major unfolded form of ribonuclease A is known to show well-populated structural intermediates transiently during folding at 0°–10°C. We describe here how the exchange reaction between D₂O and peptide NH protons can be used to trap folding intermediates. The protons protected from exchange during folding can be characterized by ¹H-nmr after folding is complete. The feasibility of using ¹H-nmr to resolve a set of protected peptide protons is demonstrated by using a specially prepared sample of ribonuclease S in D₂O in which only the peptide protons of residues 7–14 are in the ¹H-form. All eight of these protected peptide protons are H-bonded. Resonance assignments made on isolated peptides containing these residues have been used to identify the protected protons. Other sets of protected protons trapped in the ¹H-form can also be isolated by differential exchange, using either ribonuclease A or S. Earlier model compound studies have indicated that H-bonded folding intermediates should be unstable in water unless stabilized by additional interactions. Nevertheless, peptides derived from ribonuclease A that contain residues 3–13 do show partial helix formation in water at low temperatures. We discuss the possibility that specific interactions between side chains can stabilize short α-helixes by nucleating the helix, and that specific interactions may also define the helix boundaries at early stages in folding.     

Characterization of the molten globule conformation of V26A ubiquitin by far-UV circular dichroic spectroscopy and amide hydrogen/deuterium exchange

The molten globular conformation of V26A ubiquitin (valine to alanine mutation at residue 26) was studied by nuclear magnetic resonance spectroscopy in conjunction with amide hydrogen/deuterium exchange. Most of the amide protons that are involved in the native secondary structures were observed to be protected in the molten globule state with the protection factors from 1.2 to 6.7. These protection factors are about 2 to 6 orders of magnitude smaller than those of the native state. These observations indicate that V26A molten globule has native-like backbone structure with marginal stability. The comparison of amide protection factors of V26A ubiquitin molten globule state with those of initial collapsed state of the wild type ubiquitin suggests that V26A ubiquitin molten globule state is located close to unfolded state in the folding reaction coordinate. It is considered that V26A ubiquitin molten globule is useful model to study early events in protein folding reaction.     

Stein and Moore Award address. The molten globule intermediate of apomyoglobin and the process of protein folding.

The molten globule model for the beginning of the folding process, which originated with Kuwajima's studies of alpha-lactalbumin (Kuwajima, K., 1989, Proteins Struct. Funct. Genet. 6, 87-103, and references therein), states that, for those proteins that exhibit equilibrium molten globule intermediates, the molten globule is a major kinetic intermediate near the start of the folding pathway. Pulsed hydrogen-deuterium exchange measurements confirm this model for apomyoglobin (Jennings, P.A. & Wright, P.E., in prep.). The energetics of the acid-induced unfolding transition, which have been determined by fitting a minimal three-state model (N<-->I<-->U; N = native, I = molten globule intermediate, U = unfolded) show that I is more stable than U at neutral pH (Barrick, D. & Baldwin, R.L., 1993, Biochemistry 32, in press), which provides an explanation for why I is formed from U at the start of folding. Hydrogen exchange rates measured by two-dimensional NMR for individual peptide NH protons, taken together with the CD spectrum of I, indicate that moderately stable helices are present in I at the locations of the A, G, and H helices of native myoglobin (Hughson, F.M., Wright, P.E., & Baldwin, R.L., 1990, Science 249, 1544-1548). Directed mutagnesis experiments indicate that the interactions between the A, G, and H helices in I are loose (Hughson, F.M., Barrick, D., & Baldwin, R.L., 1991, Biochemistry 30, 4113-4118), which can explain why I is formed rapidly from U at the start of folding.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Direct evidence for a two-state protein unfolding transition from hydrogen-deuterium exchange, mass spectrometry, and NMR.

We use mass spectrometry in conjunction with hydrogen-deuterium exchange and NMR to characterize the conformational dynamics of the 62-residue IgG binding domain of protein L under conditions in which the native state is marginally stable. Mass spectra of protein L after short incubations in D2O reveal the presence of two distinct populations containing different numbers of protected protons. NMR experiments indicate that protons in the hydrophobic core are protected in one population, whereas all protons are exchanged for deuterons in the other. As the exchange period is increased, molecules are transferred from the former population to the latter. The absence of molecules with a subset of the core protons protected suggests that exchange occurs in part via a highly concerted transition to an excited state in which all protons exchange rapidly with deuterons. A steady increase in the molecular weight of the population with protected protons, and variation in the exchange rates of the individual protected protons indicates the presence of an additional exchange mechanism. A simple model in which exchange results from rapid (> 10(5)/s) local fluctuations around the native state superimposed upon transitions to an unfolded excited state at approximately 0.06/s is supported by qualitative agreement between the observed mass spectra and the mass spectra simulated according to the model using NMR-derived estimates of the proton exchange rates.     

Consequences of stabilizing the natively disordered f helix for the folding pathway of apomyoglobin

The F helix region of sperm whale apomyoglobin is disordered, undergoing conformational fluctuations between a folded helical conformation and one or more locally unfolded states. To examine the effects of F helix stabilization on the folding pathway of apomyoglobin, we have introduced mutations to augment intrinsic helical structure in the F helix of the kinetic folding intermediate and to increase its propensity to fold early in the pathway, using predictions based on plots of the average area buried upon folding (AABUF) derived from the primary sequence. Two mutant proteins were prepared: a double mutant, P88K/S92K (F2), and a quadruple mutant, P88K/A90L/S92K/A94L (F4). Whereas the AABUF for F2 predicts that the F helix will not fold early in the pathway, the F helix in F4 shows a significantly increased AABUF and is therefore predicted to fold early. Protection of amide protons by formation of hydrogen-bonded helical structure during the early folding events has been analyzed by pH-pulse labeling. Consistent with the AABUF prediction, many of the F helix residues for F4 are significantly protected in the kinetic intermediate but are not protected in the F2 mutant. F4 folds via a kinetically trapped burst-phase intermediate that contains stabilized secondary structure in the A, B, F, G, and H helix regions. Rapid folding of the F helix stabilizes the central core of the misfolded intermediate and inhibits translocation of the H helix back to its native position, thereby decreasing the overall folding rate.     

Equilibrium amide hydrogen exchange and protein folding kinetics

The classical Linderstrøm-Lang hydrogen exchange (HX) model is extended to describe the relationship between the HX behaviors (EX1 and EX2) and protein folding kinetics for the amide protons that can only exchange by global unfolding in a three-state system including native (N), intermediate (I), and unfolded (U) states. For these slowly exchanging amide protons, it is shown that the existence of an intermediate (I) has no effect on the HX behavior in an off-pathway three-state system (IUN). On the other hand, in an on-pathway three-state system (UIN), the existence of a stable folding intermediate has profound effect on the HX behavior. It is shown that fast refolding from the unfolded state to the stable intermediate state alone does not guarantee EX2 behavior. The rate of refolding from the intermediate state to the native state also plays a crucial role in determining whether EX1 or EX2 behavior should occur. This is mainly due to the fact that only amide protons in the native state are observed in the hydrogen exchange experiment. These new concepts suggest that caution needs to be taken if one tries to derive the kinetic events of protein folding from equilibrium hydrogen exchange experiments.     

Investigating the refolding pathway of human acidic fibroblast growth factor (hFGF-1) from the residual structure(s) obtained by denatured-state hydrogen/deuterium exchange

Human fibroblast growth factor 1 (hFGF-1) consists of 12 anti-parallel β-strands arranged into a β-trefoil architecture. We directly measured hydrogen/deuterium exchange rates on the urea-denatured hFGF-1 to obtain the information with regard to the persistent residual interaction(s) in the unfolded hFGF-1. Thirty-eight residues whose heteronuclear single quantum coherence cross-peaks can be observed after exchange show higher protections than those predicted for the same residues in a random coil conformation, suggesting the existence of residual structure(s). The urea-denaturation of hFGF-1 tested by both circular dichroism and fluorescence spectroscopy indicated that the unfolding process is a cooperative two-state process and that the residual structures observed did not originate from the existence of a partially structured intermediate. The coincident disappearance of the native heteronuclear single quantum coherence cross-peaks during the urea-denaturation process suggests that the residual structures observed contain no nativelike interactions. The protected residues (fold ons) in the urea-denatured state are mostly those that exchange slowly in the native state H/D exchange. The distribution of these fold ons in the native structure of hFGF-1 suggests that the refolding starts by collisions between the residual structures (microdomains) between the β-strands VI and VII, and between the β-strands II and III, which appear to be two independent refolding coordinates during the refolding process.     

Protein folding kinetics by combined use of rapid mixing techniques and NMR observation of individual amide protons

A method to be used for experimental studies of protein folding introduced by Schmid and Baldwin (J. Mol. Biol. 135: 199-215, 1979), which is based on the competition between amide hydrogen exchange and protein refolding, was extended by using rapid mixing techniques and 1H NMR to provide site-resolved kinetic information on the early phases of protein structure acquisition. In this method, a protonated solution of the unfolded protein is rapidly mixed with a deuterated buffer solution at conditions assuring protein refolding in the mixture. This simultaneously initiates the exchange of unprotected amide protons with solvent deuterium and the refolding of protein segments which can protect amide groups from further exchange. After variable reaction times the amide proton exchange is quenched while folding to the native form continues to completion. By using 1H NMR, the extent of exchange at individual amide sites is then measured in the refolded protein. Competition experiments at variable reaction times or variable pH indicate the time at which each amide group is protected in the refolding process. This technique was applied to the basic pancreatic trypsin inhibitor, for which sequence-specific assignments of the amide proton NMR lines had previously been obtained. For eight individual amide protons located in the beta-sheet and the C-terminal alpha-helix of this protein, apparent refolding rates in the range from 15 s-1 to 60 s-1 were observed. These rates are on the time scale of the fast folding phase observed with optical probes.     

GuHCl and NaCl-dependent hydrogen exchange in MerP reveals a well-defined core with an unusual exchange pattern

We have analysed hydrogen exchange at amide groups to characterise the energy landscape of the 72 amino acid residue protein MerP. From the guanidine hydrochloride (GuHCl) dependence of exchange in the pre-transitional region we have determined free energy values of exchange (DeltaG(HX)) and corresponding m-values for individual amide protons. Detailed analysis of the exchange patterns indicates that for one set of amide protons there is a weak dependence on denaturant, indicating that the exchange is dominated by local fluctuations. For another set of amide protons a linear, but much stronger, denaturant dependence is observed. Notably, the plots of free energy of exchange versus [GuHCl] for 16 amide protons show pronounced upward curvature, and a close inspection of the structure shows that these residues form a well-defined core in the protein. The hydrogen exchange that was measured at various concentrations of NaCl shows an apparent selective stabilisation of this core. Detailed analysis of this exchange pattern indicates that it may originate from selective destabilisation of the unfolded state by guanidinium ions and/or selective stabilisation of the core in the native state by chloride ions.     

Structural and kinetic characterization of early folding events in beta-lactoglobulin

We have defined the structural and dynamic properties of an early folding intermediate of beta-lactoglobulin known to contain non-native alpha-helical structure. The folding of beta-lactoglobulin was monitored over the 100 micros--10 s time range using ultrarapid mixing techniques in conjunction with fluorescence detection and hydrogen exchange labeling probed by heteronuclear NMR. An initial increase in Trp fluorescence with a time constant of 140 micros is attributed to formation of a partially helical compact state. Within 2 ms of refolding, well protected amide protons indicative of stable hydrogen bonded structure were found only in a domain comprising beta-strands F, G and H, and the main alpha-helix, which was thus identified as the folding core of beta-lactoglobulin. At the same time, weak protection (up to approximately 10-fold) of amide protons in a segment spanning residues 12--21 is consistent with formation of marginally stable non-native alpha-helices near the N-terminus. Our results indicate that efficient folding, despite some local non-native structural preferences, is insured by the rapid formation of a native-like alpha/beta core domain.     

Exchange behavior of the H-bonded amide protons in the 3 to 13 helix of ribonuclease S

The preceding article shows that there are eight highly protected amide protons in the S-peptide moiety of RNAase S at pH 5, 0 degrees C. The residues with protected NH protons are 7 to 13, whose amide protons are H-bonded in the 3 to 13 alpha-helix, and Asp 14, whose NH proton is H-bonded to the CO group of Val47. We describe here the exchange behavior of these eight protected protons as a function of pH. Exchange rates of the individual NH protons are measured by 1H nuclear magnetic resonance in D2O. A procedure is used for specifically labeling with 1H only these eight NH protons. The resonance assignments of the eight protons are made chiefly by partial exchange, through correlating the resonance intensities in spectra taken when the peptide is bound and when it is dissociated from S-protein in 3.5 M-urea-d4, in D2O, pH 2.3, -4 degrees C. The two remaining assignments are made and some other assignments are checked by measurements of the nuclear Overhauser effect between adjacent NH protons of the alpha-helix. There is a transition in exchange behavior between pH 3, where the helix is weakly protected against exchange, and pH 5 where the helix is much more stable. At pH 3.1, 20 degrees C, exchange rates are uniform within the helix within a factor of two, after correction for different intrinsic exchange rates. The degree of protection within the helix is only 10 to 20-fold at this pH. At pH 5.1, 20 degrees C, the helix is more stable by two orders of magnitude and exchange occurs preferentially from the N-terminal end. At both pH values the NH proton of Asp 14, which is just outside the helix, is less protected by an order of magnitude than the adjacent NH protons inside the helix. Opening of the helix can be observed below pH 3.7 by changes in chemical shifts of the NH protons in the helix. At pH 2.4 the changes are 25% of those expected for complete opening. Helix opening is a fast reaction on the n.m.r. time scale (tau much less than 1 ms) unlike the generalized unfolding of RNAase S which is a slow reaction. Dissociation of S-peptide from S-protein in native RNAase S at pH 3.0 also is a slow reaction. Opening of the helix below pH 3.7 is a two-state reaction, as judged by comparing chemical shifts with exchange rates. The exchange rates at pH 3.1 are predicted correctly from the changes in chemical shift by assuming that helix opening is a two-state reaction. At pH values above 3.7, the nature of the helix opening reaction changes. These results indicate that at least one partially unfolded state of RNAase S is populated in the low pH unfolding transition.     

Methanol-induced conformations of myoglobin at pH 4.0

The equilibrium methanol-induced conformation changes of holomyoglobin (hMb) at pH 4.0 have been studied by circular dichroism, tryptophan fluorescence, and Soret band absorption and by electrospray ionization mass spectrometry (ESI-MS). Optical spectra show the following: (1) In 35-40% (v/v) methanol/water, the native-like secondary structure remains, the tertiary structure is lost, the heme protein interactions are decreased, and a folding intermediate is formed. (2) In 50% methanol, heme is lost from the protein, and there is a small decrease in helicity together with a loss of tertiary structure. (3) At >60% methanol, the helicity increases and the apoprotein goes into a helical denatured state. The conformations are also probed by the charge states produced in ESI-MS and by hydrogen/deuterium (H/D) exchange with mass measurement by ESI-MS. At 0-30% methanol, native hMb produces relatively low charge states (9(+)-13(+)) in ESI-MS and exchanges relatively few hydrogens. In 35-40% methanol, at which an intermediate is formed, there is a bimodal distribution of hMb ions with both low (9(+)-13(+)) and high (14(+)-23(+)) charge states and also a high charge state distribution (12(+)-26(+)) of apomyoglobin (aMb) ions. Low and high charge states of hMb and a high charge state of aMb all show the same H/D exchange rate, indicating that an unfolded hMb intermediate interconverts between folded hMb and unfolded aMb. The charge state distribution for the unfolded hMb intermediate observed here is similar to that of the recently reported transient intermediate formed during the acid denaturation of hMb. At 50% alcohol the protein produces predominantly high charge states of aMb ions and shows H/D exchange rates close to those of the acid-denatured protein. H/D exchange of the helical denatured protein at alcohol concentrations >60%, at which high charge states of aMb are produced, shows that the protein structure is more protected than at approximately 50% methanol.     

Folding subdomains of thioredoxin characterized by native-state hydrogen exchange

Native-state hydrogen exchange (HX) studies, used in conjunction with NMR spectroscopy, have been carried out on Escherichia coli thioredoxin (Trx) for characterizing two folding subdomains of the protein. The backbone amide protons of only the slowest-exchanging 24 amino acid residues, of a total of 108 amino acid residues, could be followed at pH 7. The free energy of the opening event that results in an amide hydrogen exchanging with solvent (DeltaG(op)) was determined at each of the 24 amide hydrogen sites. The values of DeltaG(op) for the amide hydrogens belonging to residues in the helices alpha(1), alpha(2), and alpha(4) are consistent with them exchanging with the solvent only when the fully unfolded state is sampled transiently under native conditions. The denaturant-dependences of the values of DeltaG(op) provide very little evidence that the protein samples partially unfolded forms, lower in energy than the unfolded state. The amide hydrogens belonging to the residues in the beta strands, which form the core of the protein, appear to have higher values of DeltaG(op) than amide hydrogens belonging to residues in the helices, suggesting that they might be more stable to exchange. This apparently higher stability to HX of the beta strands might be either because they exchange out their amide hydrogens in a high energy intermediate preceding the globally unfolded state, or, more likely, because they form residual structure in the globally unfolded state. In either case, the central beta strands-beta(3,) beta(2), and beta(4)-would appear to form a cooperatively folding subunit of the protein. The native-state HX methodology has made it possible to characterize the free energy landscape that Trx can sample under equilibrium native conditions.