Role of methionine-1 in ubiquitin conformation and activity

Methionine-1 of ubiquitin was oxidized to the sulfone without significant effect on biological activity or conformation at neutral pH. However, at low pH, the oxidized protein expanded to a more open conformation, similar in gel sieving properties to denatured ubiquitin but similar in secondary structure to native ubiquitin. This conformational transition was absent in the native protein. Interpretation of these results in the light of X-ray data suggests that ubiquitin contains two independently folded domains that are held together in part by a hydrogen bond between Met-1 and Lys-63 and which can be separated when this bond is broken. It is suggested that separation of these domains may occur upon ubiquitin conjugation.     

Kinetics of unfolding and folding from amide hydrogen exchange in native ubiquitin

Amide hydrogen (NH) exchange is one of the few experimental techniques with the potential for determining the thermodynamics and kinetics of conformational motions at nearly every residue in native proteins. Quantitative interpretation of NH exchange in terms of molecular motions relies on a simple two-state kinetic model: at any given slowly exchanging NH, a closed or exchange-incompetent conformation is in equilibrium with an open or exchange-competent conformation. Previous studies have demonstrated the accuracy of this model in measuring conformational equilibria by comparing exchange data with the thermodynamics of protein unfolding. We report here a test of the accuracy of the model in determining the kinetics of conformational changes in native proteins. The kinetics of folding and unfolding for ubiquitin have been measured by conventional methods and compared with those derived from a comprehensive analysis of the pH dependence of exchange in native ubiquitin. Rate constants for folding and unfolding from these two very different types of experiments show good agreement. The simple model for NH exchange thus appears to be a robust framework for obtaining quantitative information about molecular motions in native proteins.     

Molecular dynamics simulations of the native and partially folded states of ubiquitin: influence of methanol cosolvent, pH, and temperature on the protein structure and dynamics

A series of explicit-solvent molecular dynamics simulations of the protein ubiquitin are reported, which investigate the effect of environmental factors (presence of methanol cosolvent in the aqueous solution, neutral or low pH value, room or elevated temperature) on the structure, stability, and dynamics of the protein. The simulations are initiated either from the native structure of the protein or from a model of a partially folded state (A-state) that is known to exist at low pH in methanol-water mixtures. The main results of the simulations are: (1) The ubiquitin native structure is remarkably stable at neutral pH in water; (2) the addition of the methanol cosolvent enhances the stability of the secondary structure but weakens tertiary interactions within the protein; (3) this influence of methanol on the protein structure is enhanced at low pH, while the effect of lowering the pH in pure water is limited; and (4) the A-state of ubiquitin can be described as a set of relatively rigid secondary structure elements (a native-like beta-sheet and native-like alpha-helix plus two nonnative alpha-helices) connected by flexible linkers.     

Methionine oxidation perturbs the structural core of the prion protein and suggests a generic misfolding pathway

Oxidative stress and misfolding of the prion protein (PrP(C)) are fundamental to prion diseases. We have therefore probed the effect of oxidation on the structure and stability of PrP(C). Urea unfolding studies indicate that H(2)O(2) oxidation reduces the thermodynamic stability of PrP(C) by as much as 9 kJ/mol. (1)H-(15)N NMR studies indicate methionine oxidation perturbs key hydrophobic residues on one face of helix-C as follows: Met-205, Val-209, and Met-212 together with residues Val-160 and Tyr-156. These hydrophobic residues pack together and form the structured core of the protein, stabilizing its ternary structure. Copper-catalyzed oxidation of PrP(C) causes a more significant alteration of the structure, generating a monomeric molten globule species that retains its native helical content. Further copper-catalyzed oxidation promotes extended β-strand structures that lack a cooperative fold. This transition from the helical molten globule to β-conformation has striking similarities to a misfolding intermediate generated at low pH. PrP may therefore share a generic misfolding pathway to amyloid fibers, irrespective of the conditions promoting misfolding. Our observations support the hypothesis that oxidation of PrP destabilizes the native fold of PrP(C), facilitating the transition to PrP(Sc). This study gives a structural and thermodynamic explanation for the high levels of oxidized methionine in scrapie isolates.     

Oxidation of methionine residues in recombinant human interleukin-1 receptor antagonist: implications of conformational stability on protein oxidation kinetics

Oxidation of methionine residues is involved in several biochemical processes and in degradation of therapeutic proteins. The relationship between conformational stability and methionine oxidation in recombinant human interleukin-1 receptor antagonist (rhIL-1ra) was investigated to document how thermodynamics of unfolding affect methionine oxidation in proteins. Conformational stability of rhIL-1ra was monitored by equilibrium urea denaturation, and thermodynamic parameters of unfolding (DeltaGH2O, m, and Cm) were estimated at different temperatures. Methionine oxidation induced by hydrogen peroxide at varying temperatures was monitored during "coincubation" of rhIL-1ra with peptides mimicking specific regions of the reactive methionine residues in the protein. The coincubation study allowed estimation of oxidation rates in protein and peptide at each temperature from which normalized oxidation rate constants and activation energies were calculated. The rate constants for buried Met-11 in the protein were lower than for methionine in the peptide with an associated increase in activation energy. The rate constants and activation energy of solvent exposed methionines in protein and peptide were similar. The results showed that conformational stability, monitored using the Cm value, has an effect on oxidation rates of buried methionines. The rate constant of buried Met-11 correlated well with the Cm value but not DeltaGH2O. No correlation was observed for the oxidation rates of solvent-exposed methionines with any thermodynamic parameters of unfolding. The findings presented have implications in protein engineering, in design of accelerated stability studies for protein formulation development, and in understanding disease conditions involving protein oxidation.     

Conformation and stability of thiol-modified bovine beta-lactoglobulin

Bovine beta-lactoglobulin A assumes a dimeric native conformation at neutral pH, while the conformation at pH 2 is monomeric but still native. Beta-lactoglobulin A has a free thiol at Cys121, which is buried between the beta-barrel and the C-terminal major alpha-helix. This thiol group was specifically reacted with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) in the presence of 1.0 M Gdn-HCI at pH 7.5, producing a modified beta-lactoglobulin (TNB-bIg) containing a mixed disulfide bond with 5-thio-2-nitrobenzoic acid (TNB). The conformation and stability of TNB-bIg were studied by circular dichroism (CD), tryptophan fluorescence, analytical ultracentrifugation, and one-dimensional 1H-NMR. The CD spectra of TNB-bIg indicated disordering of the native secondary structure at pH 7.5, whereas a slight increase in the alpha-helical content was observed at pH 2.0. The tryptophan fluorescence of TNB-bIg was significantly quenched compared with that of the intact protein, probably by the energy transfer to TNB. Sedimentation equilibrium analysis indicated that, at neutral pH, TNB-bIg is monomeric while the intact protein is dimeric. In contrast, at pH 2.0, both the intact beta-lactoglobulin and TNB-bIg were monomeric. The unfolding transition of TNB-bIg induced by Gdn-HCl was cooperative in both pH regions, although the degree of cooperativity was less than that of the intact protein. The 1H-NMR spectrum for TNB-bIg at pH 3.0 was native-like, whereas the spectrum at pH 7.5 was similar to that of the unfolded proteins. These results suggest that modification of the buried thiol group destabilizes the rigid hydrophobic core and the dimer interface, producing a monomeric state that is native-like at pH 2.0 but is molten globule-like at pH 7.5. Upon reducing the mixed disulfide of TNB-bIg with dithiothreitol, the intact beta-lactoglobulin was regenerated. TNB-bIg will become a useful model to analyze the conformation and stability of the intermediate of protein folding.     

Conformational analysis of peptides corresponding to all the secondary structure elements of protein L B1 domain: secondary structure propensities are not conserved in proteins with the same fold.

The solution conformation of three peptides corresponding to the two beta-hairpins and the alpha-helix of the protein L B1 domain have been analyzed by circular dichroism (CD) and nuclear magnetic resonance spectroscopy (NMR). In aqueous solution, the three peptides show low populations of native and non-native locally folded structures, but no well-defined hairpin or helix structures are formed. In 30% aqueous trifluoroethanol (TFE), the peptide corresponding to the alpha-helix adopts a high populated helical conformation three residues longer than in the protein. The hairpin peptides aggregate in TFE, and no significant conformational change occurs in the NMR observable fraction of molecules. These results indicate that the helical peptide has a significant intrinsic tendency to adopt its native structure and that the hairpin sequences seem to be selected as non-helical. This suggests that these sequences favor the structure finally attained in the protein, but the contribution of the local interactions alone is not enough to drive the formation of a detectable population of native secondary structures. This pattern of secondary structure tendencies is different to those observed in two structurally related proteins: ubiquitin and the protein G B1 domain. The only common feature is a certain propensity of the helical segments to form the native structure. These results indicate that for a protein to fold, there is no need for large native-like secondary structure propensities, although a minimum tendency to avoid non-native structures and to favor native ones could be required.     

Effects of acid exposure on the conformation, stability, and aggregation of monoclonal antibodies

Exposure of antibodies to low pH is often unavoidable for purification and viral clearance. The conformation and stability of two humanized monoclonal antibodies (hIgG4-A and -B) directed against different antigens and a mouse monoclonal antibody (mIgG1) in 0.1M citrate at acidic pH were studied using circular dichroism (CD), differential scanning calorimetry (DSC), and sedimentation velocity. Near- and far-UV CD spectra showed that exposure of these antibodies to pH 2.7-3.9 induced only limited conformational changes, although the changes were greater at the lower pH. However, the acid conformation is far from unfolded or so-called molten globule structure. Incubation of hIgG4-A at pH 2.7 and 3.5 at 4 degrees C over the course of 24 h caused little change in the near-UV CD spectra, indicating that the acid conformation is stable. Sedimentation velocity showed that the hIgG4-A is largely monomeric at pH 2.7 and 3.5 as well as at pH 6.0. No time-dependent changes in sedimentation profile occurred upon incubation at these low pHs, consistent with the conformational stability observed by CD. The sedimentation coefficient of the monomer at pH 2.7 or 3.5 again suggested that no gross conformational changes occur at these pHs. DSC analysis of the antibodies showed thermal unfolding at pH 2.7-3.9 as well as at pH 6.0, but with decreased melting temperatures at the lower pH. These results are consistent with the view that the antibodies undergo limited conformational change, and that incubation at 4 degrees C at low pH results in no time-dependent conformational changes. Titration of hIgG4-A from pH 3.5 to 6.0 resulted in recovery of native monomeric proteins whose CD and DSC profiles resembled those of the original sample. However, titration from pH 2.7 resulted in lower recovery of monomeric antibody, indicating that the greater conformational changes observed at this pH cannot be fully reversed to the native structure by a simple pH titration.     

Preparation and properties of three specific active derivatives of ribonuclease A obtained by methylation of methionine residues in 8 M urea.

In 8 M urea at low pH, CH3I reacts specifically with the four methionine residues of ribonuclease A, and all four residues react at the same rate. Uon removal of the denaturant, only unmodified ribonuclease and 3 of the 15 possible derivatives modified on methionine refold to regenerate activity. All the enzymatic activity is recored after chromatography on IRC-50 and the four active proteins separate from each other and from the 12 inactive derivatives, which are not eluted from the resin under the conditions used. By the use of 14CH3I, performic acid oxidation, chymotryptic digestion, and separation of the resulting peptides by ion exchange, the active species were determined to be unmodified ribonuclease, CH3Met-29-RNase, CH3Met-79-RNase, and CH3Met-29, CH3Met-79-RNase. these proteins have melting temperatures of 63, 58, 43, and 36 degrees, respectively, at pH 6.3-70. Methylation at methionine-29 or -79 has no effect on enzymatic activity. Conversely, methylation at methionine-13 or -30 prevents refolding to an active conformation at 25 degrees elution from IRC-50. These results are consistent with the positions of the four methionine residues in crystals of ribonuclease A and ribonuclease S as determined by X-ray diffraction.     

The alpha-helix to beta-sheet transition in poly(L-lysine): effects of anesthetics and high pressure.

Poly(L-lysine) exists in a random-coil formation at a low pH, alpha-helix at a pH above 10.6, and transforms into beta-sheet when the alpha-helix polylysine is heated. Each conformation is clearly distinguishable in the amide-I band of the infrared spectrum. The thermotropic alpha-to-beta transition was studied by using differential scanning calorimetry. At pH 10.6, the transition temperature was 43.5 degrees C and the transition enthalpy was 170 cal/mol residue. At pH 11.85, the measurements were 36.7 degrees C and 910 cal/mol residue, respectively. Volatile anesthetics (chloroform, halothane, isoflurane and enflurane) partially transformed alpha-helix polylysine into beta-sheet. The transformation was reversed by the application of hydrostatic pressure in the range of 100-350 atm. Apparently, the alpha-to-beta transition was induced by anesthetics through partial dehydration of the peptide side-chains (beta-sheet surface is less hydrated than alpha-helix). High pressure reversed this process by re-hydrating the peptide. Because the membrane spanning domains of channel and receptor proteins are predominantly in the alpha-helix conformation, anesthetics may suppress the activity of excitable cells by transforming them into a less than optimal structure for electrogenic ion transport and neurotransmission. Proteins and lipid membranes maintain their structural integrity by interaction with water. That which attenuates the interaction will destabilize the structure. These data suggest that anesthetics alter macromolecular conformations essentially by a solvent effect, thereby destroying the solvation water shell surrounding macromolecules.     

Effects of pH on conformational properties related to the toxicity of Bacillus thuringiensis delta-endotoxin.

The delta-endotoxin of Bacillus thuringiensis subspecies kurstaki is an intracellular crystalline proteinaceous inclusion which, upon ingestion, is toxic to lepidopteran insects. Upon dissolution at pH > 9 it yields a protein subunit called protoxin. Under appropriate conditions, protoxin is hydrolyzed to a toxin molecule, which is responsible for killing the insect. It is known that this toxic activity decreases considerably above pH 10. In this study, circular dichroism spectroscopy has been used to examine the secondary structures of the protoxin and toxin molecules at different pH values to determine if there are detectable conformational changes associated with their pH-dependent functional properties. At pH 10, where toxic activity is approximately maximal, both the protoxin and toxin molecules were found to assume a conformation that is on an average approx. 26% alpha-helix and approx. 45% beta-structure. As the pH was increased above 10, where the insecticidal activity decreases, the magnitude of the CD spectrum at 222 nm decreased for protoxin and the calculated alpha-helix contents of both protoxin and toxin molecules decreased. The net secondary structure did not change significantly at pH values below 10. Significant conformational differences are observed between the secondary structure of the protoxin and toxin molecules at different pH values. The pH-dependent changes in secondary structure of the protoxin and toxin can be correlated with the effects of pH on the insecticidal activity of these proteins.     

Optimization of hydrophobic domains in peptides that undergo transformation from alpha-helix to beta-fibril

Recent studies on peptide fibrillogenesis by the de novo method as well as amyloidogenic proteins including prion proteins and Alzheimer's beta-peptides have provided insights into the conformational changes, such as alpha-helix to beta-structure, involved in folding and misfolding processes. We have found that an exposed hydrophobic nucleation domain at N-terminal causes a structural transition of a peptide from alpha-helix to beta-fibril. It became clear that N-terminal acyl groups of particular lengths in a 2alpha-helix peptide caused the peptide to undergo an alpha-to-beta transition. The peptide with the octanoyl group (C8-2alpha) showed the highest rate of transformation. The study of the designed peptides revealed that these alpha-to-beta transitions were closely related to the initial alpha-helix conformation and its stability. Engineering peptides that undergo alpha-to-beta transitions are attractive not only to the study of pathogenic proteins such as prion proteins, but also to the control of self-assembly of peptides, which will lead to the development of peptidyl self-assembling materials.     

Alcohol-induced conformational changes of ubiquitin

Ubiquitin has been found to be soluble in ethylene glycol and alcohols as the perchlorate or hydrochloride salt. When the effect of alcohol on the structure of ubiquitin is examined, two reversible conformational transitions are observed. Upon lowering the dielectric constant of aqueous alcohol solutions of ubiquitin from 80 to 45, the native structure of ubiquitin is converted to a form consistent with 50% helical structure. This conformational change results in a change in exposure to solvent of the single methionine and the single tyrosine residues of ubiquitin. In agreement with crystallographic results, these residues are buried in the native conformation but become fully exposed to solvent upon undergoing this transition. Further lowering of the dielectric constant to 20 results in the accumulation of a conformation with almost complete helical structure. Thus, hydrophobic interactions cause facile conformational changes in the ubiquitin structure. These results are discussed in terms of a preferential solvation model. It is shown that the results obtained with different alcohols can be normalized by the use of a dielectric constant scale. This normalization corrects for the different molar volumes of different alcohols, allows comparison of results obtained with different alcohols, and should be useful in studying this phenomenon with different proteins.     

Conformational stability of apoflavodoxin.

Flavodoxins are alpha/beta proteins that mediate electron transfer reactions. The conformational stability of apoflavodoxin from Anaboena PCC 7119 has been studied by calorimetry and urea denaturation as a function of pH and ionic strength. At pH > 12, the protein is unfolded. Between pH 11 and pH 6, the apoprotein is folded properly as judged from near-ultraviolet (UV) circular dichroism (CD) and high-field 1H NMR spectra. In this pH interval, apoflavodoxin is a monomer and its unfolding by urea or temperature follows a simple two-state mechanism. The specific heat capacity of unfolding for this native conformation is unusually low. Near its isoelectric point (3.9), the protein is highly insoluble. At lower pH values (pH 3.5-2.0), apoflavodoxin adopts a conformation with the properties of a molten globule. Although apoflavodoxin at pH 2 unfolds cooperatively with urea in a reversible fashion and the fluorescence and far-UV CD unfolding curves coincide, the transition midpoint depends on the concentration of protein, ruling out a simple two-state process at acidic pH. Apoflavodoxin constitutes a promising system for the analysis of the stability and folding of alpha/beta proteins and for the study of the interaction between apoflavoproteins and their corresponding redox cofactors.     

Metal ions- and pH-induced conformational changes of acutolysin A from Agkistrodon acutus venom probed by fluorescent spectroscopy

Acutolysin A isolated from the venom of Agkistrodon acutus is a protein of 22 kDa with marked haemorrhagic and proteolytic activities. The metal ions- and pH-induced conformational changes of acutolysin A have been studied by following fluorescence and activity measurements. Here, we provide evidence for the fact that native holo-acutolysin A adopts two subtly different conformations, native state a (Na) stable in the weak acidic pH range from 6.0 to 7.0 with low activity and native state b (Nb) stable in the weak alkaline pH range from 7.5 to 9.0 with high activity. Holo-acutolysin A has an optimum pH of 8.5 for caseinolytic activity, and the protein adopts the most stable conformation with the maximum fluorescence at pH 8.5. The Ca2+ and Zn2+ ions have significant effects on both the pH-induced denaturing transition curve and the pH-dependent activity curve. Addition of 1 mM Ca2+ to holo-acutolysin A shifts both the acid-induced denaturing transition curve and the end zone of acid-induced inactivation curve towards lower pH value, and shifts both the alkali-induced denaturing transition curve and the end zone of alkali-induced inactivation curve towards higher pH value. Addition of 1 mM Zn2+ also shifts both the alkali-induced denaturing transition curve and the end zone of alkali-induced inactivation curve towards higher pH value and shifts the acid-induced denaturing transition curve to lower pH value, but has little effect on the acid-induced inactivation. Removal of Ca2+ and Zn2+ from the protein enhances its sensitivity to pH and significantly reduces its overall stability during acid-induced denaturation. It is also evident from the present work that the free Zn2+ -induced inactivation in the pH range from 8.0 to 9.0 should be attributed to the effect of Zn(OH)2 precipitation on the protein.     

The conformation and stability of recombinant-derived granulocyte-macrophage colony stimulating factors

The conformation and stability of recombinant-derived human and murine granulocyte-macrophage colony stimulating factors produced in Escherichia coli have been investigated by analytical ultracentrifugation, urea-gradient polyacrylamide gel electrophoresis and several spectroscopic methods. The proteins were demonstrated to be physically homogeneous monomeric proteins with compact globular shapes and shown to have similar secondary structures containing both alpha-helix and beta-sheet structure. The intramolecular disulphide linkages of both proteins were shown to be essential for maintaining native conformation as reduction with dithiothreitol resulted in protein unfolding. Comparison of the human E. coli-derived (non-glycosylated) and mammalian cell culture-derived (glycosylated) proteins by urea-gradient electrophoresis indicated that glycosylation had no major effect on the conformational stability and kinetics of urea induced unfolding and refolding.     

Structural basis for the remarkable stability of Bacillus subtilis lipase (Lip A) at low pH

Understanding the structural basis of altered properties of proteins due to changes in temperature or pH provides useful insights in designing proteins with improved stability. Here we report the basis for the pH-dependent thermostability of the Bacillus subtilis lipase (Lip A) using spectroscopic and X-ray crystallographic studies. At pH values above 7, lipase denatures and aggregates when heated at temperatures above 45 degrees C. However, at pH below 6 lipase denatures upon heating but the activity and its native structure is completely recovered upon cooling. In order to obtain the structural basis of this unusual stability of lipase, we determined high-resolution crystal structures of the lipase in two different crystal forms at pH 4.5 and 5. These structures show linear oligomerization of lipase using only two types of dimeric associations and these inter-molecular interactions are completely absent in several crystal forms of wild-type and mutant proteins obtained at basic pH. In accordance with the crystallographic studies, spectroscopic investigations reveal an invariant secondary structure in the pH range of 4-10. Quaternary organization of lipase at low pH resulted in changes in the tryptophan environment and binding of 1-anilino-8-naphthalene sulfate (ANS) at low pH. Low pH stability of the lipase is not observed in the presence of sodium chloride (>0.2 M) indicating the importance of ionic interactions at low pH. Inter- and intra-molecular ionic interactions that occur at pH below 6.0 are proposed to trap the molecule in a conformation that allows its complete refolding upon cooling.     

Context-dependent effects of proline residues on the stability and folding pathway of ubiquitin.

Substitution of trans-proline at three positions in ubiquitin (residues 19, 37 and 38) produces significant context-dependent effects on protein stability (both stabilizing and destabilizing) that reflect changes to a combination of parameters including backbone flexibility, hydrophobic interactions, solvent accessibility to polar groups and intrinsic backbone conformational preferences. Kinetic analysis of the wild-type yeast protein reveals a predominant fast-folding phase which conforms to an apparent two-state folding model. Temperature-dependent studies of the refolding rate reveal thermodynamic details of the nature of the transition state for folding consistent with hydrophobic collapse providing the overall driving force. Br?nsted analysis of the refolding and unfolding rates of a family of mutants with a variety of side chain substitutions for P37 and P38 reveals that the two prolines, which are located in a surface loop adjacent to the C terminus of the main alpha-helix (residues 24-33), are not significantly structured in the transition state for folding and appear to be consolidated into the native structure only late in the folding process. We draw a similar conclusion regarding position 19 in the loop connecting the N-terminal beta-hairpin to the main alpha-helix. The proline residues of ubiquitin are passive spectators in the folding process, but influence protein stability in a variety of ways.     

Regeneration of native ovalbumin conformation disrupted by citraconylation.

Ovalbumin was reacted with a 960-fold molar excess of citraconic anhydride, and 91% of the epsilon-amino groups, representing 18 of the 20 lysine residues, were citraconylated. As detected by fluorescence and far-ultraviolet circular dichroic (CD) measurements, the modified protein displayed significant disruption of the native conformation. Treatment at pH 2.2 for 5 h resulted in the hydrolysis of 10 of the 18 citraconyl groups, but when subjected to the acid conditions for 12 h, all 18 modifying groups were removed. Electrophoretically, the 5-h and the 12-h acid-treated proteins were homogeneous and showed decreasing anodic mobility at pH 8.3; indeed, the anodic mobility of the 12-h acid-treated protein was identical to that of the native protein. Similarly, the 12-h acid-treated protein possessed conformational properties almost indistinguishable from the native protein. These properties included similar emission fluorescence spectra and far-ultraviolet CD spectra, similar resistance to undergoing helix-to-coil transition at pH 12.2, and identical urea unfolding curves, and thus identical urea transition mid-point of about 8.0 M. These observations indicate that the protein with all the epsilon-amino groups regenerated by acid treatment has the same conformational stability as the native ovalbumin.     

Structural and dynamic characteristics of a partially folded state of ubiquitin revealed by hydrogen exchange mass spectrometry

Structural and dynamic properties of a partially folded conformation (A-state) of ubiquitin are studied using amide hydrogen exchange in solution (HDX) and mass spectrometric detection. A clear distinction between the native state of the protein and the A-state can be made when HDX is carried out in a semicorrelated regime. Convoluted exchange patterns are interpreted with the aid of HDX simulations in a three-state system (highly structured, partially unstructured, and fully unstructured states). The data clearly indicate a highly dynamic character of the non-native state. Furthermore, combination of HDX and protein ion fragmentation in the gas phase [by means of collision-induced dissociation (CAD)] is used to evaluate the conformational stability of various protein segments specifically in the molten globular state. Chain flexibility appears to be distributed very unevenly in this non-native conformation. The highest degree of structural disorder is displayed by the C-terminal segment (Gly(53)-Gly(76)), which was previously suggested to form a transient alpha-helix. The least dynamic segment of ubiquitin in the A-state is Thr(9)-Glu(18) (which was previously suggested to form a stable nativelike beta-strand), with the adjacent segments exhibiting somewhat diminished conformational stability. The study also demonstrates the power of mass spectrometry as a tool in providing conformer-specific information about the structure and dynamics of both native and non-native protein states coexisting in solution under equilibrium.