Publications with Abstract


Maki, K., Cheng, H., Dolgikh, D. A., Shastry, M. C. & Roder, H. (2004). Early Events During Folding of Wild-type Staphylococcal Nuclease and a Single-tryptophan Variant Studied by Ultrarapid Mixing. J. Mol. Biol. 338, 383-400. (pdf)


Roder, H. (2004). Stepwise helix formation and chain compaction during protein folding. Proc. Natl. Acad. Sci. USA 101, 1793-1794. (pdf) 


Zhu, Y., Alonso, D.O.V., Maki, K., Cheng-Yen Huang, C.-Y., Lahr, S.J., Daggett, V., Roder, H., DeGrado, W.F. & Gai, F. (2003) Ultrafast folding of a3D, a de novo designed three-helix bundle protein. Proc. Natl. Acad. Sci. USA 100, 15486-91. (pdf)


Kuwata, K., Matumoto, T., Cheng, H., Nagayama, K., James, T. L. & Roder, H. NMR-detected hydrogen exchange and molecular dynamics simulations provide structural insight into fibril formation of prion protein fragment 106-126. Proc. Natl. Acad. Sci. USA 100, 14790-14795, 2003.

PrP106-126, a peptide corresponding to residues 107-127 of the human prion protein, induces neuronal cell death by apoptosis and causes proliferation and hypertrophy of glia, reproducing the main neuropathological features of prion-related transmissible spongiform encephalopathies, such as bovine spongiform encephalopathy and Creutzfeldt-Jakob disease. Although PrP106-126 has been shown to form amyloid-like fibrils in vitro, their structural properties have not been elucidated. Here, we investigate the conformational characteristics of a fibril-forming fragment of the mouse prion protein, MoPrP106-126, by using electron microscopy, CD spectroscopy, NMR-detected hydrogen-deuterium exchange measurements, and molecular dynamics simulations. The fibrils contain approximately 50% beta-sheet structure, and strong amide exchange protection is limited to the central portion of the peptide spanning the palindromic sequence VAGAAAAGAV. Molecular dynamics simulations indicate that MoPrP106-126 in water assumes a stable structure consisting of two four-stranded parallel beta-sheets that are tightly packed against each other by methyl-methyl interactions. Fibril formation involving polyalanine stacking is consistent with the experimental observations.

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Gianni S, Travaglini-Allocatelli C, Cutruzzola F, Brunori M, Shastry MC, Roder H. Parallel Pathways in Cytochrome c(551) Folding. J. Mol. Biol. 330, 1145-1152, 2003.

The folding of cytochrome c(551) from Pseudomonas aeruginosa was previously thought to follow a simple sequential mechanism,
consistent with the lack of histidine residues, other than the native His16 heme ligand, that can give rise to mis-coordinated species. However, further kinetic analysis reveals complexities indicative of a folding mechanism involving parallel pathways. Double-jump interrupted refolding experiments at low pH indicate that approximately 50% of the unfolded cytochrome c(551) population can reach the native state via a fast (10 ms) folding track, while the rest follows a slower folding path with populated intermediates. Stopped-flow experiments using absorbance at 695 nm to monitor refolding confirm the presence of a rapidly folding species containing the native methionine-iron bond while measurements on carboxymethylated cytochrome c(551) (which lacks the Met-Fe coordination bond) indicate that methionine ligation occurs late during folding along the fast folding track, which appears to be dominant at physiological pH. Continuous-flow measurements of tryptophan-heme energy transfer, using a capillary mixer with a dead time of about 60 micros, show evidence for a rapid chain collapse within 100 micros preceding the rate-limiting folding phase on the milliseconds time scale. A third process with a time constant in the 10-50 ms time range is consistent with a minor population of molecules folding along a parallel channel, as confirmed by quantitative kinetic modeling. These findings indicate the presence of two or more slowly inter-converting ensembles of denatured states that give rise to pH-dependent partitioning among fast and slow-folding pathways.

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Teilum, K., K., Maki, K., Kragelund, B.B., Poulsen, F.M. & Roder, H. Early kinetic intermediate in the folding of Acyl-Coenzyme A Binding Protein detected by fluorescence labeling and ultra rapid mixing. Proc. Natl. Acad. Sci. USA 99, 9807-12, 2002.

Early conformational events during folding of acyl-coenzyme A binding protein (ACBP), an 86-residue a-helical protein, were explored by using a continuous-flow mixing apparatus with a dead time of 70 ms to measure changes in intrinsic tryptophan fluorescence and tryptophan-dansyl fluorescence energy transfer.  Although the folding of ACBP was initially described as a concerted two-state process, the tryptophan fluorescence measurements revealed a previously unresolved phase with a time constant τ = 80 μs, indicating formation of an intermediate with only slightly enhanced florescence of Trp55 and Trp58 relative to the unfolded state. To amplify this phase, a dansyl fluorophore was introduced at the C-terminus by labeling an I86C mutant of ACBP with 5-IAEDANS. Continuous-flow refolding of guanidine HCl-denatured ACBP showed a major increase in tryptophan-dansyl fluorescence energy transfer indicating formation of a partially collapsed ensemble of states on the 100 ms time scale. A subsequent decrease in dansyl fluorescence is attributed to intramolecular quenching of donor fluorescence on formation of the native state.  The kinetic data are fully accounted for by three-state mechanisms with either on-pathway or off-pathway intermediates. The intermediate accumulates to a maximum population of 40%, and its stability depends only weakly on denaturant concentration, which is consistent with a marginally stable ensemble of partially collapsed states with ~1/3 of the solvent accessible surface buried. The findings indicate that ultrafast mixing methods combined with sensitive conformational probes can reveal transient accumulation of intermediate states in proteins with apparent two-state folding mechanisms.

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Abdullaev, Z., Bodrova, M.E., Chernyak, B.V., Dolgikh, D.A., Kluck, R.M., Pereverzev, M.O., Arseniev, A.S., Efremov, R.G., Kirpichnikov, M.P., Mokhova, E.N., Newmeyer, D.D., Roder, H. & Skulachev, V.P. A cytochrome c mutant with high electron transfer and antioxidant activities but devoid of apoptogenic effect. Biochem. J. 362, 749-54, 2002.

A cytochrome c mutant lacking apoptogenic function but competent in electron transfer and antioxidant activities has been constructed. To this end, mutant species of horse and yeast cytochromes c with substitutions in the N-terminal alpha-helix or position 72 were obtained. It was found that yeast cytochrome c was much less effective than the horse protein in activating respiration of rat liver mitoplasts deficient in endogenous cytochrome c as well as in inhibition of H(2)O(2) production by the initial segment of the respiratory chain of intact rat heart mitochondria. The major role in the difference between the horse and yeast proteins was shown to be played by the amino acid residue in position 4 (glutamate in horse, and lysine in yeast; horse protein numbering). A mutant of the yeast cytochrome c containing K4E and some other "horse" modifications in the N-terminal alpha-helix, proved to be (i) much more active in electron transfer and antioxidant activity than the wild-type yeast cytochrome c and (ii), like the yeast cytochrome c, inactive in caspase stimulation, even if added in 400-fold excess compared with the horse protein. Thus this mutant seems to be a good candidate for knock-in studies of the role of cytochrome c-mediated apoptosis, in contrast with the horse K72R, K72G, K72L and K72A mutant cytochromes that at low concentrations were less active in apoptosis than the wild-type, but were quite active when the concentrations were increased by a factor of 2-12.


Yi., Cheng, H., Andrake, M.D., Dunbrack, R.L., Jr., Roder, H. & Skalka, A.M. Mapping the epitope of an inhibitory mAb to the C-terminal DNA binding domain of HIV-1 integrase. J. Biol. Chem., 2002.

Integrase (IN) catalyzes the insertion of retroviral DNA into chromosomal DNA of a host cell and is one of three virus-encoded enzymes that are required for replication. A library of monoclonal antibodies against human immunodeficiency virus-type 1 (HIV-1) IN was raised and characterized in our laboratory. Among them, mAb33 and mAb32 compete for binding to the C-terminal domain of the HIV-1 IN protein. Here we show that mAb33 is a strong inhibitor of IN catalytic activity whereas mAb32 is only weakly inhibitory. Furthermore, as the Fab fragment of mAb32 has no effect on IN activity, inhibition by this mAb may result solely from its bivalency. In contrast, Fab33 does inhibit IN catalytic activity, although bivalent binding by mAb33 may enhance the inhibition. Interaction with Fab33 also prevents DNA binding to the isolated C-terminal domain of IN. Results from size exclusion chromatography, gel electrophoresis, and MALDI-TOF MS analyses reveal that multiple Fab33:IN C-terminal domain complexes exits in solution. Studies using heteronuclear NMR show a steep decrease in 15N-1H cross peak intensity for 8 residues in the isolated C-terminal domain upon binding of Fab33, indicating that these residues become immobilized in the complex. Among them, Ala239 and Ile251 are buried in the interior of the domain while the remaining residues (Phe223, Arg224, Tyr226, Lys244, Ile267, and Ile268) form a contiguous, solvent-accessible patch on the surface of the protein likely including the epitope of Fab33. Molecular modeling of Fab33 followed by computer-assisted docking with the IN C-terminal domain suggest a structure for the antibody-antigen complex that is consistent with our experimental data and suggest a potential target for anti-AIDS drug design.


Hagen, S.J., Latypov, R.F., Dolgikh, D.A. & Roder, H. Rapid intrachain binding of histidine-26 and histidine-33 to heme in unfolded ferrocytochrome c. Biochemistry 41, 1372-80, 2002.

Time-resolved spectroscopic studies of unfolded horse iron(II) cytochrome c have suggested that the imidazole side chains of His26 and His33 bind transiently to the heme iron on microsecond time scales, after photodissociation of a carbon monoxide ligand from the heme. Our studies of four variants of cytochrome c (horse wild type, horse H33N, horse H33N/H26Q, and tuna wild type), unfolded in guanidine hydrochloride at pH 6.5, demonstrate that these side chains are responsible for the observed microsecond spectral changes. As His33 and then His26 are eliminated from the horse wild-type sequence, transient optical absorption spectra show systematic suppression of a rapid (approximately 10-100 micros) Soret absorbance change that follows photolysis of CO. Transient binding of these histidine side chains to the heme therefore generates one of the fast kinetic phases observed in previous photochemically triggered spectroscopic studies of dynamics in unfolded iron(II) cytochrome c. Furthermore, both His33 and His26 appear to contribute to a similar extent in these early kinetics. Thus, the stiffness of the polypeptide chain creates a deviation from Gaussian chain behavior by impeding, although not preventing, the formation of short (<10 peptide bonds) intrachain loops around the heme group.

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Walkenhorst, W.F., Edwards, J.A., Markley, J.L. & Roder, H. Early formation of a beta hairpin during folding of staphylococcal nuclease H124L as detected by pulsed hydrogen exchange. Protein Sci 11, 82-91, 2002.

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.

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Cheng, R.P., Suich, D.J., Cheng, H., Roder, H. & DeGrado, W.F. Template-constrained somatostatin analogues: a biphenyl linker induces a type-V' turn. J. Am. Chem. Soc. 123, 12710-1, 2001.

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Kuwata, K., Shastry, M. C. R., Cheng, H., Hoshima, M., Batt, C. A., Goto, Y., & Roder, H. Structural and kinetic characterization of early folding events in b-lactoglobulin. Nature Struct. Biol., 2001. 8:151-155.

To define the structural and dynamic properties of an early folding intermediate in b -lactoglobulin known to contain non-native a -helical structure, the kinetics of folding was measured over the 100 m s to 10 s time range, using ultra-rapid mixing techniques in conjunction with fluorescence detection and hydrogen exchange labeling probed by heteronuclear NMR. An initial increase in tryptophan fluorescence with a time constant of 140 m s 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 are found only in a domain comprising b -strands F, G and H, and the main a -helix, which is thus identified as the folding core of b -lactoglobulin. At the same time, weak protection (up to ~10-fold) of amide protons in a segment spanning residues 12-21 is consistent with formation of marginally stable non-native a -helices near the N-terminus. The results indicate that efficient folding, despite some local non-native structural preferences, is insured by the rapid formation of a native-like a /b core domain.

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Capaldi, A.P., Shastry, M.C.R., Kleanthous, C., Roder, H. & Radford, S.E. Ultra-rapid mixing experiments reveal that Im7 folds via an on-pathway intermediate. Nature Struct. Biol., 2001. 8:68-72.

Many proteins populate partially organized structures during folding. Since these intermediates often accumulate within the dead-time (2-5 ms) of conventional stopped- and quench-flow devices it has been difficult to determine their role in the formation of the native state. Here we use a microcapillary mixing apparatus, with a time resolution of ~150 ms, to directly follow the formation of an intermediate in the folding of the four-helix protein, Im7. Quantitative kinetic modeling of folding and unfolding data acquired over a wide range of urea concentrations demonstrates that this intermediate ensemble lies on a direct path from the unfolded to the native state.

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Talaga, D. S., Lau, W. L., Roder, H., Tang, J., Jia, Y., DeGrado, W. F. & Hochstrasser, R. M. Dynamics and folding of single two-stranded coiled-coil peptides studied by fluorescent energy transfer confocal microscopy. PNAS, 2000. 97:13021-6.

We report single-molecule measurements on the folding and unfolding conformational equilibrium distributions and dynamics of a disulfide crosslinked version of the two-stranded coiled coil from GCN4. The peptide has a fluorescent donor and acceptor at the N termini of its two chains and a Cys disulfide near its C terminus. Thus, folding brings the two N termini of the two chains close together, resulting in an enhancement of fluorescent resonant energy transfer. End-to-end distance distributions have thus been characterized under conditions where the peptide is nearly fully folded (0 M urea), unfolded (7.4 M urea), and in dynamic exchange between folded and unfolded states (3.0 M urea). The distributions have been compared for the peptide freely diffusing in solution and deposited onto aminopropyl silanized glass. As the urea concentration is increased, the mean end-to-end distance shifts to longer distances both in free solution and on the modified surface. The widths of these distributions indicate that the molecules are undergoing millisecond conformational fluctuations. Under all three conditions, these fluctuations gave nonexponential correlations on 1- to 100-ms time scale. A component of the correlation decay that was sensitive to the concentration of urea corresponded to that measured by bulk relaxation kinetics. The trajectories provided effective intramolecular diffusion coefficients as a function of the end-to-end distances for the folded and unfolded states. Single-molecule folding studies provide information concerning the distributions of conformational states in the folded, unfolded, and dynamically interconverting states.

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Pinheiro, T.J., Cheng, H., Seeholzer, S.H. & Roder, H. Direct evidence for the cooperative unfolding of cytochrome c in lipid membranes from H-(2)H exchange kinetics. J. Mol. Biol, 2000. 303:617-26.

The interaction of cytochrome c (cyt c) with anionic lipid membranes is known to disrupt the tightly packed native structure of the protein. This process leads to a lipid-inserted denatured state, which retains a native-like a -helical structure but lacks any specific tertiary interactions. The structural and dynamic properties of cyt c bound to vesicles containing an anionic phospholipid (DOPS) were investigated by amide H-D exchange using 2D NMR spectroscopy and ESI mass spectrometry. The H-D exchange kinetics of the core amide protons in cyt c, which in the native protein undergo exchange via an uncorrelated EX2 mechanism, exchange in the lipid vesicles via a highly concerted global transition that exposes these protected amide groups to solvent. The lack of pH dependence and the observation of distinct populations of deuterated and protonated species by mass spectrometry confirms that exchange occurs via an EX1 mechanism with a common rate of 1 ( 0.5) h-1, which reflects the rate of transition from the lipid-inserted state Hl to an unprotected conformation Di associated with the lipid interface.

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Roder, H., Elöve, G.A. & Shastry, M.C.R. Early stages of protein folding. in Protein folding: Frontiers in Molecular Biology (ed. Pain, R.H.) Oxford University Press, New York, 2000. pp. 65-104.

SUMMARY: The recent development of highly efficient devices for turbulent and diffusive mixing of solutions coupled with fluorescence, Raman or small angle x-ray scattering measurements has made it possible to extend kinetic measurements on protein folding reactions into the submillisecond time regime, which opened a new window for exploring the dynamics of conformational events during the initial stages of protein folding. Hydrogen exchange labeling combined with NMR spectroscopy or mass spectrometry continues to be an essential tool for the structural characterization of partially folded states. Although hydrogen labeling methods are best suited for the analysis of the more highly organized states populated during the late stages of folding, the method can be adapted to detect the formation of marginally hydrogen bonded structures at early stages of folding. By combining continuous-flow fluorescence experiments with a dead time of less than 50 m s with conventional stopped-flow measurements, we were able to account for the entire time course of folding of cytochrome c over six orders of magnitude in time, including the previously unresolved initial collapse of the polypeptide chain. The observation of a major exponential fluorescence decay with a time constant of about 50 m s indicates that the first large-scale conformational event in folding represents the barrier-limited formation of compact conformations. The presence of a free energy barrier with a significant enthalpic component clearly shows that the initial collapse of the cytochrome c polypeptide chain is a cooperative (two-state) process, in contrast to the behavior predicted for homopolymers, and shows that the resulting ensemble of compact conformations represents a thermodynamic state distinct from the initial population of denatured states. More detailed structural information was obtained in NMR-detected H/D exchange labeling experiments. Amide protons involved in a -helical hydrogen bonds of the native cytochrome c structure were found to acquire weak but significant protection against solvent exchange within 2 ms of refolding, consistent with rapid formation of a core domain with loosely packed a -helices. Kinetic measurements on the microsecond time scale for a number of proteins have shown that important conformational events involving both secondary and tertiary structural interactions can occur long before the rate-limiting step in folding. Although much remains to be learned about the structural and kinetic role of these early events, there is little doubt that they represent genuine folding events. While some proteins can reach the native state in a single cooperative transition, for many others one can resolve two or more physically distinct stages, even in the absence of slow extraneous steps, such as heme ligand exchange or peptide bond isomerization. These findings are consistent with a minimal three-state folding mechanism in which a directed collapse of the chain into a loosely packed intermediate with some native-like structural features precedes and facilitates the rate-limiting acquisition of the tightly packed native structure.


Walsh, S.T.R., Cheng, H., Bryson, J.W., Roder, H. & DeGrado, W.F. Solution structure and dynamics of a de novo designed three helix bundle protein. PNAS, 1999. 96:5486-5491.

Although de novo protein design is an important endeavor with implications for understanding protein folding, until now, structures have been determined for only a few 25 to 30 residue designed mini-proteins. Here, the NMR solution structure of a complex 73 residue three-helix bundle protein, a3D, is reported. The structure of a3D was not based on any natural protein, and yet it shows thermodynamic and spectroscopic properties typical of native proteins. A variety of features contribute to its unique structure, including electrostatics, the packing of a diverse set of hydrophobic side chains, and a loop that incorporates common capping motifs. Thus, it is now possible to design a complex protein with a well-defined and predictable three-dimensional structure.

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Roder, H. & M.C.R. Shastry. Methods for exploring early events in protein folding. Curr. Opin. Struct. Biol., 1999. 9:620-626.

Progress in understanding dynamic aspects of protein folding relies on the continuing development of methods for obtaining more detailed structural information on the transient conformational ensembles that often appear within microseconds of initiating refolding. Advances in rapid mixing and other time-resolved spectroscopic methods have made it possible to explore some of the earliest stages of folding, including the initial formation of compact states, which is determined by the presence of a sequence-specific kinetic barrier, as well as the 'downhill' folding kinetics after the rate-limiting barrier has been crossed.


Park, S-H., Shastry, M.C.R. & Roder, H. Folding dynamics of the B1 domain of protein G explored by ultrarapid mixing. Nature Struct. Biol., 1999. 6:943-947.

For many proteins, compact conformations are known to accumulate in advance of the rate-limiting step in folding. To understand the nature and significance of these early conformational events, we employed ultrarapid mixing methods to fully characterize the kinetics of folding of the 57-residue B1 domain of protein G. Continuous-flow fluorescence measurements exhibit a major exponential phase on the submillisecond time scale (600-700 us), which is followed by a slower phase with a denaturant-dependent time constant (2-30 ms) observable by conventional stopped-flow measurements. The combined kinetic traces quantitatively account for the total change in Trp 43 fluorescence upon folding, including the previously unresolved 'burst phase' signal. The denaturant dependence of the two rate constants and their relative amplitudes are fully consistent with a three-state mechanism, U <=> I <=> N, where I is a productive intermediate with native-like fluorescence properties. The relatively slow rate and exponential time course of the initial folding phase indicates that a substantial free energy barrier is encountered during chain condensation, resulting in a partially organized ensemble of states distinct from the initial unfolded conformations.

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Rankin, S.E., Watts, A., Roder, H. & Pinheiro, T.J.T. Folding of apocytochrome c induced by the interaction with negatively charged lipid micelles proceeds via a collapsed intermediate state. Protein Science, 1999. 8:381-393.

Unfolded apocytochrome c acquires an alpha-helical conformation upon interaction with lipid. Folding kinetic results below and above the lipid's CMC, together with energy transfer measurements of lipid bound states, and salt-induced compact states in solution, show that the folding transition of apocytochrome c from the unfolded state in solution to a lipid-inserted helical conformation proceeds via a collapsed intermediate state (Ic). This initial compact state is driven by a hydrophobic collapse of the polypeptide chain in the absence of the heme group and may represent a heme-free analogue of an early compact intermediate detected on the folding pathway of cytochrome c in solution. Insertion into the lipid phase occurs via an unfolding step of Ic through a more extended state associated with the membrane surface (IS). While Ic appears to be as compact as salt-induced compact states in solution with substantial alpha-helix content, the final lipid-inserted state (Hmic) is as compact as the unfolded state in solution at pH 5 and has an alpha-helix content which resembles that of native cytochrome c.


Sauder, J.M. & Roder, H. Amide protection in an early folding intermediate of cytochrome c. Folding & Design, 1998. 3:293-301.

Background: For many proteins, compact states appear long before the rate-limiting step in formation of the native structure. However, the structural and kinetic role of these intermediate states remains poorly understood. A key issue is whether the initial collapse of the chain is driven by random or more specific hydrophobic interactions.
Results: Hydrogen-exchange labeling coupled with NMR was used to monitor the formation of stable hydrogen-bonded and solvent-excluded structure in horse cytochrome c (cyt c). Protection was measured using a hydrogen exchange/folding competition protocol at variable pH and short competition time (2 ms). Protection factors of threefold to eightfold were observed in all three alpha-helices of cyt c, whereas other regions showed no significant protection. This suggests that the compact states that are present contain segments of marginally stable hydrogen-bonded structure. When the intermediate(s) are destabilized, only amide protons from Cys14, Ala15 and His18 show significant protection, indicating a region of persistent residual structure near the covalently bound heme group in the unfolded protein. Fluorescence-detected stopped-flow studies showed that the maximum protection factor in the early intermediate is consistent with its unfolding equilibrium constant, which is pH-independent under the conditions of the competition experiment (pH 7-11).
Conclusions: Together with previous fluorescence and circular dichroism results, the observed pattern of amide protection is consistent with the early formation of an alpha-helical core domain in an ensemble of compact states, indicating that efficient folding is facilitated by stepwise acquisition of native structural elements. These specific early interactions are established on the sub-millisecond time scale, prior to the rate-limiting step for folding.


Shastry, M.C.R., Sauder, J.M. & Roder, H. Kinetic and structural analysis of submillisecond folding events in cytochrome c. Accts. Chem. Res., 1998. 31:717-725.

The continuous-flow mixing method recently developed in our laboratory (Shastry, M.C.R., Luck, S.D. and Roder, H., Biophys. J. 74: 2714-2721, 1998), which combines an efficient capillary mixer with a novel detection method employing a CCD camera, enabled us to monitor tryptophan fluorescence changes during refolding of cyt c down to tens of microseconds. In combination with conventional stopped-flow measurements, we were thus able to account for the entire time course of folding over six orders of magnitude in time, including the previously unresolved initial collapse of the polypeptide chain (Shastry, M.C.R. and Roder, H., Nature Struct. Biol. 5: 385-392, 1998). Our observation of a major exponential fluorescence decay with a time constant of about 50 microseconds indicates that the first large-scale conformational event in folding represents the barrier-limited formation of an ensemble of compact states. This free energy barrier has only a small enthalpic component, and its height varies little with initial and final refolding conditions, both in the presence and absence of non-native heme ligands, indicating that it reflects a general entropic bottleneck encountered during the initial compaction of the polypeptide chain. In a recent hydrogen exchange labeling study (Sauder, J.M. and Roder, H., Fold. Des., 1998, 3: 293-301), we found that amide protons involved in alpha-helical hydrogen bonds of the native structure acquired weak but significant protection against solvent exchange within 2 ms of refolding, consistent with rapid formation of a core domain with loosely packed alpha-helices. This partially structured ensemble of states appears long before the rate-limiting folding step, even in the absence of slow extraneous steps, such as heme ligand exchange or peptide bond isomerization, indicating that folding occurs in at least two physically distinct stages. The results are fully consistent with a minimal three-state folding mechanism where a directed collapse of the chain into a loosely packed intermediate with some native-like structural features precedes and facilitates the rate-limiting acquisition of the tightly packed native structure.


Shastry, M.C.R. & Roder, H. Evidence for barrier-limited protein folding kinetics on the microsecond time scale. Nature Struct. Biol., 1998. 5: 385-392.

Important structural events in protein folding, including both local and long-range conformational changes, are known to occur on the submillisecond time scale. However, the limited time resolution of conventional kinetic methods has precluded direct observation of the initial collapse of the polypeptide chain, and the mechanism of this critical event remains poorly understood. In previous kinetic studies on horse cytochrome c, a major fraction of the heme-induced quenching of Trp59 fluorescence indicative of chain collapse was found to occur within the first millisecond of refolding. We recently developed a continuous-flow capillary mixing method, which enabled us to quantitatively account for the entire fluorescence change associated with refolding of cytochrome c over the time window from tens of microseconds to to minutes. The kinetics of folding under various conditions exhibits a major exponential process with a time constant of about 50 microseconds, indicating a common free energy barrier encountered during the initial collapse of the polypeptide chain. The initial phase is independent of the heme ligation state, confirming that it reflects an intrinsic conformational event. The barrier-limited collapse of the chain into a loosely packed intermediate occus long before the rate-limiting formation of specific tertiary interactions, indicating that folding occurs in at least two stages.


Houry, W.A., Sauder, J.M., Roder, H. and Scheraga, H.A. Definition of amide protection factors for early kinetic intermediates in protein folding. PNAS, 1998. 95: 4299-4302. (PDF)

Hydrogen-deuterium exchange experiments have been used previously to investigate the structures of well-defined states of a given protein. These include the native state, the unfolded state, and any intermediate states that can be stably populated at equilibrium. More recently, the hydrogen-deuterium exchange technique has been applied in kinetic labeling experiments in order to probe the structures of transiently formed intermediates on the kinetic folding pathway of a given protein. From these equilibrium and non-equilibrium studies, protection factors are usually obtained. These protection factors are defined as the ratio of the rate of exchange of a given backbone amide when it is in a fully solvent- exposed state (usually obtained from model peptides) to the rate of exchange of that amide in some state of the protein or in some intermediate on the folding pathway of the protein. This definition is straightforward for the case of equilibrium studies; however, it is less clear-cut for the case of transient kinetic intermediates. In order to clarify the concept for the case of burst-phase intermediates, we have introduced and mathematically defined two different types of protection factors: one is Pstruc, which is more related to the structure of the intermediate, and the other is Papp, which is more related to the stability of the intermediate. Kinetic hydrogen-exchange data from disulfide-intact ribonuclease A and from cytochrome c are discussed in order to explain the use and implications of these two definitions.


Shastry, M.C.R., Luck, S.D., Roder, H. A continuous-flow capillary mixer to monitor reactions on the microsecond time scale. Biophys. J., 1998. 74: 2714-2721. (PDF)

A continuous-flow capillary mixing apparatus, based on the original design of Regenfuss et al. (Regenfuss, P., R.M. Clegg, M.J. Fulwyler, F.J. Barrantes, and T.M. Jovin. 1985. Rev. Sci. Instrum. 56: 283-290), has been developed with significant advances in mixer design, detection method and data analysis. To overcome the problems associated with the free-flowing jet used for observation in the original design (instability, optical artifacts due to scattering, poor definition of the geometry), the solution emerging from the capillary is injected directly into a flow-cell joined to the tip of the outer capillary via a ground-glass joint. The reaction kinetics are followed by measuring fluorescence versus distance down-stream from the mixer, using an Hg(Xe) arc lamp for excitation and a digital camera with a UV-sensitized CCD detector for detection. Test reactions involving fluorescent dyes indicate that mixing is completed within 15 microseconds of its initiation and that the dead-time of the measurement is 45 ± 5 microseconds, which represents a >30-fold improvement in time resolution over conventional stopped-flow instruments. The high sensitivity and linearity of the CCD camera has been instrumental in obtaining artifact-free kinetic data over the time window from ~45 microseconds to a few milliseconds with signal-to-noise levels comparable to conventional methods. The scope of the method is discussed and illustrated with an example of a protein folding reaction.

See also the New and Notable section in the same issue by J.M. Beechem, Expanding time-regimes usher-in a new era for kinetic studies. 74: 2141. (PDF)


Milne, J.S., Mayne, L., Roder, H., Wand, A.J., and Englander, S.W. Determinants of protein hydrogen exchange studied in equine cytochrome c. Protein Science, 1998. 7: 739-745.

The exchange of a large number of amide hydrogens in oxidized equine cytochrome c was measured by NMR and compared with structural parameters. Hydrogens known to exchange through local structural fluctuations and through larger unfolding reactions were separately considered. All hydrogens protected from exchange by factors greater than 103 are in defined H-bonds, and almost all H-bonded hydrogens including those at the protein surface were measured to exchange slowly. H-exchange rates do not correlate with H-bond strength (length) or crystallographic B factors. It appears that the transient structural fluctuation necessary to bring an exchangeable hydrogen into H-bonding contact with the H-exchange catalyst (OH--ion) involves a fairly large separation of the H-bond donor and acceptor, several angstroms at least, and therefore depends on the relative resistance to distortion of immediately neighboring structure. Accordingly, H-exchange by way of local fluctuational pathways tends to be very slow for hydrogens that are neighbored by tightly anchored structure and for hydrogens that are well buried. The slowing of buried hydrogens may also reflect the need for additional motions that allow solvent access once the protecting H-bond is separated, although it is noteworthy that burial in a protein like cytochrome c does not exceed 4 Å. When local fluctuational pathways are very slow, exchange can become dominated by a different category of larger, cooperative, segmental unfolding reactions reaching up to global unfolding.


Park, S-H., O'Neil, K. T., and Roder, H. An early intermediate in the folding reaction of the B1 domain of protein G contains a native-like core. Biochemistry, 1997. 36: 14277-14283.

The folding kinetics of a 57-residue IgG binding domain of streptococcal protein G has been studied under varying solvent conditions, using stopped-flow fluorescence methods. Although GB1 has been cited as an example of a protein that obeys a two-state folding mechanism, the following kinetic observations suggest the presence of an early folding intermediate. Under stabilizing conditions (low denaturant concentrations, especially in the presence of sodium sulfate), the kinetics of folding shows evidence for a major unresolved fluorescence change during the 1.5 ms dead-time of the stopped-flow experiment (burst phase). Together with some curvature in the rate profile for the single observable folding phase, this provides clear evidence for the rapid formation of compact states with native- like fluorescence for the single tryptophan at position 43. In refolding experiments at increasing denaturant concentration, the amplitude of the sub-millisecond phase decreases sharply and the corresponding slope (m-value) is only about 30% lower than that of the equilibrium unfolding curve indicative of a pre-equilibrium transition involving cooperative unfolding of an ensemble of compact intermediates. The dependence on guanidine hydrochloride concentrations of both rates and amplitudes (including the equilibrium transition) is described quantitatively by a sequential three-state mechanism, U <=> I <=> N, where an intermediate (I) in rapid equilibrium with the unfolded state (U) precedes the rate-limiting formation of the native state (N). A 66-residue fragment of GB1 with an N-terminal extension containing five apolar side chains exhibits virtually identical three-state kinetic behavior as the 57-residue fragment. This is consistent with the presence of a well shielded native-like core excluding the N-terminal tail in the early folding intermediate and argues against a mechanism involving random hydrophobic collapse, which would predict a correlation between overall hydrophobicity and stability of compact states.


Pinheiro, T. J. T., Elöve, G. A., Watts, A., and Roder, H. Structural and kinetic description of cytochrome c unfolding induced by the interaction with lipid vesicles. Biochemistry, 1997. 36: 13122-13132.

The interaction of cytochrome c with anionic lipid vesicles of DOPS induces an extensive disruption of the native structure of the protein. The kinetics of this lipid-induced unfolding process were investigated in a series of fluorescence- and absorbance-detected stopped-flow measurements. The results show that the tightly packed native structure of cytochrome c is disrupted at a rate of ~ 1.5 s-1 (independent of protein and lipid concentration), leading to the formation of a lipid-inserted denatured state (DL). Comparison with the expected rate of unfolding in solution (~ 2 x 10-3 s-1 at pH 5.0 in the absence of denaturant), suggests that the lipid environment dramatically accelerates the structural unfolding process of cytochrome c. We propose that this acceleration is in part due to the low effective pH in the vicinity of the lipid headgroups. This hypothesis was tested by comparative kinetic measurements of acid-unfolding of cytochrome c in solution. Our absorbance and fluorescence kinetic data, combined with a well-characterised mechanism for folding/unfolding of cytochrome c in solution, allow us to propose a kinetic mechanism for cytochrome c unfolding at the membrane surface. Binding of native cytochrome c in water (NW) to DOPS vesicles is driven by the electrostatic interaction between positively charged residues in the protein and the negatively charged lipid headgroups on the membrane surface. This binding step occurs within the dead time of the stopped-flow experiments (< 2 ms), where a membrane-associated native state (NS) is formed. Unfolding of NS driven by the acidic environment at the membrane surface is proposed to occur via a native-like intermediate lacking Met 80 ligation MS, as previously observed during unfolding in solution. The overall unfolding process (NS -> DL) is limited by the rate of disruption of the hydrophobic core in MS. Equilibrium spectroscopic measurements by near-IR and Soret absorbance, fluorescence and circular dichroism showed that DL has native-like helical secondary structure, but shows no evidence for specific tertiary interactions. This lipid denatured equilibrium state (DL) is clearly more extensively unfolded than the A-state in solution, but is distinct from the unfolded protein in water (UW), which has no stable secondary structure.


Colón, W., Wakem, L.P., Sherman, F., Roder, H. Identification of the predominant non-native histidine ligand in unfolded cytochrome c. Biochemistry, 1997. 36: 12535-12541.

The heme and its two axial ligands, His18 and Met80, play a central role in the folding/unfolding mechanism of cytochrome c. Because of the covalent heme attachment, His18 remains bound under typical denaturing conditions, while the more labile Met80 ligand is replaced by an alternate histidine ligand. To distinguish between the two possible nono-native histidine ligands in horse cytochrome c, variants with a His26 to Gln or His33 to Asn substitution were prepared using a yeast expression system. Protonation of the non-native histidine ligand in the GuHCl-denatured state results in a pronounced blue shift of the Soret heme absorbance band (low-spin to high-spin transition). While substitution of His26 has no effect on the apparent pKa of this transition (5.7 ± 0.05), the H33N variant exhibits a substantially higher pKa (6.1 ± 0.05), indicating that His33 is the dominant sixth heme ligand in denatured cytochrome c and that His26 (or another nitrogenous group) acts as a ligand in the absence of a histidine at position 33. The kinetics of the pH-induced ligand dissociation shows two phases which were assigned to each of the two histidine ligands on the basis of their distinct temperature dependence. Despite their nearly identical equilibrium unfolding transitions, the two histidine mutants show differences in their folding kinetics. While the kinetic behavior of H26Q cyt c is very similar to that of the wild-type, the H33N mutation leads to loss of a kinetic phase with a rate in the 2-10 s-1 range that has previously been attributed to the rate-limiting dissociation of a trapped non-native histidine, which is thus identified as His33.


Walkenhorst, W. F., Green, S. M., and Roder, H. Kinetic evidence for folding and unfolding intermediates in Staphylococcal nuclease. Biochemistry, 1997. 36: 5795-5805.

The complex kinetic behavior commonly observed in protein folding studies suggests that a heterogeneous population of molecules exists in solution and that a number of discrete steps are involved in the conversion of unfolded molecules to the fully native form. A central issue in protein folding is whether any of these kinetic events represent conformational steps important for efficient folding rather than side reactions caused by slow steps such as proline isomerization or misfolding of the polypeptide chain. In order to address this question, we used stopped-flow fluorescence techniques to characterize the kinetic mechanism of folding and unfolding for a Pro- variant of SNase in which all six proline residues were replaced by glycines or alanines. Compared to the wild-type protein, which exhibits a series of proline-dependent slow folding phases, the folding kinetics of Pro- SNase were much simpler, which made quantitative kinetic analysis possible. Despite the absence of prolines or other complicating factors, the folding kinetics still contain several phases and exhibit a complex denaturant dependence. The GuHCl dependence of the major observable folding phase and a distinct lag in the appearance of the native state provide clear evidence for an early folding intermediate. The fluorescence of Trp140 in the alpha-helical domain is insensitive to the formation of this early intermediate, which is consistent with a partially folded state with a stable beta-domain and a largely disordered alpha-helical region. A second intermediate is required to model the kinetics of unfolding for the Pro- variant, which shows evidence for a denaturant-induced change in the rate-limiting unfolding step. With the inclusion of these two intermediates, we are able to completely model the major phase(s) in both folding and unfolding across a wide range of denaturant concentrations using a sequential four-state folding mechanism. In order to model the minor slow phase observed for the Pro- mutant, a six-state scheme containing a parallel pathway originating from a distinct unfolded state was required. The properties of this alternate unfolded conformation are consistent with those expected due to the presence of a non-prolyl cis peptide bond. To test the kinetic model, we used simulations based on the six-state scheme and were able to completely reproduce the folding kinetics for Pro- SNase across a range of denaturant concentrations.


Roder, H. and Colón. Kinetic role of early intermediates in protein folding (Review) Current Opinion in Struct. Biology, 1997. 7: 15-28.

The traditional view that partly folded intermediates are important for directing a protein toward the native state has been challenged by the notion that proteins can intrinsically fold rapidly in a single step if kinetic complications due to slow conformational events are avoided. Intermediates that accumulate within the first few milliseconds of folding are, however, a common observation even for small single-domain proteins. Recent spectroscopic studies, coupled with quantitative kinetic analysis, suggest that folding is facilitated by the rapid formation of compact intermediates with some native-like structural features.


Sauder, J. M., MacKenzie, N. E. and Roder, H. Kinetic mechanism of folding and unfolding of Rhodobacter capsulatus cytochrome c2. Biochemistry, 1996. 35: 16852-16862. (PDF)

In spite of marginal sequence homology, cytochrome c2 from photosynthetic bacteria and the mitochondrial cytochromes c exhibit some striking structural similarities, including the tertiary arrangement of the three main helices. To compare the folding mechanisms for these two distantly related groups of proteins, equilibrium and kinetic measurements of the folding/unfolding reaction of cytochrome c2 from Rhodobacter capsulatus were performed as a function of guanidine hydrochloride (GuHCl) concentration in the absence and presence of a stabilizing salt, sodium sulfate. Quenching of the fluorescence of Trp67 by the heme was used as a conformational probe. Kinetic complexities due to non-native histidine ligation are avoided, since cytochrome c2 contains only one histidine, His17, which forms the axial heme ligand under native and denaturing conditions. Quantitative kinetic modeling showed that both equilibrium and kinetic results are consistent with a minimal four-state mechanism with two sequential intermediates. The observation of a large decrease in fluorescence during the 2-ms dead-time of the stopped-flow measurement (burst phase) at low GuHCl concentration, followed by a sigmoidal recovery of the initial amplitude toward the unfolding transition region, is attributed to a well-populated compact folding intermediate in rapid exchange with unfolded molecules. A nearly denaturant-independent process at low GuHCl concentrations reflects the rate-limiting conversion of a compact intermediate to the native state. At high GuHCl concentrations a process with little denaturant dependence is attributed to the rate-limiting Met96-iron deligation process during unfolding, which is supported by the kinetics of imidazole binding. The strong GuHCl-dependence of folding and unfolding rates near the midpoint of the equilibrium transition is attributed to destabilization of each intermediate and their transition states in folding and unfolding. Addition of sodium sulfate shifts the rate profile to higher denaturant concentration, which can be understood in terms of the relative stabilizing effect of the salt on partially and fully folded states.


Colón, W. and Roder, H. Kinetic intermediates in the formation of the cytochrome c molten globule. Nature Struct. Biology, 1996. 3: 1019-1025.

The relationship between molten globules and transient intermediates in protein folding has been explored by equilibrium and kinetic analysis of the compact acid-denatured A-state of cytochrome c. The chloride-induced formation of the A-state is a complex reaction with structural intermediates resembling those found under native refolding conditions, including a rapidly formed compact state and a subsequent intermediate with interacting N- and C-terminal helices. Together with mutational evidence for specific helix-helix packing interactions, this shows that the A-state is a stable analog of a late folding intermediate. The L94A mutation blocks all folding steps after the initial collapse and its equilibrium state resembles early kinetic intermediates.


Colón, W., Elöve, G.A., Wakem, L.P., Sherman, F., Roder, H. Side chain packing of the N- and C-terminal helices plays a critical role in the kinetics of cytochrome c folding. Biochemistry, 1996. 35: 5538-5549. (PDF)

The pairing of two alpha-helices at opposite ends of the chain is a highly conserved structural motif found throughout the cytochrome c family of proteins. It has previously been shown that association of the N- and C-terminal helices is a critical early event in the folding process of horse cytochrome c and is responsible for the formation of a partially folded intermediate (INC). In order to gain further insight into the structural and energetic basis of helix packing interactions and their role in folding, we prepared a series of horse cytochrome c variants in which Leu94, a critical residue at the helix contact site, was replaced by Ile, Val, or Ala. The Ile and Val substitutions resulted in minor changes in the stability of the native state, indicating that conservative mutations can be accommodated at the helix interface with only minor structural perturbations. In contrast, the L94A mutation resulted in a 3.5 kcal/mol decrease in unfolding free energy, suggesting that the smaller Ala side chain causes severe packing defects at the helix interface. The effect of these mutations on the kinetics of folding and unfolding as a function of denaturant concentration was studied by a systematic series of stopped-flow fluorescence measurements. The proteins with Leu, Ile, or Val at position 94 exhibit a major unresolved fluorescence change during the 1-ms dead time of the stopped-flow refolding measurements, while this effect is less pronounced in L94A, indicating that the rapid formation of a compact state (IC) with largely quenched Trp59 fluorescence is favored by a large hydrophobic side chain at the helix-helix interface. Despite their small effects on overall stability, the L94I and L94V mutations result in a substantial reduction in the relative amplitude of the fastest observable folding phase (formation of INC) consistent with a strong decrease in the population of INC compared to the wild-type protein. This effect is amplified in the case of the destabilizing L94A variant, which exhibits slower folding kinetics and negligible accumulation of INC. Whereas the presence of a large hydrophobic side chain at position 94 is sufficient for the stabilization of IC, the subsequent partially folded intermediate, INC, is stabilized by specific interactions that are responsible for the proper packing of the two alpha-helices.


Khorasanizadeh, S., Peters, I.D., and Roder, H. Evidence for a three-state model of protein folding from kinetic analysis of ubiquitin variants with altered core residues. Nature Structural Biology, 1996. 3(2): 193-205.

The widely held view that partially folded intermediates are important for efficient protein folding has recently been challenged by the suggestion that proteins can fold rapidly without apparent intermediates, unless they encounter misfolded conformations. We address the role of early intermediates in protein folding by exploring the effect of amino acid changes in the hydrophobic core of ubiquitin on the structure and stability of the native state, and on the kinetics of folding and unfolding. For wild-type ubiquitin and two stable variants (V26I and V26L), a folding intermediate with native-like fluorescence properties accumulates during the first few milliseconds of refolding, whereas two destabilizing mutations (V26A, V26G) show apparent two-state behavior. The denaturant dependence of the rates and amplitudes for all variants can be modeled quantitatively, using a three-state mechanism with an obligatory folding intermediate. A denaturant-independent process that limits the rate of folding at low denaturant concentrations is attributed to the conversion of the intermediate into the native state. The observation that disruptive mutations and/or addition of denaturants result in apparent two-state kinetics with slower rates is explained by the destabilization of the intermediate, which is in rapid equilibrium with the unfolded state. The kinetic effects of mutations and solvent conditions (denaturing and stabilizing) indicate that the core regions around residue 26 and the fluorescence probe (Trp45) are already formed in the intermediate and in the subsequent transition state. The model predicts that proteins showing apparent two-state kinetics, even in the absence of denaturant, may exhibit well-populated kinetic intermediates under sufficiently stabilizing conditions. This is the case for the V26A variant of ubiquitin, which shows clear evidence for an intermediate in the presence of a stabilizing salt.


Roder, H., Watching protein folding unfold. Nature Structural Biology, 1995. 2: 817-820.

Direct NMR observation of a transient folding intermediate provides new evidence for the importance of molten globule intermediates as general intermediates in protein folding [News and Views].


Zhang, Y.Z., Y. Paterson, and H. Roder, Rapid amide proton exchange rates in peptides and proteins measured by solvent quenching and two-dimensional NMR. Protein Science, 1995. 4: 804-14.

In an effort to develop a more versatile quenched hydrogen exchange method for studies of peptide conformation and protein-ligand interactions, the mechanism of amide proton exchange for model peptides in DMSO-D2O mixtures was investigated by NMR methods. As in water, H-D exchange rates in the presence of 90% or 95% DMSO exhibit characteristic acid- and base-catalyzed processes and negligible water catalysis. However, the base-catalyzed rate is suppressed by as much as four orders of magnitude in 95% DMSO. As a result, the pH at which the exchange rate goes through a minimum is shifted up by about two pH units and the minimum exchange rate is approximately 100-fold reduced relative to that in D2O. The solvent-dependent decrease in base-catalyzed exchange rates can be attributed primarily to a large increase in pKa values for the NH group, whereas solvent effects on pKW seem less important. Addition of toluene and cyclohexane resulted in improved proton NMR chemical shift dispersion. The dramatic reduction in exchange rates observed in the solvent mixture at optimal pH makes it possible to apply 2D NMR for NH exchange measurements on peptides under conditions where rates are too rapid for direct NMR analysis. To test this solvent-quenching method, melittin was exchanged in D2O (pH 3.2, 12 degrees C), aliquots were quenched by rapid freezing, lyophilized, and dissolved in quenching buffer (70% DMSO, 25% toluene, 4% D2O, 1% cyclohexane, 75 mM dichloroacetic acid) for NMR analysis. Exchange rates for 21 amide protons were measured by recording 2D NMR spectra on a series of samples quenched at different times. The results are consistent with a monomeric unfolded conformation of melittin at acidic pH. The ability to trap labile protons by solvent quenching makes it possible to extend amide protection studies to peptide ligands or labile protons on the surface of a protein involved in macromolecular interactions.


Laub, P.B., S. Khorasanizadeh, and H. Roder, Localized solution structure refinement of an F45W variant of ubiquitin using stochastic boundary molecular dynamics and NMR distance restraints. Protein Science, 1995. 4: 973-982.

The local structure within an 8-Å radius around residue 45 of a recombinant F45W variant of human ubiquitin has been determined using 67 interproton distance restraints measured by two-dimensional proton NMR. Proton chemical shift evidence indicates that structural perturbations due to the F45W mutation are minimal and limited to the immediate vicinity of the site of mutation. Simulated annealing implemented with stochastic boundary molecular dynamics was applied to refine the structure of Trp 45 and 10 neighboring residues. The stochastic boundary method allowed the entire protein to be reassembled from the refined coordinates and the outlying unrefined coordinates with little distortion at the boundary. Refinement began with four low-energy indole ring orientations of F45W-substituted wild-type (WT) ubiquitin crystal coordinates. Distance restraints were derived from mostly long-range NOE cross peaks with 51 restraints involving the Trp 45 indole ring. Tandem refinements of 64 structures were done using either (1) upper and lower bounds derived from qualitative inspection of NOE crosspeak intensities or (2) quantitative analysis of cross-peak heights using the program MARDIGRAS. Though similar to those based on qualitative restraint, structures obtained using quantitative NOE analysis were superior in terms of precision and accuracy as measured by back-calculated sixth-root R factors. The six-membered portion of the indole ring is nearly coincident with the phenyl ring of the WT and the indole NH is exposed to solvent. Accommodation of the larger ring is accompanied by small perturbations in the backbone and a 120 degrees rotation of the chi 2 dihedral angle of Leu 50.


Gochin, M. and H. Roder, Protein structure refinement based on paramagnetic NMR shifts: applications to wild-type and mutant forms of cytochrome c. Protein Science, 1995. 4: 296-305.

A new approach to NMR solution structure refinement is introduced that uses paramagnetic effects on nuclear chemical shifts as constraints in energy minimization or molecular dynamics calculations. Chemical shift differences between oxidized and reduced forms of horse cytochrome c for more than 300 protons were used as constraints to refine the structure of the wild-type protein in solution and to define the structural changes induced by a Leu 94 to Val mutation. A single round of constrained minimization, using the crystal structure as the starting point, converged to a low-energy structure with an RMS deviation between calculated and observed pseudo-contact shifts of 0.045 ppm, 7.5-fold lower than the starting structure. At the same time, the procedure provided stereospecific assignments for more than 45 pairs of methylene protons and methyl groups. Structural changes caused by the mutation were determined to a precision of better than 0.3 Å. Structure determination based on dipolar paramagnetic (pseudocontact) shifts is applicable to molecules containing anisotropic paramagnetic centers with short electronic relaxation times, including numerous naturally occurring metalloproteins, as well as proteins or nucleic acids to which a paramagnetic metal ion or ligand may be attached. The long range of paramagnetic shift effects (up to 20 Å from the iron in the case of cytochrome c) provides global structural constraints, which, in conjunction with conventional NMR distance and dihedral angle constraints, will enhance the precision of NMR solution structure determination.


Elöve, G.A., A.K. Bhuyan, and H. Roder, Kinetic mechanism of cytochrome c folding: involvement of the heme and its ligands. Biochemistry, 1994. 33: 6925-35.

The covalently attached heme and its axial ligands not only are essential for the structure and function of cytochrome c but they also play an important role in the folding process. Under typical denaturing conditions (concentrated guanidine hydrochloride or urea near pH 7), one of the axial ligands, His 18, remains bound to the oxidized heme iron, but the second ligand, Met 80, is replaced by a non-native histidine ligand (His 26 or His 33 in horse cytochrome c). Using quenched-flow and NMR methods, hydrogen exchange rates were measured for several individual amide protons in guanidine-denatured horse cytochrome c. The observation of a single highly protected (140-fold) backbone amide, that of His 18, suggests the presence of a persistent H-bond consistent with heme ligation of the His 18 side chain in the unfolded state. Heme absorbance changes induced by rapid acidification of oxidized cytochrome c in 4.5 M guanidine hydrochloride from pH 7.8 to 4.6 or below exhibit two kinetic phases with rates of 110 and 25 s-1, attributed to the dissociation of non-native histidine ligands from the heme in the unfolded state. The kinetics of folding from guanidine-denatured cytochrome c under a variety of initial and final conditions was investigated by stopped-flow methods, using tryptophan fluorescence as a conformational probe and Soret absorbance as a probe for the ligation state of the heme. A fast kinetic phase (80 s-1) accompanied by a major decrease in fluorescence and a minor absorbance change coincides with the formation of a partially folded intermediate with interacting chain termini detected in earlier pulsed NH exchange measurements [Roder, H., Elove, G. A., & Englander, S. W. (1988) Nature 335, 700]. At neutral pH, an intermediate kinetic phase (1.8 s-1) accounts for 78% of the absorbance change and 47% of the fluorescence change. In contrast, the folding kinetics at pH 5 is dominated by the fast phase, and the amplitude of the intermediate phase is reduced to approximately 10%. The pH-dependent amplitude changes show titration behavior with an apparent pK of approximately 5.7, consistent with the protonation of a single histidine residue. The intermediate phase can also be suppressed by the addition of 20 mM imidazole. Since both of these conditions interfere with histidine ligation, the intermediate kinetic phase is attributed to the presence of a non-native histidine ligand (His 26 or His 33) that can become trapped in a partially folded intermediate. In order to investigate the formation of H-bonded structure without interference from heme ligation events, quenched-flow and two-dimensional NMR methods were used to measure the time course of protection against NH exchange during the folding reaction at pH 5. In contrast to earlier results at higher pH, all amide protons had already acquired extensive protection during the fast folding phase, indicating a more cooperative structural transition. All except the N- and C-terminal amide protons exhibit a minor protection phase on the 100-ms time scale, suggesting that some preferential interaction of N- and C-terminal helices is also found at lower pH. A kinetic mechanism is presented that accounts for most of the observed structural and kinetic data on the cytochrome c folding process under various conditions. The model predicts that distinct populations of unfolded molecules with alternative axial ligands give rise to multiple parallel folding pathways, as previously observed [Elove, G.A. & Roder, H. (1991) ACS Symp. Series 470, 50].


Roder, H. and G.A. Elöve, Early stages of protein folding, in Mechanisms of Protein Folding: Frontiers in Molecular Biology, R.H. Pain, Editor. 1994, Oxford University Press: New York. p. 26-55.

Introduction, Hydrogen exchange and NMR approaches, Folding events on the millisecond time-scale, Intermediates with partially assembled tertiary structure, Late stages, Concluding remarks.


Khorasanizadeh, S., Peters, I.D., Butt, T.R., Roder, H. Folding and stability of a tryptophan-containing mutant of ubiquitin. Biochemistry, 1993. 32: 7054-63.

To provide a fluorescence probe for equilibrium and kinetic folding studies on ubiquitin, cassette mutagenesis in an Escherichia coli expression plasmid was used to replace the largely buried Phe 45 by a tryptophan. Under native conditions, the tryptophan fluorescence spectrum of this F45W mutant exhibits a blue-shifted emission maximum at 336 nm indicative of a largely solvent-shielded tryptophan environment. In contrast, the unfolded protein in 6 M guanidine hydrochloride (GuHCl) shows a 4-fold more intense emission band at 353 nm matching that of free tryptophan. The two-dimensional 1H NMR spectrum of F45W ubiquitin was assigned by comparison with published assignments of the wild type. The mutation results in only limited chemical shift changes for residues in the immediate vicinity of residue 45. The structural similarity of F45W with wild-type ubiquitin was confirmed by a preliminary analysis of the nuclear Overhauser spectrum. NMR and circular dichroism measurements of the reversible GuHCl-induced unfolding transition show that the F45W mutation lowers the stability of the folded ubiquitin structure by less than 0.4 kcal/mol. The biological activity of the mutant was found to be indistinguishable from that of wild-type in terms of its reaction with the ubiquitin activating enzyme E1 and an in vitro assay of ATP-dependent protein degradation. The kinetics of folding and unfolding of F45W ubiquitin was studied at two temperatures (8 and 25 degrees C) in a series of fluorescence-detected stopped-flow measurements over a wide range of GuHCl concentrations (0.5-6 M). The measurements at 25 degrees C are consistent with a two-state model with strongly denaturant-dependent folding and unfolding rates above about 2 M GuHCl. However, at lower denaturant concentrations, the rate of the major folding phase becomes GuHCl-independent, and up to 60% of the total fluorescence change occurs during the 2-ms dead time of the stopped-flow measurement. These observations provide clear evidence for the formation of an early folding intermediate during the first few milliseconds of refolding with a partially developed hydrophobic core involving Trp 45. The sigmoid denaturant dependence of the initial amplitude with an apparent midpoint of 1.3 M GuHCl suggests the presence of a discrete state that is destabilized at higher denaturant concentrations. In contrast, there is no evidence for an early intermediate in the folding kinetics at 8 degrees C. The destabilization of the intermediate at low temperature is consistent with a collapsed state stabilized primarily by hydrophobic interactions.


Wu, L.C., Laub, P.B., Elöve, G.A., Carey, J. Roder, H. A noncovalent peptide complex as a model for an early folding intermediate of cytochrome c. Biochemistry, 1993. 32: 10271-6.

Horse heart cytochrome c is one of a small number of proteins for which the folding pathway has been elucidated in structural detail by pulsed hydrogen exchange and NMR. Those studies indicated that a partially folded intermediate with interacting N- and C-terminal helices is formed at an early stage of folding when most of the chain is still disordered. This report describes a peptide model for this early intermediate, consisting of a noncovalent complex between a heme-containing N-terminal fragment (residues 1-38) and a synthetic peptide corresponding to the C-terminal helix (residues 87-104). Far-UV circular dichroism and proton NMR indicate that the isolated peptides are largely disordered, but when combined, they form a flexible, yet tightly bound complex with enhanced helical structure. These results emphasize the importance of interactions between marginally stable elements of secondary structure in forming tertiary subdomains in protein folding.


Jones, C.M., Henry, E.R., Hu, Y., Chan, C-K., Luck, S.D., Bhuyan, A., Roder, H., Hofrichter, J., Eaton, W.A. Fast events in protein folding initiated by nanosecond laser photolysis. PNAS, 1993. 90: 11860-4.

Initiation of protein folding by light can dramatically improve the time resolution of kinetic studies. Here we present an example of an optically triggered folding reaction by using nanosecond photodissociation of the heme-carbon monoxide complex of reduced cytochrome c. The optical trigger is based on the observation that under destabilizing conditions cytochrome c can be unfolded by preferential binding of carbon monoxide to the covalently attached heme group in the unfolded state. Photodissociation of the carbon monoxide thus triggers the folding reaction. We used time-resolved absorption spectroscopy to monitor binding at the heme. Before folding begins we observe transient binding of both nonnative and native ligands from the unfolded polypeptide on a microsecond time scale. Kinetic modeling suggests that the intramolecular binding of methionine-65 and -80 is faster than that of histidine-26 and -33, even though the histidines are closer to the heme. This optical trigger should provide a powerful method for studying chain collapse and secondary structure formation in cytochrome c without any limitations in time resolution.


Elöve, G.A., Chaffotte, A.F., Roder, H., Goldberg, M.E. Early steps in cytochrome c folding probed by time-resolved circular dichroism and fluorescence spectroscopy. Biochemistry, 1992. 31: 6876-83.

The kinetics of protein folding for horse ferricytochrome c was investigated by stopped-flow methods, using far-UV circular dichroism (CD), near-UV CD, and tryptophan fluorescence to probe the formation of secondary structure and tertiary interactions. In the far-UV region of the CD spectrum (222 nm), 44% of the total change associated with refolding occurs within the dead time of the stopped-flow experiment, indicating that a significant amount of helical secondary structure is formed in less than 4 ms. The remaining changes in the ellipticity at 222 nm occur in two kinetic phases with time constants of about 40 ms and 0.7 s, respectively. In contrast, there is no evidence for rapid changes in the ellipticity at 289 nm: an aromatic CD band, which is indicative of the formation of a tightly packed core, only begins to appear in a 400-ms step and is completed in a final 10-s phase. The fluorescence of a single tryptophan at position 59, which becomes quenched upon folding via nonradiative energy transfer to the heme group, provides complementary information on the condensation of the polypeptide chain during refolding. The fluorescence-detected stopped-flow folding kinetics of ferricytochrome c exhibits a 35% decrease in fluorescence during the dead time, suggesting that a substantial decrease in the average tryptophan-heme distance occurs on a submillisecond time scale. The subsequent fluorescence changes exhibit two prominent phases with time constants of about 20 and 300 ms, followed by a minor 5-s phase. Transient peptide CD spectra measured at different folding times (4 ms to 5 s) show no evidence for non-native elements of secondary structure at any stage of folding. Together with previous pulsed amide proton exchange data measured under identical folding conditions [Roder, Elove, & Englander (1988) Nature 335, 700-704], the results suggest that during the early stages of folding (<4 ms), a partially condensed intermediate with a fluctuating core is formed that contains a significant amount of helical secondary structure but no stable hydrogen bonds. A second intermediate, populated on the 20-100 ms time scale, exhibits stable hydrogen-bonded structure in two helical segments near the chain termini and increased compactness, but the aromatic side chains are still in a dynamic or exposed environment. Other helical segments contribute to the helical character of the far-UV CD spectrum but lack sufficiently stable hydrogen bonding to provide protection against NH exchange.


Briggs, M.S. and H. Roder, Early hydrogen-bonding events in the folding reaction of ubiquitin. PNAS, 1992. 89: 2017-21.

The formation of hydrogen-bonded structure in the folding reaction of ubiquitin, a small cytoplasmic protein with an extended beta-sheet and an alpha-helix surrounding a pronounced hydrophobic core, has been investigated by hydrogen-deuterium exchange labeling in conjunction with rapid mixing methods and two-dimensional NMR analysis. The time course of protection from exchange has been measured for 26 back-bone amide protons that form stable hydrogen bonds upon refolding and exchange slowly under native conditions. Amide protons in the beta-sheet and the alpha-helix, as well as protons involved in hydrogen bonds at the helix/sheet interface, become 80% protected in an initial 8-ms folding phase, indicating that the two elements of secondary structure form and associate in a common cooperative folding event. Somewhat slower protection rates for residues 59, 61, and 69 provide evidence for the subsequent stabilization of a surface loop. Most probes also exhibit two minor phases with time constants of about 100 ms and 10 s. Only two of the observed residues, Gln-41 and Arg-42, display significant slow folding phases, with amplitudes of 37% and 22%, respectively, which can be attributed to native-like folding intermediates containing cis peptide bonds for Pro-37 and/or Pro-38. Compared with other proteins studied by pulse labeling, including cytochrome c, ribonuclease, and barnase, the initial formation of hydrogen-bonded structure in ubiquitin occurs at a more rapid rate and slow-folding species are less prominent.


Baldwin, R.L. and H. Roder, Characterizing protein folding intermediates. Current Biology, 1991. 1: 218-220.

Exchange rates of individual amide protons in the polypeptide backbone of a protein can be measured by two-dimensional NMR. They serve to locate structure in folding intermediates.


Elöve, G.A. and H. Roder, Structure and stability of cytochrome c folding intermediates. ACS Symposium Series, 1991. 470: 50-63.

Hydrogen exchange labeling and nuclear magnetic resonance (NMR) approaches were used to elucidate the folding mechanism of horse cytochrome c (cyt c). The development of hydrogen bonded structure during refolding was observed by pulse labeling at variable refolding times, and the degree of protection from NH exchange was probed by systematic variation of the labeling pH. The results show that the folding reaction involves both sequential and parallel pathways. About 50% of the molecules follow a sequential pathway where a partially folded intermediate is formed in a 20 ms folding phase. This intermediate has two native-like helices near the chain termini, but lacks stable H-bonded structure in other parts of the molecule. Amide sites on either helix are not only protected at the same rate, but they also exhibit the same degree of protection, confirming previous evidence that association of the N- and C-terminal helices is an important early event in cyt c folding. Subsequent folding events on the 100 ms time scale involve replacement of a non-native histidine ligand by the native methionine ligand. In addition, there is evidence for a minor species of very rapidly folding molecules that form native-like structure within the mixing dead-time, as well as slow-folding forms that take several seconds to fold.


Paterson, Y., S.W. Englander, and H. Roder, An antibody binding site on cytochrome c defined by hydrogen exchange and two-dimensional NMR. Science, 1990. 249: 755-9.

The interaction of a protein antigen, horse cytochrome c (cyt c), with a monoclonal antibody has been studied by hydrogen-deuterium (H-D) exchange labeling and two-dimensional nuclear magnetic resonance (2D NMR) methods. The H-exchange rate of residues in three discontiguous regions of the cyt c polypeptide backbone was slowed by factors up to 340-fold in the antibody-antigen complex compared with free cyt c. The protected residues, 36 to 38, 59, 60, 64 to 67, 100, and 101, and their hydrogen-bond acceptors, are brought together in the three-dimensional structure to form a contiguous, largely exposed protein surface with an area of about 750 square angstroms. The interaction site determined in this way is consistent with prior epitope mapping studies and includes several residues that were not previously identified. The hydrogen exchange labeling approach can be used to map binding sites on small proteins in antibody-antigen complexes and may be applicable to protein-protein and protein-ligand interactions in general.


Jeng, M.F., Englander, S.W., Elöve, G.A., Wand, A.J., Roder, H. Structural description of acid-denatured cytochrome c by hydrogen exchange and 2D NMR [published erratum appears in Biochemistry 1991 Mar 19;30(11):2988]. [Review]. Biochemistry, 1990. 29: 10433-7.

Hydrogen exchange and two-dimensional nuclear magnetic resonance (2D NMR) techniques were used to characterize the structure of oxidized horse cytochrome c at acid pH and high ionic strength. Under these conditions, cytochrome c is known to assume a globular conformation (A state) with properties resembling those of the molten globule state described for other proteins. In order to measure the rate of hydrogen-deuterium exchange for individual backbone amide protons in the A state, samples of oxidized cytochrome c were incubated at 20 degrees C in D2O buffer (pD 2.2, 1.5 M NaCl) for time periods ranging from 2 min to 500 h. The exchange reaction was then quenched by transferring the protein to native conditions (pD 5.3). The extent of exchange for 44 amide protons trapped in the refolded protein was measured by 2D NMR spectroscopy. The results show that this approach can provide detailed information on H-bonded secondary and tertiary structure in partially folded equilibrium forms of a protein. All of the slowly exchanging amide protons in the three major helices of native cytochrome c are strongly protected from exchange at acid pH, indicating that the A state contains native-like elements of helical secondary structure. By contrast, a number of amide protons involved in irregular tertiary H-bonds of the native structure (Gly37, Arg38, Gln42, Ile57, Lys79, and Met80) are only marginally protected in the A state, indicating that these H-bonds are unstable or absent. The H-exchange results suggest that the major helices of cytochrome c and their common hydrophobic domain are largely preserved in the globular acidic form while the loop region of the native structure is flexible and partly disordered.


Roder, H., Structural characterization of protein folding intermediates by proton magnetic resonance and hydrogen exchange. Methods in Enzymology, 1989. 176: 446-73.

Introduction, Protein Folding-Unfolding Transitions at Equilibrium; Hydrogen Exchange and Protein Stability; Structural Characterization of Unfolded Proteins; Dynamics of Folding and Unfolding at Equilibrium; Characterization of Folding Pathways by Hydrogen Exchange Labeling.


Roder, H., G.A. Elöve, and S.W. Englander, Structural characterization of folding intermediates in cytochrome c by H-exchange labelling and proton NMR. Nature, 1988. 335: 700-4.

To understand the process of protein folding, it will be necessary to obtain detailed structural information on folding intermediates. This difficult problem is being studied by using hydrogen exchange and rapid mixing to label transient structural intermediates, with subsequent analysis of the proton-labelling pattern by two-dimensional nuclear magnetic resonance spectroscopy. Results for cytochrome c show that the method provides the spatial and temporal resolution necessary to monitor structure formation at many defined sites along the polypeptide chain on a timescale ranging from milliseconds to minutes.


Wand, A.J., H. Roder, and S.W. Englander, Two-dimensional 1H NMR studies of cytochrome c: hydrogen exchange in the N-terminal helix. Biochemistry, 1986. 25: 1107-14.

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.


Roder, H. and K. Wüthrich, Protein folding kinetics by combined use of rapid mixing techniques and NMR observation of individual amide protons. Proteins, 1986. 1: 34-42.

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.


Roder, H., G. Wagner, and K. Wüthrich, Individual amide proton exchange rates in thermally unfolded basic pancreatic trypsin inhibitor. Biochemistry, 1985. 24: 7407-11.

A novel experiment is described for measurements of amide proton exchange rates in proteins with a time resolution of about 1 s. A flow apparatus was used to expose protein solutions in 2H2O first to high temperature for a predetermined time period, during which 1H-2H exchange proceeded, and then to ice-water. The technique was applied for exchange studies in thermally unfolded, selectively reduced basic pancreatic trypsin inhibitor. Measurements were made by 1H nuclear magnetic resonance after the exchange was quenched by rapid cooling. Thereby, the sequence-specific resonance assignments for the folded protein could be used, which had been previously obtained. The results of this study indicate that the exchange rates in the thermally unfolded protein are close to those expected for a random chain and that the NH exchange is catalyzed by 2H+ and O2H- up to high temperature, with no significant contributions from p2H-independent catalysis. We conclude that the parameters derived by Molday et al. [Molday, R. S., Englander, S. W., & Kallen, R. G. (1972) Biochemistry 11, 150-158] from measurements with small model peptides can be used to calculate intrinsic exchange rates in unfolded proteins and thus provide a reliable reference for the interpretation of exchange rates measured under native conditions.


Roder, H., G. Wagner, and K. Wüthrich, Amide proton exchange in proteins, by EX1 kinetics: Studies of the basic pancreatic trypsin inhibitor at variable p2H and temperature. Biochemistry, 1985. 24: 7396-7407.

With the use of one-dimensional 1H nuclear magnetic resonance, two-dimensional correlated spectroscopy, and two-dimensional nuclear Overhauser enhancement spectroscopy, the exchange mechanisms for numerous individual amide protons in the basic pancreatic trypsin inhibitor (BPTI) were investigated over a wide range of p2H and temperature. Correlated exchange under an EX1 regime was observed only for the most slowly exchanging protons in the central hydrogen bonds of the antiparallel beta-sheet and only over a narrow range of temperature and p2H, i.e., above ca. 55 degrees C and between p2H 7 and 9, where the opening rates of the structure fluctuations which promote the exchange of these protons are of the order 0.1 min-1. At p2H below 7, the exchange of this most stable group of protons is uncorrelated and is governed by an EX2 mechanism. At p2H above 9, the exchange is also uncorrelated and occurs via either EX2 or EX1 processes promoted by strictly local structure fluctuations. For all other backbone amide protons in BPTI, the exchange was found to be uncorrelated and by an EX2 mechanism under all conditions of p2H and temperature where quantitative measurements could be obtained with the methods used, i.e., for kex approximately less than 5 min-1. From these observations with BPTI it can be concluded that the amide proton exchange in globular proteins is quite generally via EX2 processes, with rare exceptions for measurements with extremely stable protons at high temperature and basic p2H. This emphasizes the need for further development of suitable concepts for the structural interpretation of EX2 amide proton exchange [Wagner, G. (1983) Q. Rev. Biophys. 16, 1-57; Wagner, G., Stassinopoulou, C. I., & Wuthrich, K. (1984) Eur. J. Biochem. 145, 431-436] and for more detailed investigations of the intrinsic exchange rates for solvent-exposed amide protons in the "open" states of a protein [Roder, H., Wagner, G., & Wuthrich, K. (1985) Biochemistry (following paper in this issue)].


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