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

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

Cooperativity of a protein folding reaction probed at multiple chain positions by real-time 2D NMR spectroscopy

The refolding reaction of S54G/P55N ribonuclease T1 is a two-step process, where fast formation of a partly folded intermediate is followed by the slow reaction to the native state, limited by a trans --> cis isomerization of Pro39. The hydrodynamic radius of this kinetic folding intermediate was determined by real-time diffusion NMR spectroscopy. Its folding to the native state was monitored by a series of 128 very fast 2D (15)N-HMQC spectra, to observe the kinetics of 66 individual backbone amide probes. We find that the intermediate is as compact as the native protein with many native chemical shifts. All 66 analyzed amide probes follow the rate-limiting prolyl isomerization, which indicates that this cooperative refolding reaction is fully synchronized. The stability of the folding intermediate was determined from the protection factors of 45 amide protons derived from a competition between refolding and H/D exchange. The intermediate has already gained 40% of the Gibbs free energy of refolding with many protected amides in not-yet-native regions.     

A globular protein with slower amide proton exchange from an alpha helix than from antiparallel beta sheets

In proteinase inhibitor IIA from bull seminal plasma, which is a small globular protein with 57 amino acid residues, measurements of individual amide proton exchange rates by two-dimensional correlated 1H NMR spectroscopy (COSY) showed that the exchange was slowest for some hydrogen bonded amide groups in an alpha-helix. This contrasts with all other proteins which were so far studied in detail, where the slowest exchange rates were observed for hydrogen bonded amide protons in antiparallel beta-sheets.     

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

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

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

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

Hydrogen exchange in native and alcohol forms of ubiquitin.

Ubiquitin adopts a non-native folded structure in 60% methanol solution at low pH. Two-dimensional nuclear magnetic resonance (2D NMR) was used to measure the hydrogen-exchange rates of backbone amide protons of ubiquitin in both native and methanol forms, and to characterize the structure of ubiquitin in the methanol state. Protection factors (the ratios of experimentally determined exchange rates to the rates calculated for an unfolded polypeptide) for protons in the native form of ubiquitin range from less than 10 to greater than 10(5). Most of the protons that are protected from exchange are located in regions of hydrogen-bonded secondary structure. The most strongly protected backbone amide protons are those of residues comprising the hydrophobic core. Hydrogen exchange from ubiquitin in methanol solution was too rapid to measure directly by 2D NMR, so a labeling scheme was employed, in which exchange with solvent occurred while the protein was in methanol solution. Exchange was quenched by dilution with aqueous buffer after the desired labeling time, and proton occupancies were measured by 1H NMR of the native form of the protein. Protection factors for protons in the methanol form of ubiquitin range from 2.6 to 42, with all protected protons located in hydrogen-bonded structure in the native form. Again, the most strongly protected protons are those of residues in the hydrophobic core. Comparison of the patterns of the hydrogen-exchange rates in the native and methanol forms indicates that almost all of the native secondary structure persists in the methanol form, but that it is almost uniformly destabilized by 4-6 kcal/mol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stabilization of native protein fold by intein-mediated covalent cyclization

A mutant version of the N-terminal domain of Escherichia coli DnaB helicase was used as a model system to assess the stabilization against unfolding gained by covalent cyclization. Cyclization was achieved in vivo by formation of an amide bond between the N and C termini with the help of a split mini-intein. Linear and circular proteins were constructed to be identical in amino acid sequence. Mutagenesis of Phe102 to Glu rendered the protein monomeric even at high concentration. A difference in free energy of unfolding, DeltaDeltaG, between circular and linear protein of 2.3(+/-0.5) kcal mol(-1) was measured at 10 degrees C by circular dichroism. A theoretical estimate of the difference in conformational entropy of linear and circular random chains in a three-dimensional cubic lattice model predicted DeltaDeltaG=2.3 kcal mol(-1), suggesting that stabilization by protein cyclization is driven by the reduced conformational entropy of the unfolded state. Amide-proton exchange rates measured by NMR spectroscopy and mass spectrometry showed a uniform, approximately tenfold decrease of the exchange rates of the most slowly exchanging amide protons, demonstrating that cyclization globally decreases the unfolding rate of the protein. The amide proton exchange was found to follow EX1 kinetics at near-neutral pH, in agreement with an unusually slow refolding rate of less than 4 min(-1) measured by stopped-flow circular dichroism. The linear and circular proteins differed more in their unfolding than in their folding rates. Global unfolding of the N-terminal domain of E.coli DnaB is thus promoted strongly by spatial separation of the N and C termini, whereas their proximity is much less important for folding.     

Backbone dynamics of a model membrane protein: measurement of individual amide hydrogen-exchange rates in detergent-solubilized M13 coat protein using 13C NMR hydrogen/deuterium isotope shifts

Hydrogen-exchange rates have been measured for individual assigned amide protons in M13 coat protein, a 50-residue integral membrane protein, using a 13C nuclear magnetic resonance (NMR) equilibrium isotope shift technique. The locations of the more rapidly exchanging amides have been determined. In D2O solutions, a peptide carbonyl resonance undergoes a small upfield isotope shift (0.08-0.09 ppm) from its position in H2O solutions; in 1:1 H2O/D2O mixtures, the carbonyl line shape is determined by the exchange rate at the adjacent nitrogen atom. M13 coat protein was labeled biosynthetically with 13C at the peptide carbonyls of alanine, glycine, phenylalanine, proline, and lysine, and the exchange rates of 12 assigned amide protons in the hydrophilic regions were measured as a function of pH by using the isotope shift method. This equilibrium technique is sensitive to the more rapidly exchanging protons which are difficult to measure by classical exchange-out experiments. In proteins, structural factors, notably H bonding, can decrease the exchange rate of an amide proton by many orders of magnitude from that observed in the freely exposed amides of model peptides such as poly(DL-alanine). With corrections for sequence-related inductive effects [Molday, R. S., Englander, S. W., & Kallen, R. G. (1972) Biochemistry 11, 150-158], the retardation of amide exchange in sodium dodecyl sulfate solubilized coat protein has been calculated with respect to poly(DL-alanine). The most rapidly exchanging protons, which are retarded very little or not at all, are shown to occur at the N- and C-termini of the molecule.(ABSTRACT TRUNCATED AT 250 WORDS)     

A water-lipid interface induces a highly dynamic folded state in apocytochrome c and cytochrome c, which may represent a common folding intermediate.

In this study, we have used CD and NMR techniques to investigate the secondary structure of (apo-) cytochrome c both in solution and when associated with micelles. In aqueous solution, the holoprotein cytochrome c is tightly folded at secondary and tertiary levels and differs strongly from its random-coiled precursor. However, in the presence of 12-PN/12-Pglycol (9:1) micelles, we observed a remarkable resemblance between the CD spectra of these partially helical proteins. The water-lipid interface induces a secondary folding of apocytochrome c, whereas cytochrome c is suggested to partially lose its tertiary structure. The exchange of all amide protons and, using deuterium-labeled proteins, of all amide deuterons with the solvent was monitored by NMR. A rapid exchange rate was observed, indicating that these folding states are highly dynamic. Saturation-transfer NMR of micelle-associated apocytochrome c showed that the exchange takes place at the (sub-) second time scale. The holoprotein in the presence of micelles was found to have two distinct exchange rates: (1) a fast rate, comparable to that found for the micelle-associated precursor and 4.5 times slower than that of the random-coiled apocytochrome c, and (2) a slow rate which is 75 times slower than the precursor in solution. Urea denaturation studies showed the micelle-bound proteins to have a low helix stability, which explains the inability of the lipid-induced secondary structure to prevent its labile protons from rapid exchange. The uniqueness of this lipid-induced highly dynamic folding state of (apo-) cytochrome c is demonstrated by comparison with amphiphilic polypeptides like melittin, and its implications for membrane translocation and functioning are discussed.     

Amide proton hydrogen exchange rates for sperm whale myoglobin obtained from 15N-1H NMR spectra

The hydrogen exchange behavior of exchangeable protons in proteins can provide important information for understanding the principles of protein structure and function. The positions and exchange rates of the slowly-exchanging amide protons in sperm whale myoglobin have been mapped using 15N-1H NMR spectroscopy. The slowest-exchanging amide protons are those that are hydrogen bonded in the longest helices, including members of the B, E, and H helices. Significant protection factors were observed also in the A, C, and G helices, and for a few residues in the D and F helices. Knowledge of the identity of slowly-exchanging amide protons forms the basis for the extensive quench-flow kinetic folding experiments that have been performed for myoglobin, and gives insights into the tertiary interactions and dynamics in the protein.     

Investigation of the structural stability of the human acidic fibroblast growth factor by hydrogen-deuterium exchange

The conformational stability of the human acidic fibroblast growth factor (hFGF-1) is investigated using amide proton exchange and temperature-dependent chemical shifts, monitored by two-dimensional NMR spectroscopy. The change in free energy of unfolding (DeltaG(u)) of hFGF-1 is estimated to be 5.00 +/- 0.09 kcal.mol(-)(1). Amide proton-exchange rates of 74 residues (in hFGF-1) have been unambiguously measured, and the exchange process occurs predominately according to the conditions of the EX2 limit. The exchange rates of the fast-exchanging amide protons exposed to the solvent have been measured using the clean SEA-HSQC technique. The amide proton protection factor and temperature coefficient estimates show reasonably good correlation. Residues in beta-strands II and VI appear to constitute the stability core of the protein. Among the 12 beta-strands constituting the beta-barrel architecture of hFGF-1, beta-strand XI, located in the heparin binding domain, exhibits the lowest average protection factor value. Amide protons involved in the putative folding nucleation site in hFGF-1, identified by quench-flow NMR studies, do not represent the slow-exchanging core. Residues in portions of hFGF-1 experiencing high conformational flexibility mostly correspond to those involved in receptor recognition and binding.     

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

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

The 1H nuclear-magnetic-resonance spectra of Neurotoxin I and cardiotoxin Vii4 from Naja mossambica mossambica.

Two toxins from the venom of Naja mossambica mossambica, neurotoxin I and cardiotoxin VII4, were investigated in aqueous solution by high-resolution 1H nuclear magnetic resonance (NMR) techniques at 360 MHz. The spectral characterization of the proteins included determination of the number of slowly exchanging amide protons which can be observed in 2H2O solution, measurement of the amide proton chemical shifts and exchange rates, characterization of the aromatic spin systems and the internal mobilities of aromatic rings, and studies of the pH dependence of the NMR spectra. For numerous resonances of labile and non-labile protons quite outstanding pH titration shifts were observed. It is suggested that these NMR parameters provide a useful basis for comparative structural studies of different proteins in the large group of homologous snake toxins. As a first application the NMR data presently available in the literature on neurotoxin II from Naja naja oxiana, toxin alpha from Naja nigricollis and erabutoxin a and b from Laticauda semifasciata have been used to compare these three proteins with neurotoxin I from Naja mossambica mossambica. This preliminary comparative study provides evidence that the same type of spatial structure prevails for these four homologous neurotoxins and that the folding of the backbone corresponds quite closely to that observed in the crystal structure of erabutoxin b. A second application is the comparison of cardiotoxin VII4 from Naja mossambica mossambica with the neurotoxins. The experimental data indicate that the folding of the polypeptide backbone is closely similar, but that the cardiotoxin molecule is markedly more flexible than the neurotoxins.     

Unfolding and refolding of bovine beta-lactoglobulin monitored by hydrogen exchange measurements.

Bovine beta-lactoglobulin (beta-LG) is a widely studied protein belonging to the lipocalin family, whose structural characterisation has been reported by X-ray crystallography and NMR studies at physiological and acidic pH, respectively. Bovine beta-LG consists of nine antiparallel beta-sheets and a terminal alpha-helix segment. The beta-sheets form a calyx structure with a hydrophobic buried cluster conferring stability to the protein while a hydrophobic surface patch provides stabilising interactions between the barrel and the flanking terminal helix. Here, the stability and the folding properties of bovine beta-LG in the presence of a chemical denaturant is probed. The analysis of the NMR spectra recorded in aqueous solution with increasing amounts of urea revealed that the intensities of the backbone cross-peaks decrease upon increasing urea concentration, while their secondary shifts do not change significantly on going from 0 to 5 M urea, thus suggesting the presence of slow exchange between native and unfolded protein. Hydrogen exchange measurements at different urea concentrations were performed in order to evaluate the exchange rates of individual backbone amide protons. The opening reactions that determine protein exchange can be computed for the most slowly exchanging hydrogen atoms, and the measured exchange rates and the corresponding free energies can be expressed in terms of the equilibrium energetic for the global transition and the local fluctuations. Most of the residues converge to define a common isotherm identifying a unique cooperative folding unit, encompassing all the strands, except strand betaI, and the terminal region of the helix. The amides that do not join the same global unfolding isotherm are characterised by low DeltaGH20op and especially by low m values, indicating that they are already substantially exposed in the native state. A two-state unfolding model N <==> U is therefore proposed for this rather big protein, in agreement with CD data. Renaturation studies show that the unfolding mechanism is reversible up to 6 M urea and suggest a similar unfolding and refolding pathway. Present results are discussed in light of the hypothesis of an alpha-->beta transition proposed for bovine beta-LG refolding.     

Investigation of the structural stability of cardiotoxin analogue III from the Taiwan cobra by hydrogen-deuterium exchange kinetics.

The conformational stability of a small ( approximately 7 kDa), all beta-sheet protein, cardiotoxin analogue III (CTX III), from the venom of the Taiwan cobra has been investigated by hydrogen-deuterium (H/D) exchange using two-dimensional NMR spectroscopy. The H/D exchange kinetics of backbone amide protons in CTX III has been monitored at pD 3.6 and 6.6 (at 25 degrees C), for over 5000 h. Examination of H/D exchange kinetics in the protein showed that a number of slowly exchanging residues are in the hydrophobic core of the protein. The average protection factor of the amide protons of residues belonging to the triple-stranded beta-sheet domain is about 20 times greater than that of those in the double-stranded beta-sheet segment. The residues in the C-terminal tail of the molecule, though structureless, have been found to exhibit significant protection against H/D exchange. Comparison of the quenched-flow H/D exchange data on CTX III with those obtained in the present study reveals that the most slowly exchanging portion constitutes the folding core of the protein.     

Amide-proton exchange studies by two-dimensional correlated 1H NMR in two chemically modified analogs of the basic pancreatic trypsin inhibitor

The backbone amide proton exchange with the solvent was investigated in 2H2O solutions of the basic pancreatic trypsin inhibitor and two chemical modifications thereof, which were obtained by transamination of the N-terminus and by cleavage of the disulfide bond 14-38, respectively. The three proteins have nearly identical conformations, but the stability with respect to thermal denaturation is markedly different. Exchange rates for a large number of individually assigned amide protons located both in central and peripheral parts of the protein structures were measured by two-dimensional correlated spectroscopy (COSY). From analysis of the individual proton exchange rates in the three proteins at different temperatures, an interplay of global and local structure fluctuations was characterized, which promote hydrogen exchange in distinct regions of the molecules. The exchange of particular amide protons may be governed by different motional processes at different temperatures. As a general trend, global fluctuations involving breakage of numerous hydrophilic secondary bonds appear to be dominant at higher temperatures, whereas at lower temperatures the influence of local fluctuations in hydrophobic regions of the protein structures is also clearly noticeable.     

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

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

NMR spectroscopy of hydroxyl protons in aqueous solutions of peptides and proteins

Summary Hydroxyl groups of serine and threonine, and to some extent also tyrosine are usually located on or near the surface of proteins. NMR observations of the hydroxyl protons is therefore of interest to support investigations of the protein surface in solution, and knowledge of the hydroxyl NMR lines is indispensable as a reference for studies of protein hydration in solution. In this paper, solvent suppression schemes recently developed for observation of hydration water resonances were used to observe hydroxyl protons of serine, threonine and tyrosine in aqueous solutions of small model peptides and the protein basic pancreatic trypsin inhibitor (BPTI). The chemical shifts of the hydroxyl protons of serine and threonine were found to be between 5.4 and 6.2 ppm, with random-coil shifts at 4°C of 5.92 ppm and 5.88 ppm, respectively, and those of tyrosine between 9.6 and 10.1 ppm, with a random-coil shift of 9.78 ppm. Since these spectral regions are virtually free of other polypeptide¹H NMR signals, cross peaks with the hydroxyl protons are usually well separated even in homonuclear two-dimensional¹H NMR spectra. To illustrate the practical use of hydroxyl proton NMR in polypeptides, the conformations of the side-chain hydroxyl groups in BPTI were characterized by measurements of nuclear Overhauser effects and scalar coupling constants involving the hydroxyl protons. In addition, hydroxyl proton exchange rates were measured as a function of pH, where simple first-order rate processes were observed for both acid- and base-catalysed exchange of all but one of the hydroxyl-bearing residues in BPTI. For the conformations of the individual Ser, Thr and Tyr side chains characterized in the solution structure with the use of hydroxyl proton NMR, both exact coincidence and significant differences relative to the corresponding BPTI crystal structure data were observed.[/p]     

Hydrogen exchange kinetics in a membrane protein determined by 15N NMR spectroscopy: use of the INEPT experiment to follow individual amides in detergent-solubilized M13 coat protein

The coat protein of the filamentous coliphage M13 is a 50-residue polypeptide which spans the inner membrane of the Escherichia coli host upon infection. Amide hydrogen exchange kinetics have been used to probe the structure and dynamics of M13 coat protein which has been solubilized in sodium dodecyl sulfate (SDS) micelles. In a previous 1H nuclear magnetic resonance (NMR) study [O'Neil, J. D. J., & Sykes, B. D. (1988) Biochemistry 27, 2753-2762], multiple exponential analysis of the unresolved amide proton envelope revealed the existence of two slow "kinetic sets" containing a total of about 30 protons. The slower set (15-20 amides) originates from the hydrophobic membrane-spanning region and exchanges at least 10(5)-fold slower than the unstructured, non-H-bonded model polypeptide poly(DL-alanine). Herein we use 15N NMR spectroscopy of biosynthetically labeled coat protein to follow individual, assigned, slowly exchanging amides in or near the hydrophobic segment. The INEPT (insensitive nucleus enhancement by polarization transfer) experiment [Morris, G. A., & Freeman, R. (1979) J. Am. Chem. Soc. 101, 760-762] can be used to transfer magnetization to the 15N nucleus from a coupled proton; when 15N-labeled protonated protein is dissolved in 2H2O, the INEPT signal disappears with time as the amide protons are replaced by solvent deuterons. Amide hydrogen exchange is catalyzed by both H+ and OH- ions. Base catalysis is significantly more effective, resulting in a characteristic minimum rate in model peptides at pH approximately equal to 3. Rate versus pH profiles have been obtained by using the INEPT experiment for the amides of leucine-14, leucine-41, tyrosine-21, tyrosine-24, and valines-29, -30, -31, and -33 in M13 coat protein. The valine residues exchange most slowly and at very similar rates, showing an apparent 10(6)-fold retardation over poly(DL-alanine). A substantial basic shift in the pH of the minimum rate (up to 1.5 pH units) was also observed for some residues. Possible reasons for the shift include accumulation of catalytic H+ ions at the negatively charged micelle surface or destabilization of the negatively charged transition state of the base-catalyzed reaction by either charge or hydrophobic effects within the micelle. The time-dependent exchange-out experiment is suitable for slow exchange rates (kex), i.e., less than (1-2) x 10(-4) s-1.(ABSTRACT TRUNCATED AT 400 WORDS)