Chemical Conformation of the Essential Glutamate Site of the c-Ring within Thermophilic Bacillus FoF1-ATP Synthase Determined by Solid-State NMR Based on its Isolated c-Ring Structure.

Proton translocation through the membrane-embedded Fo component of F-type ATP synthase (FoF1) is facilitated by the rotation of the Fo c-subunit ring (c-ring), carrying protons at essential acidic amino acid residues. Cryo-electron microscopy (Cryo-EM) structures of FoF1 suggest a unique proton translocation mechanism. To elucidate it based on the chemical conformation of the essential acidic residues of the c-ring in FoF1, we determined the structure of the isolated thermophilic Bacillus Fo (tFo) c-ring, consisting of 10 subunits, in membranes by solid-state NMR. This structure contains a distinct proton-locking conformation, wherein Asn23 (cN23) CγO and Glu56 (cE56) CδOH form a hydrogen bond in a closed form. We introduced stereo-array-isotope-labeled (SAIL) Glu and Asn into the tFoc-ring to clarify the chemical conformation of these residues in tFoF1-ATP synthase (tFoF1). Two well-separated 13C signals could be detected for cN23 and cE56 in a 505 kDa membrane protein complex, respectively, thereby suggesting the presence of two distinct chemical conformations. Based on the signal intensity and structure of the tFoc-ring and tFoF1, six pairs of cN23 and cE56 surrounded by membrane lipids take the closed form, whereas the other four in the a-c interface employ the deprotonated open form at a proportion of 87%. This indicates that the a-c interface is highly hydrophilic. The pKa values of the four cE56 residues in the a-c interface were estimated from the cN23 signal intensity in the open and closed forms and distribution of polar residues around each cE56. The results favor a rotation of the c-ring for ATP synthesis.

Evolution and diversification of the plant gibberellin receptor GID1.

The plant gibberellin (GA) receptor GID1 shows sequence similarity to carboxylesterase (CXE). Here, we report the molecular evolution of GID1 from establishment to functionally diverse forms in eudicots. By introducing 18 mutagenized rice GID1s into a rice gid1 null mutant, we identified the amino acids crucial for GID1 activity in planta. We focused on two amino acids facing the C2/C3 positions of ent-gibberellane, not shared by lycophytes and euphyllophytes, and found that adjustment of these residues resulted in increased GID1 affinity toward GA4, new acceptance of GA1 and GA3 carrying C13-OH as bioactive ligands, and elimination of inactive GAs. These residues rendered the GA perception system more sophisticated. We conducted phylogenetic analysis of 169 GID1s from 66 plant species and found that, unlike other taxa, nearly all eudicots contain two types of GID1, named A- and B-type. Certain B-type GID1s showed a unique evolutionary characteristic of significantly higher nonsynonymous-to-synonymous divergence in the region determining GA4 affinity. Furthermore, these B-type GID1s were preferentially expressed in the roots of Arabidopsis, soybean, and lettuce and might be involved in root elongation without shoot elongation for adaptive growth under low-temperature stress. Based on these observations, we discuss the establishment and adaption of GID1s during plant evolution.

Pressure dependence of side chain 1H and 15N-chemical shifts in the model peptides Ac-Gly-Gly-Xxx-Ala-NH2.

For interpreting the pressure induced shifts of resonance lines of folded as well as unfolded proteins the availability of data from well-defined model systems is indispensable. Here, we report the pressure dependence of 1H and 15N chemical shifts of the side chain atoms in the protected tetrapeptides Ac-Gly-Gly-Xxx-Ala-NH2 (Xxx is one of the 20 canonical amino acids) measured at 800 MHz proton frequency. As observed earlier for other nuclei the chemical shifts of the side chain nuclei have a nonlinear dependence on pressure in the range from 0.1 to 200 MPa. The pressure response is described by a second degree polynomial with the pressure coefficients B1 and B2 that are dependent on the atom type and type of amino acid studied. A number of resonances could be assigned stereospecifically including the 1H and 15N resonances of the guanidine group of arginine. In addition, stereoselectively isotope labeled SAIL amino acids were used to support the stereochemical assignments. The random-coil pressure coefficients are also dependent on the neighbor in the sequence as an analysis of the data shows. For Hα and HN correction factors for different amino acids were derived. In addition, a simple correction of compression effects in thermodynamic analysis of structural transitions in proteins was derived on the basis of random-coil pressure coefficients.

Aromatic Ring Dynamics, Thermal Activation, and Transient Conformations of a 468 kDa Enzyme by Specific 1H-13C Labeling and Fast Magic-Angle Spinning NMR.

Aromatic residues are located at structurally important sites of many proteins. Probing their interactions and dynamics can provide important functional insight but is challenging in large proteins. Here, we introduce approaches to characterize the dynamics of phenylalanine residues using 1H-detected fast magic-angle spinning (MAS) NMR combined with a tailored isotope-labeling scheme. Our approach yields isolated two-spin systems that are ideally suited for artifact-free dynamics measurements, and allows probing motions effectively without molecular weight limitations. The application to the TET2 enzyme assembly of ∼0.5 MDa size, the currently largest protein assigned by MAS NMR, provides insights into motions occurring on a wide range of time scales (picoseconds to milliseconds). We quantitatively probe ring-flip motions and show the temperature dependence by MAS NMR measurements down to 100 K. Interestingly, favorable line widths are observed down to 100 K, with potential implications for DNP NMR. Furthermore, we report the first 13C R1ρ MAS NMR relaxation-dispersion measurements and detect structural excursions occurring on a microsecond time scale in the entry pore to the catalytic chamber and at a trimer interface that was proposed as the exit pore. We show that the labeling scheme with deuteration at ca. 50 kHz MAS provides superior resolution compared to 100 kHz MAS experiments with protonated, uniformly 13C-labeled samples.

Perspective: next generation isotope-aided methods for protein NMR spectroscopy.

In this perspective, we describe our efforts to innovate the current isotope-aided NMR methodology to investigate biologically important large proteins and protein complexes, for which only limited structural information could be obtained by conventional NMR approaches. At the present time, it is widely believed that only backbone amide and methyl signals are amenable for investigating such difficult targets. Therefore, our primary mission is to disseminate our novel knowledge within the biological NMR community; specifically, that any type of NMR signals other than methyl and amide groups can be obtained, even for quite large proteins, by optimizing the transverse relaxation properties by isotope labeling methods. The idea of "TROSY by isotope labeling" has been cultivated through our endeavors aiming to improve the original stereo-array isotope labeling (SAIL) method (Kainosho et al., Nature 440:52-57, 2006). The SAIL TROSY methods subsequently culminated in the successful observations of individual NMR signals for the side-chain aliphatic and aromatic 13CH groups in large proteins, as exemplified by the 82 kDa single domain protein, malate synthase G. Meanwhile, the expected role of NMR spectroscopy in the emerging integrative structural biology has been rapidly shifting, from structure determination to the acquisition of biologically relevant structural dynamics, which are poorly accessible by X-ray crystallography or cryo-electron microscopy. Therefore, the newly accessible NMR probes, in addition to the methyl and amide signals, will open up a new horizon for investigating difficult protein targets, such as membrane proteins and supramolecular complexes, by NMR spectroscopy. We briefly introduce our latest results, showing that the protons attached to 12C-atoms give profoundly narrow 1H-NMR signals even for large proteins, by isolating them from the other protons using the selective deuteration. The direct 1H observation methods exhibit the highest sensitivities, as compared to heteronuclear multidimensional spectroscopy, in which the 1H-signals are acquired via the spin-coupled 13C- and/or 15N-nuclei. Although the selective deuteration method was launched a half century ago, as the first milestone in the following prosperous history of isotope-aided NMR methods, our results strongly imply that the low-dimensional 1H-direct observation NMR methods should be revitalized in the coming era, featuring ultrahigh-field spectrometers beyond 1 GHz.

Pressure dependence of side chain 13C chemical shifts in model peptides Ac-Gly-Gly-Xxx-Ala-NH2.

For evaluating the pressure responses of folded as well as intrinsically unfolded proteins detectable by NMR spectroscopy the availability of data from well-defined model systems is indispensable. In this work we report the pressure dependence of 13C chemical shifts of the side chain atoms in the protected tetrapeptides Ac-Gly-Gly-Xxx-Ala-NH2 (Xxx, one of the 20 canonical amino acids). Contrary to expectation the chemical shifts of a number of nuclei have a nonlinear dependence on pressure in the range from 0.1 to 200 MPa. The size of the polynomial pressure coefficients B 1 and B 2 is dependent on the type of atom and amino acid studied. For HN, N and Cα the first order pressure coefficient B 1 is also correlated to the chemical shift at atmospheric pressure. The first and second order pressure coefficients of a given type of carbon atom show significant linear correlations suggesting that the NMR observable pressure effects in the different amino acids have at least partly the same physical cause. In line with this observation the magnitude of the second order coefficients of nuclei being direct neighbors in the chemical structure also are weakly correlated. The downfield shifts of the methyl resonances suggest that gauche conformers of the side chains are not preferred with pressure. The valine and leucine methyl groups in the model peptides were assigned using stereospecifically 13C enriched amino acids with the pro-R carbons downfield shifted relative to the pro-S carbons.

(13)C-NMR studies on disulfide bond isomerization in bovine pancreatic trypsin inhibitor (BPTI).

Conformational isomerization of disulfide bonds is associated with the dynamics and thus the functional aspects of proteins. However, our understanding of the isomerization is limited by experimental difficulties in probing it. We explored the disulfide conformational isomerization of the Cys14-Cys38 disulfide bond in bovine pancreatic trypsin inhibitor (BPTI), by performing an NMR line-shape analysis of its Cys carbon peaks. In this approach, 1D (13)C spectra were recorded at small temperature intervals for BPTI samples selectively labeled with site-specifically (13)C-enriched Cys, and the recorded peaks were displayed in the order of the temperature after the spectral scales were normalized to a carbon peak. Over the profile of the line-shape, exchange broadening that altered with temperature was manifested for the carbon peaks of Cys14 and Cys38. The Cys14-Cys38 disulfide bond reportedly exists in equilibrium between a high-populated (M) and two low-populated states (m c14 and m c38). Consistent with the three-site exchange model, biphasic exchange broadening arising from the two processes was observed for the peak of the Cys14 α-carbon. As the exchange broadening is maximized when the exchange rate equals the chemical shift difference in Hz between equilibrating sites, semi-quantitative information that was useful for establishing conditions for (13)C relaxation dispersion experiments was obtained through the carbon line-shape profile. With respect to the m c38 isomerization, the (1)H-(13)C signals at the ß-position of the minor state were resolved from the major peaks and detected by exchange experiments at a low temperature.

Highly efficient residue-selective labeling with isotope-labeled Ile, Leu, and Val using a new auxotrophic E. coli strain.

We recently developed a practical protocol for preparing proteins bearing stereo-selectively (13)C-methyl labeled leucines and valines, instead of the commonly used (13)C-methyl labeled precursors for these amino acids, by E. coli cellular expression. Using this protocol, proteins with any combinations of isotope-labeled or unlabeled Leu and Val residues were prepared, including some that could not be prepared by the precursor methods. However, there is still room for improvement in the labeling efficiencies for Val residues, using the methods with labeled precursors or Val itself. This is due to the fact that the biosynthesis of Val could not be sufficiently suppressed, even by the addition of large amounts of Val or its precursors. In this study, we completely solved this problem by using a mutant strain derived from E. coli BL21(DE3), in which the metabolic pathways depending on two enzymes, dihydroxy acid dehydratase and ß-isopropylmalate dehydrogenase, are completely aborted by deleting the ilvD and leuB genes, which respectively encode these enzymes. The ΔilvD E. coli mutant terminates the conversion from α,ß-dihydroxyisovalerate to α-ketoisovalerate, and the conversion from α,ß-dihydroxy-α-methylvalerate to α-keto-ß-methylvalerate, which produce the preceding precursors for Val and Ile, respectively. By the further deletion of the leuB gene, the conversion from Val to Leu was also fully terminated. Taking advantage of the double-deletion mutant, ΔilvDΔleuB E. coli BL21(DE3), an efficient and residue-selective labeling method with various isotope-labeled Ile, Leu, and Val residues was established.

Differential Large-Amplitude Breathing Motions in the Interface of FKBP12-Drug Complexes.

The tight complexes FKBP12 forms with immunosuppressive drugs, such as FK506 and rapamycin, are frequently used as models for developing approaches to structure-based drug design. Although the interfaces between FKBP12 and these ligands are well-defined structurally and are almost identical in the X-ray crystallographic structures of various complexes, our nuclear magnetic resonance studies have revealed the existence of substantial large-amplitude motions in the FKBP12-ligand interfaces that depend on the nature of the ligand. We have monitored these motions by measuring the rates of Tyr and Phe aromatic ring flips, and hydroxyl proton exchange for residues clustered within the FKBP12-ligand interface. The results show that the rates of hydroxyl proton exchange and ring flipping for Tyr26 are much slower in the FK506 complex than in the rapamycin complex, whereas the rates of ring flipping for Phe48 and Phe99 are significantly faster in the FK506 complex than in the rapamycin complex. The apparent rate differences observed for the interfacial aromatic residues in the two complexes confirm that these dynamic processes occur without ligand dissociation. We tentatively attribute the differential interface dynamics for these complexes to a single hydrogen bond between the ζ-hydrogen of Phe46 and the C32 carbonyl oxygen of rapamycin, which is not present in the KF506 complex. This newly identified Phe46 ζ-hydrogen bond in the rapamycin complex imposes motional restriction on the surrounding hydrophobic cluster and subsequently regulates the dynamics within the protein-ligand interface. Such information concerning large-amplitude dynamics at drug-target interfaces has the potential to provide novel clues for drug design.

Molecular bases of multimodal regulation of a fungal transient receptor potential (TRP) channel.

Multimodal activation by various stimuli is a fundamental characteristic of TRP channels. We identified a fungal TRP channel, TRPGz, exhibiting activation by hyperosmolarity, temperature increase, cytosolic Ca(2+) elevation, membrane potential, and H2O2 application, and thus it is expected to represent a prototypic multimodal TRP channel. TRPGz possesses a cytosolic C-terminal domain (CTD), primarily composed of intrinsically disordered regions with some regulatory modules, a putative coiled-coil region and a basic residue cluster. The CTD oligomerization mediated by the coiled-coil region is required for the hyperosmotic and temperature increase activations but not for the tetrameric channel formation or other activation modalities. In contrast, the basic cluster is responsible for general channel inhibition, by binding to phosphatidylinositol phosphates. The crystal structure of the presumed coiled-coil region revealed a tetrameric assembly in an offset spiral rather than a canonical coiled-coil. This structure underlies the observed moderate oligomerization affinity enabling the dynamic assembly and disassembly of the CTD during channel functions, which are compatible with the multimodal regulation mediated by each functional module.

Expression and purification of a GRAS domain of SLR1, the rice DELLA protein.

GRAS proteins belong to a plant specific protein family that participates in diverse and important functions in growth and development. GRAS proteins are typically composed of a variable N-terminal domain and highly conserved C-terminal GRAS domain. Despite the importance of the GRAS domain, little biochemical or structural analyses have been reported, mainly due to difficulties with purification of sufficient quality and quantity of protein. This study is focused on one of the most extensively studied GRAS proteins, the rice DELLA protein (SLR1), which is known to be involved in gibberellin (GA) signaling. Using a baculovirus-insect cell expression system we have achieved overproduction and purification of full-length SLR1. Limited proteolysis of the full-length SLR1 indicated that a region including the entire GRAS domain (SLR1(206-625)) is protease resistant. Based on those results, we have constructed an expression and purification system of the GRAS domain (SLR1(206-625)) in Escherichia coli. Several physicochemical assays have indicated that the folded structure of the GRAS domain is rich in secondary structural elements and that alanine substitutions for six cysteine residues improves protein folding without impairing function. Furthermore, by NMR spectroscopy we have observed direct interaction between the purified GRAS domain and the GA receptor GID1. Taken together, our purified preparation of the GRAS domain of SLR1 is suitable for further structural and functional studies that will contribute to precise understanding of the plant regulation mechanism through DELLA and GRAS proteins.

Structural analysis of ATP bound to the F1-ATPase ß-subunit monomer by solid-state NMR- insight into the hydrolysis mechanism in F1.

ATP-hydrolysis-associated conformational change of the ß-subunit during the rotation of F1-ATPase (F1) has been discussed using cryo-electron microscopy (cryo-EM). Since it is worthwhile to further investigate the conformation of ATP at the catalytic subunit through an alternative approach, the structure of ATP bound to the F1ß-subunit monomer (ß) was analyzed by solid-state NMR. The adenosine conformation of ATP-ß was similar to that of ATP analog in F1 crystal structures. 31P chemical shift analysis showed that the Pα and Pß conformations of ATP-ß are gauche-trans and trans-trans, respectively. The triphosphate chain is more extended in ATP-ß than in ATP analog in F1 crystals. This appears to be in the state just before ATP hydrolysis. Furthermore, the ATP-ß conformation is known to be more closed than the closed form in F1 crystal structures. In view of the cryo-EM results, ATP-ß would be a model of the most closed ß-subunit with ATP ready for hydrolysis in the hydrolysis stroke of the F1 rotation.

Differential isotope-labeling for Leu and Val residues in a protein by E. coli cellular expression using stereo-specifically methyl labeled amino acids.

The (1)H-(13)C HMQC signals of the (13)CH3 moieties of Ile, Leu, and Val residues, in an otherwise deuterated background, exhibit narrow line-widths, and thus are useful for investigating the structures and dynamics of larger proteins. This approach, named methyl TROSY, is economical as compared to laborious methods using chemically synthesized site- and stereo-specifically isotope-labeled amino acids, such as stereo-array isotope labeling amino acids, since moderately priced, commercially available isotope-labeled α-keto acid precursors can be used to prepare the necessary protein samples. The Ile δ1-methyls can be selectively labeled, using isotope-labeled α-ketobutyrates as precursors. However, it is still difficult to prepare a residue-selectively Leu and Val labeled protein, since these residues share a common biosynthetic intermediate, α-ketoisovalerate. Another hindering drawback in using the α-ketoisovalerate precursor is the lack of stereo-selectivity for Leu and Val methyls. Here we present a differential labeling method for Leu and Val residues, using four kinds of stereo-specifically (13)CH3-labeled [U-(2)H;(15)N]-leucine and -valine, which can be efficiently incorporated into a protein using Escherichia coli cellular expression. The method allows the differential labeling of Leu and Val residues with any combination of stereo-specifically isotope-labeled prochiral methyls. Since relatively small amounts of labeled leucine and valine are required to prepare the NMR samples; i.e., 2 and 10 mg/100 mL of culture for leucine and valine, respectively, with sufficient isotope incorporation efficiency, this approach will be a good alternative to the precursor methods. The feasibility of the method is demonstrated for 82 kDa malate synthase G.

Changes in the hydrophobic network of the FliGMC domain induce rotational switching of the flagellar motor.

The FliG protein plays a pivotal role in switching the rotational direction of the flagellar motor between clockwise and counterclockwise. Although we previously showed that mutations in the Gly-Gly linker of FliG induce a defect in switching rotational direction, the detailed molecular mechanism was not elucidated. Here, we studied the structural changes in the FliG fragment containing the middle and C-terminal regions, named FliGMC, and the switch-defective FliGMC-G215A, using nuclear magnetic resonance (NMR) and molecular dynamics simulations. NMR analysis revealed multiple conformations of FliGMC, and the exchange process between these conformations was suppressed by the G215A residue substitution. Furthermore, changes in the intradomain orientation of FliG were induced by changes in hydrophobic interaction networks throughout FliG. Our finding applies to FliG in a ring complex in the flagellar basal body, and clarifies the switching mechanism of the flagellar motor.

Conformational analysis by quantitative NOE measurements of the ß-proton pairs across individual disulfide bonds in proteins.

NOEs between the ß-protons of cysteine residues across disulfide bonds in proteins provide direct information on the connectivities and conformations of these important cross-links, which are otherwise difficult to investigate. With conventional [U-(13)C, (15)N]-proteins, however, fast spin diffusion processes mediated by strong dipolar interactions between geminal ß-protons prohibit the quantitative measurements and thus the analyses of long-range NOEs across disulfide bonds. We describe a robust approach for alleviating such difficulties, by using proteins selectively labeled with an equimolar mixture of (2R, 3S)-[ß-(13)C; α,ß-(2)H(2)] Cys and (2R, 3R)-[ß-(13)C; α,ß-(2)H(2)] Cys, but otherwise fully deuterated. Since either one of the prochiral methylene protons, namely ß2 (proS) or ß3 (proR), is always replaced with a deuteron and no other protons remain in proteins prepared by this labeling scheme, all four of the expected NOEs for the ß-protons across disulfide bonds could be measured without any spin diffusion interference, even with long mixing times. Therefore, the NOEs for the ß2 and ß3 pairs across each of the disulfide bonds could be observed at high sensitivity, even though they are 25% of the theoretical maximum for each pair. With the NOE information, the disulfide bond connectivities can be unambiguously established for proteins with multiple disulfide bonds. In addition, the conformations around disulfide bonds, namely χ(2) and χ(3), can be determined based on the precise proton distances of the four ß-proton pairs, by quantitative measurements of the NOEs across the disulfide bonds. The feasibility of this method is demonstrated for bovine pancreatic trypsin inhibitor, which has three disulfide bonds.

Optimal isotope labelling for NMR protein structure determinations.

Nuclear-magnetic-resonance spectroscopy can determine the three-dimensional structure of proteins in solution. However, its potential has been limited by the difficulty of interpreting NMR spectra in the presence of broadened and overlapping resonance lines and low signal-to-noise ratios. Here we present stereo-array isotope labelling (SAIL), a technique that can overcome many of these problems by applying a complete stereospecific and regiospecific pattern of stable isotopes that is optimal with regard to the quality and information content of the resulting NMR spectra. SAIL uses exclusively chemically and enzymatically synthesized amino acids for cell-free protein expression. We demonstrate for the 17-kDa protein calmodulin and the 41-kDa maltodextrin-binding protein that SAIL offers sharpened lines, spectral simplification without loss of information, and the ability to rapidly collect the structural restraints required to solve a high-quality solution structure for proteins twice as large as commonly solved by NMR. It thus makes a large class of proteins newly accessible to detailed solution structure determination.

Cell-free protein synthesis using E. coli cell extract for NMR studies.

The use of cell-free protein production systems for producing isotope labeled proteins generates new opportunities to perform unprecedented NMR studies. As compared with conventional cellular expression systems, the scrambling and dilution of amino acids are highly suppressed in the cell-free reaction, allowing the production of proteins with a wide variety of residue and site-specific isotope labeling patterns. In this chapter, the procedure for cell-free protein synthesis for NMR studies, using an E. coli extract, is introduced.

Stereo-array isotope labeling method for studying protein structure and dynamics.

The stereo-array isotope labeling (SAIL) method utilizes proteins with isotope labeling patterns optimized with regard to an intended NMR study. The SAIL proteins are prepared by incorporating chemically synthesized amino acids into target proteins, using a cell-free protein synthesis system or a cellular expression system. Over the past decade, the SAIL method has been facilitating a wide variety of new investigations, including high-resolution structure determinations of large proteins and investigations of protein dynamics. In this chapter, the applications of SAIL-related approaches are introduced.

SAIL--stereo-array isotope labeling.

Optimal stereospecific and regiospecific labeling of proteins with stable isotopes enhances the nuclear magnetic resonance (NMR) method for the determination of the three-dimensional protein structures in solution. Stereo-array isotope labeling (SAIL) offers sharpened lines, spectral simplification without loss of information and the ability to rapidly collect and automatically evaluate the structural restraints required to solve a high-quality solution structure for proteins up to twice as large as before. This review gives an overview of stable isotope labeling methods for NMR spectroscopy with proteins and provides an in-depth treatment of the SAIL technology.

Hydrogen exchange study on the hydroxyl groups of serine and threonine residues in proteins and structure refinement using NOE restraints with polar side-chain groups.

We recently developed new NMR methods for monitoring the hydrogen exchange rates of tyrosine hydroxyl (Tyr-OH) and cysteine sulfhydryl (Cys-SH) groups in proteins. These methods facilitate the identification of slowly exchanging polar side-chain protons in proteins, which serve as sources of NOE restraints for protein structure refinement. Here, we have extended the methods for monitoring the hydrogen exchange rates of the OH groups of serine (Ser) and threonine (Thr) residues in an 18.2 kDa protein, EPPIb, and thus demonstrated the usefulness of NOE restraints with slowly exchanging OH protons for refining the protein structure. The slowly exchanging Ser/Thr-OH groups were readily identified by monitoring the (13)C(ß)-NMR signals in an H(2)O/D(2)O (1:1) mixture, for the protein containing Ser/Thr residues with (13)C, (2)H-double labels at their ß carbons. Under these circumstances, the OH groups exist in equilibrium between the protonated and deuterated isotopomers, and the (13)C(ß) peaks of the two species are resolved when their exchange rate is slower than the time scale of the isotope shift effect. In the case of EPPIb dissolved in 50 mM sodium phosphate buffer (pH 7.5) at 40 °C, one Ser and four Thr residues were found to have slowly exchanging hydroxyl groups (k(ex) < ~40 s(-1)). With the information for the slowly exchanging Ser/Thr-OH groups in hand, we could collect additional NOE restraints for EPPIb, thereby making a unique and important contribution toward defining the spatial positions of the OH protons, and thus the hydrogen-bonding acceptor atoms.