Structural mobility in human manganese superoxide dismutase

Human manganese superoxide dismutase (MnSOD) is a homotetramer of 22 kDa subunits, a dimer of dimers containing dimeric and tetrameric interfaces. We have investigated conformational mobility at these interfaces by measuring amide hydrogen/deuterium (H/D) exchange kinetics and 19F NMR spectra, both being excellent methods for analyzing local environments. Human MnSOD was prepared in which all nine tyrosine residues in each subunit are replaced with 3-fluorotyrosine. The 19F NMR spectrum of this enzyme showed five sharp resonances that have been assigned by site-specific mutagenesis by replacing each 3-fluorotyrosine with phenylalanine; four 19F resonances not observed are near the paramagnetic manganese and extensively broadened. The temperature dependence of the line widths and chemical shifts of the 19F resonances were used to estimate conformational mobility. 3-Fluorotyrosine 169 at the dimeric interface showed little conformational mobility and 3-fluorotyrosine 45 at the tetrameric interface showed much greater mobility by these measures. In complementary studies, H/D exchange mass spectrometry was used to measure backbone dynamics in human MnSOD. Using this approach, amide hydrogen exchange kinetics were measured for regions comprising 78% of the MnSOD backbone. Peptides containing Tyr45 at the tetrameric interface displayed rapid exchange of hydrogen with deuterium while peptides containing Tyr169 in the dimeric interface only displayed moderate exchange. Taken together, these studies show that residues at the dimeric interface, such as Tyr169, have significantly less conformational freedom or mobility than do residues at the tetrameric interface, such as Tyr45. This is discussed in terms of the role in catalysis of residues at the dimeric interface.     

Periodicity of amide proton exchange rates in a coiled-coil leucine zipper peptide.

The two-stranded coiled-coil motif, which includes leucine zippers, is a simple protein structure that is well suited for studies of helix-helix interactions. The interaction between helices in a coiled coil involves packing of "knobs" into "holes", as predicted by Crick in 1953 and confirmed recently by X-ray crystallography for the GCN4 leucine zipper [O'Shea, E.K., Klemm, J.D., Kim, P.S., & Alber, T. (1991) Science 254, 539]. A striking periodicity, extending over six helical turns, is observed in the rates of hydrogen-deuterium exchange for amide protons in a peptide corresponding to the leucine zipper of GCN4. Protons at the hydrophobic interface show the most protection from exchange. The NMR chemical shifts of amide protons in the helices also show a pronounced periodicity which predicts a short H-bond followed by a long H-bond every seven residues. This variation was anticipated in 1953 by Pauling and is sufficient to give rise to a local left-handed superhelical twist characteristic of coiled coils. The amide protons that lie at the base of the "hole" in the "knobs-into-holes" packing show slow amide proton exchange rates and are predicted to have short H-bond lengths. These results suggest that tertiary interactions can lead to highly localized, but substantial, differences in stability and dynamics within a secondary structure element and emphasize the dominant nature of packing interactions in determining protein structure.     

Hydrophobic sliding: a possible mechanism for drug resistance in human immunodeficiency virus type 1 protease

Hydrophobic residues outside the active site of HIV-1 protease frequently mutate in patients undergoing protease inhibitor therapy; however, the mechanism by which these mutations confer drug resistance is not understood. From analysis of molecular dynamics simulations, 19 core hydrophobic residues appear to facilitate the conformational changes that occur in HIV-1 protease. The hydrophobic core residues slide by each other, exchanging one hydrophobic van der Waal contact for another, with little energy penalty, while maintaining many structurally important hydrogen bonds. Such hydrophobic sliding may represent a general mechanism by which proteins undergo conformational changes. Mutation of these residues in HIV-1 protease would alter the packing of the hydrophobic core, affecting the conformational flexibility of the protease. Therefore these residues impact the dynamic balance between processing substrates and binding inhibitors, and thus contribute to drug resistance.     

Methyl dynamics for understanding hydrophobic core packing of dynamically different motifs of double-stranded RNA binding domain of protein kinase R

Double-stranded RNA binding domains of human protein kinase R (dsRBD-PKR) regulate distinct cellular functions and the fate of an RNA molecule in the cell. This highly homologous domains present in multiple copies in a number of species, exhibit individual and specific functional specificity. Number of NMR and X-ray crystallographic structural studies reveals that such domains take a common alpha-beta-beta-beta-alpha tertiary fold. However, the functional specificities of these domains could be due to the dynamics of the individual amino acid residues, as has been shown earlier in the case of backbone dynamics of 15N-1H of dsRNA binding motifs (dsRBMs) of human protein kinase R (PKR) (Nanduri S, Rahman F, Williams BRG, Qin J. EMBO J 2000;19:5567-5574). To further investigate if the differences in dynamics of the two dsRBMs are restricted to only backbone, or if the side-chain motions are also different to the extent of influencing their packing of the two hydrophobic cores, we have investigated the methyl group dynamics using 13C-methyl relaxation measurements. The results show that the hydrophobic core of dsRBM1 is more tightly packed than dsRBM2, and it seems to undergo less fast scale motions in the subnanosecond regime.     

Cores and pH-dependent dynamics of ferredoxin-NADP+ reductase revealed by hydrogen/deuterium exchange

NMR-detected hydrogen/deuterium (H/D) exchange of amide protons is a powerful way for investigating the residue-based conformational stability and dynamics of proteins in solution. Maize ferredoxin-NADP(+) reductase (FNR) is a relatively large protein with 314 amino acid residues, consisting of flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADP(+))-binding domains. To address the structural stability and dynamics of FNR, H/D exchange of amide protons was performed using heteronuclear NMR at pD(r) values 8.0 and 6.0, physiologically relevant conditions mimicking inside of chloroplasts. At both pD(r) values, the exchange rate varied widely depending on the residues. The profiles of protected residues revealed that the highly protected regions matched well with the hydrophobic cores suggested from the crystal structure, and that the NADP(+)-binding domain can be divided into two subdomains. The global stability of FNR obtained by H/D exchange with NMR was higher than that by chemical denaturation, indicating that H/D exchange is especially useful for analyzing the residue-based conformational stability of large proteins, for which global unfolding is mostly irreversible. Interestingly, more dynamic conformation of the C-terminal subdomain of the NADP(+)-binding domain at pD(r) 8.0, the daytime pH in chloroplasts, than at pD(r) 6.0 is likely to be involved in the increased binding of NADP(+) for elevating the activity of FNR. In light of photosynthesis, the present study provides the first structure-based relationship of dynamics with function for the FNR-type family in solution.     

Complete resonance assignment for the polypeptide backbone of interleukin 1 beta using three-dimensional heteronuclear NMR spectroscopy

The complete sequence-specific assignment of the 15N and 1H backbone resonances of the NMR spectrum of recombinant human interleukin 1 beta (153 residues, Mr = 17,400) has been obtained by using primarily 15N-1H heteronuclear three-dimensional (3D) NMR techniques in combination with 15N-1H heteronuclear and 1H homonuclear two-dimensional NMR. The fingerprint region of the spectrum was analyzed by using a combination of 3D heteronuclear 1H Hartmann-Hahn 15N-1H multiple quantum coherence (3D HOHAHA-HMQC) and 3D heteronuclear 1H nuclear Overhauser 15N-1H multiple quantum coherence (3D NOESY-HMQC) spectroscopies. We show that the problems of amide NH and C alpha H chemical shift degeneracy that are prevalent for proteins of this size are readily overcome by using the 3D heteronuclear NMR technique. A doubling of some peaks in the spectrum was found to be due to N-terminal heterogeneity of the 15N-labeled protein, corresponding to a mixture of wild-type and des-Ala-1-interleukin 1 beta. The complete list of 15N and 1H assignments is given for all the amide NH and C alpha H resonances of all non-proline residues, as well as the 1H assignments for some of the amino acid side chains. This first example of the sequence-specific assignment of a protein using heteronuclear 3D NMR provides a basis for further conformational and dynamic studies of interleukin 1 beta.     

Thermodynamic Effects of Noncoded and Coded Methionine Substitutions in Calmodulin

The methionine residues in the calcium (Ca²⁺) regulatory protein calmodulin (CaM) are structurally and functionally important. They are buried within the N- and C-domains of apo-CaM but become solvent-exposed in Ca²⁺-CaM, where they interact with numerous target proteins. Previous structural studies have shown that methionine substitutions to the noncoded amino acids selenomethionine, ethionine, or norleucine, or mutation to leucine do not impact the main chain structure of CaM. Here we used differential scanning calorimetry to show that these substitutions enhance the stability of both domains, with the largest increase in melting temperature (19-26°C) achieved with leucine or norleucine in the apo-C-domain. Nuclear magnetic resonance spectroscopy experiments also revealed the loss of a slow conformational exchange process in the Leu-substituted apo-C-domain. In addition, isothermal titration calorimetry experiments revealed considerable changes in the enthalpy and entropy of target binding to apo-CaM and Ca²⁺-CaM, but the free energy of binding was largely unaffected due to enthalpy-entropy compensation. Collectively, these results demonstrate that noncoded and coded methionine substitutions can be accommodated in CaM because of the structural plasticity of the protein. However, adjustments in side-chain packing and dynamics lead to significant differences in protein stability and the thermodynamics of target binding.     

‘Lollipop’-shaped helical structure of a hybrid antimicrobial peptide of temporin B-lipopolysaccharide binding motif and mapping cationic residues in antibacterial activity

BackgroundTemporins are attractive templates for the development of antibiotics. However, many temporins are inactive against Gram-negative bacteria. Previously, we demonstrated conjugation of a lipopolysaccharide binding motif peptide to temporins yielded hybrid non-haemolytic AMPs that killed several Gram-negative bacteria.MethodsWe carried out a systematic Ala replacement of individual cationic and polar amino acid residues of LG21, a hybrid AMP consisted of temporin B (TB) and LPS binding motif. These Ala containing analogs of LG21 were examined for antibacterial activity, cell membrane permeabilization and liposome leakage assays using optical spectroscopic methods. Atomic resolution structure of LG21 was determined in zwitterionic dodecyl phosphocholine (DPC) micelles by NMR spectroscopy.ResultsCationic residues in the LPS binding motif of LG21 were critical for bactericidal and membrane permeabilization. Detergent bound structure of LG21 revealed helical conformation containing extensive sidechain/sidechain packing including cation/π interactions in the LPS binding motif. The helical structure of LG21 resembled a ‘lollipop’ like shape that was sustained by a compacted bulky aromatic/cationic head with a comparatively thinner ‘stick’ at the N-terminal region. The ‘head’ of the structure could be localized into micelle-water interfacial region whereas the ‘stick’ region may be inserted into the hydrophobic core of micelle.ConclusionsThe LPS binding motif of LG21 played dominant roles in broad spectrum activity and the 3-D structure provided plausible mechanistic insights for permeabilization of bacterial membrane.General significanceHybrid AMPs containing LPS binding motif could be useful for the structure based development of broad spectrum antibiotics.     

Polar residues in the protein core of Escherichia coli thioredoxin are important for fold specificity

Most globular proteins contain a core of hydrophobic residues that are inaccessible to solvent in the folded state. In general, polar residues in the core are thermodynamically unfavorable except when they are able to form intramolecular hydrogen bonds. Compared to hydrophobic interactions, polar interactions are more directional in character and may aid in fold specificity. In a survey of 263 globular protein structures, we found a strong positive correlation between the number of polar residues at core positions and protein size. To probe the importance of buried polar residues, we experimentally tested the effects of hydrophobic mutations at the five polar core residues in Escherichia coli thioredoxin. Proteins with single hydrophobic mutations (D26I, C32A, C35A, T66L, and T77V) all have cooperative unfolding transitions like the wild type (wt), as determined by chemical denaturation. Relative to wt, D26I is more stable while the other point mutants are less stable. The combined 5-fold mutant protein (IAALV) is less stable than wt and has an unfolding transition that is substantially less cooperative than that of wt. NMR spectra as well as amide deuterium exchange indicate that IAALV is likely sampling a number of low-energy structures in the folded state, suggesting that polar residues in the core are important for specifying a well-folded native structure.     

Multinuclear magnetic resonance studies of the 2Fe.2S* ferredoxin from Anabaena species strain PCC 7120. 3. Detection and characterization of hyperfine-shifted nitrogen-15 and hydrogen-1 resonances of the oxidized form

All the nitrogen signals from the amino acid side chains and 80 of the total of 98 backbone nitrogen signals of the oxidized form of the 2Fe.2S* ferredoxin from Anabaena sp. strain PCC 7120 were assigned by means of a series of heteronuclear two-dimensional experiments [Oh, B.-H. Mooberry, E. S., & Markley, J. L. (1990) Biochemistry (second paper of three in this issue )]. Two additional nitrogen signals were observed in the one-dimensional 15N NMR spectrum and classified as backbone amide resonances from residues whose proton resonances experience paramagnetic broadening. The one-dimensional 15N NMR spectrum shows nine resonances that are hyperfine shifted and broadened. From this inventory of diamagnetic nitrogen signals and the available X-ray coordinates of a related ferredoxin [Tsukihara, T., Fukuyama, K., Nakamura, M., Katsube, Y., Tanaka, N., Kakudo, M., Wada, K., Hase, T., & Matsubara, H. (1981) J. Biochem. 90, 1763-1773], the resolved hyperfine-shifted 15N peaks were attributed to backbone amide nitrogens of the nine amino acids that share electrons with the 2Fe.2S* center or to backbone amide nitrogens of two other amino acids that are close to the 2Fe.2S* center. The seven 15N signals that are missing and unaccounted for probably are buried under the envelope of amide signals. 1H NMR signals from all the amide protons directly bonded to the seven missing and nine hyperfine-shifted nitrogens were too broad to be resolved in conventional 2D NMR spectra.(ABSTRACT TRUNCATED AT 250 WORDS)     

Probing the nature of the blue-shifted intermediate of photoactive yellow protein in solution by NMR: hydrogen-deuterium exchange data and pH studies

The nature of the pB intermediate of photoactive yellow protein (PYP) from Ectothiorhodospira halophila has been probed by NMR. pH-dependent changes in the NMR spectrum of the dark state of PYP are shown to closely mimic exchange broadening effects observed previously in the NMR spectrum of the pB intermediate in solution. Amide H-D exchange data show that while pB retains a solid protected core, two regions become significantly less protected than the dark state. The amide exchange data help to rationalize why the conformational exchange process affects the N-terminal 28-residue segment of the protein, which is not close to the site of chromophore rearrangement. At very low pH (pH 1.7), the dark state NMR spectrum displays approximately 30 very sharp signals, which are characteristic of a portion of the molecule becoming unfolded. Similarities between the dark state spectra at pH approximately 3.2 and the spectra of pB suggest a model for pB in solution where the protein exists in an equilibrium between a well-ordered state and a state in which a region is unfolded. Such a two-state model accounts for the exchange phenomena observed in the NMR spectra of pB, and the hydrophobic exposure and lability inferred from thermodynamic data. It is likely that in the crystalline environment the ordered form of pB is strongly favored.     

Tyrosine replacing tryptophan as an anchor in GWALP peptides

Synthetic model peptides have proven useful for examining fundamental peptide-lipid interactions. A frequently employed peptide design consists of a hydrophobic core of Leu-Ala residues with polar or aromatic amino acids flanking each side at the interfacial positions, which serve to "anchor" a specific transmembrane orientation. For example, WALP family peptides (acetyl-GWW(LA)(n)LWWA-[ethanol]amide), anchored by four Trp residues, have received particular attention in both experimental and theoretical studies. A recent modification proved successful in reducing the number of Trp anchors to only one near each end of the peptide. The resulting GWALP23 (acetyl-GGALW(5)(LA)(6)LW(19)LAGA-[ethanol]amide) displays reduced dynamics and greater sensitivity to lipid-peptide hydrophobic mismatch than traditional WALP peptides. We have further modified GWALP23 to incorporate a single tyrosine, replacing W(5) with Y(5). The resulting peptide, Y(5)GWALP23 (acetyl-GGALY(5)(LA)(6)LW(19)LAGA-amide), has a single Trp residue that is sensitive to fluorescence experiments. By incorporating specific (2)H and (15)N labels in the core sequence of Y(5)GWALP23, we were able to use solid-state NMR spectroscopy to examine the peptide orientation in hydrated lipid bilayer membranes. The peptide orients well in membranes and gives well-defined (2)H quadrupolar splittings and (15)N/(1)H dipolar couplings throughout the core helical sequence between the aromatic residues. The substitution of Y(5) for W(5) has remarkably little influence on the tilt or dynamics of GWALP23 in bilayer membranes of the phospholipids DOPC, DMPC, or DLPC. A second analogue of the peptide with one Trp and two Tyr anchors, Y(4,5)GWALP23, is generally less responsive to the bilayer thickness and exhibits lower apparent tilt angles with evidence of more extensive dynamics. In general, the peptide behavior with multiple Tyr anchors appears to be quite similar to the situation when multiple Trp anchors are present, as in the original WALP series of model peptides.     

A dynamic bundle of four adjacent hydrophobic segments in the denatured state of staphylococcal nuclease.

In an earlier study of the denatured state of staphylococcal nuclease (Wang Y, Shortle D, 1995, Biochemistry 34:15895-15905), we reported evidence of a three-strand antiparallel beta sheet that persists at high urea concentrations and is stabilized by a local "non-native" interaction with four large hydrophobic residues. Because the amide proton resonances for all of the involved residues are severely broadened, this unusual structure is not amenable to conventional NMR analysis and must be studied by indirect methods. In this report, we present data that confirm the important role of interactions involving four hydrophobic residues (Leu 36, Leu 37, Leu 38, and Val 39) in stabilizing the structure formed by the chain segments corresponding to beta 1-beta 2-beta 3-h, interactions that are not present in the native state. Glycine substitutions for each of these large hydrophobic residues destabilizes or disrupts this beta structure, as assessed by HN line sharpening and changes in the CD spectrum. The 13C resonances of the carbonyl carbon for several of the residues in this structure indicate conformational dynamics that respond in a complex way to addition of urea or changes in sequence. Studies of hydrogen exchange kinetics in a closely related variant of staphylococcal nuclease demonstrate the absence of the stable hydrogen bonding between the strands expected for a native-like three-strand beta sheet. Instead, the data are more consistent with the three beta strand segments plus the four adjacent hydrophobic residues forming a dynamic, aligned array or bundle held together by hydrophobic interactions.     

Conformational studies of human Des-Trp1,Nle12-minigastrin in water-trifluoroethanol mixtures by 1H NMR and circular dichroism

The 1H NMR spectrum of the title peptide, H-Leu-(Glu)5-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2, in 90% H2O/10% D2O was assigned by two-dimensional methods, and the displacement of the proton resonances upon addition of 2,2,2-trifluoroethanol (TFE) was followed. This permitted the assignment of the spectrum in 90% TFE/10% D2O. While the water conformation of the minigastrin analogue is random, the CD spectrum indicates that an ordered structure is present in TFE. Variable-temperature NMR data in this medium show that six amide protons have low temperature coefficients, two of the five Glu's, Trp, Nle, Asp, and Phe. These results were interpreted in terms of an alpha-helical stretch comprising the Leu and the five Glu residues and a 3(10)-helix initiated by a beta-turn at the sequence -Ala-Tyr-Gly-Trp-. Both CD and NMR data at different solvent compositions show two regions of conformational change, between 20 and 25% water and above 60% water.     

High-resolution 1H NMR study of the solution structure of delta-hemolysin

The 26-residue toxin from Staphylococcus aureus, delta-hemolysin, is thought to act by traversing the plasma membrane. The structure of this peptide, in methanol solution, has been investigated by using high-resolution NMR in combination with molecular dynamics calculations. The 1H NMR spectrum has been completely assigned, and it is shown that residues 2-20 form a relatively stable helix while the residues at the C-terminal end appear to be more flexible. The structures were calculated only from nuclear Overhauser effect data and standard bond lengths. It is shown that the results are consistent with 3JNH-alpha CH coupling constants and amide hydrogen exchange rates.     

Paramagnetic relaxation enhancement‐assisted structural characterization of a partially disordered conformation of ubiquitin

Nuclear magnetic resonance (NMR) is a powerful tool to study three‐dimensional structures as well as protein conformational fluctuations in solution, but it is compromised by increases in peak widths and missing signals. We previously reported that ubiquitin has two folded conformations, N₁ and N₂ and plus another folded conformation, I, in which some amide group signals of residues 33–41 almost disappeared above 3 kbar at pH 4.5 and 273 K. Thus, well‐converged structural models could not be obtained for this region owing to the absence of distance restraints. Here, we reexamine the problem using the ubiquitin Q41N variant as a model for this locally disordered conformation, I. We demonstrate that the variant shows pressure‐induced loss of backbone amide group signals at residues 28, 33, 36, and 39–41 like the wild‐type, with a similar but smaller effect on CαH and CβH signals. In order to characterize this I structure, we measured paramagnetic relaxation enhancement (PRE) under high pressure to obtain distance restraints, and calculated the structure assisted by Bayesian inference. We conclude that the more disordered I conformation observed at pH 4.0, 278 K, and 2.5 kbar largely retained the N₂ conformation, although the amide groups at residues 33–41 have more heterogeneous conformations and more contact with water, which differ from the N₁ and N₂ states. The PRE‐assisted strategy has the potential to improve structural characterization of proteins that lack NMR signals, especially for relatively more open and hydrated protein conformations.     

Investigating the dynamic properties of the transmembrane segment of phospholamban incorporated into phospholipid bilayers utilizing 2H and 15N solid-state NMR spectroscopy

(2)H and (15)N solid-state NMR spectroscopic techniques were used to investigate the membrane composition, orientation, and side-chain dynamics of the transmembrane segment of phospholamban (TM-PLB), a sarcoplasmic Ca(2+)-regulator protein. (2)H NMR spectra of (2)H-labeled leucine (deuterated at one terminal methyl group) incorporated at different sites (CD(3)-Leu28, CD(3)-Leu39, and CD(3)-Leu51) along the TM-PLB peptide exhibited line shapes characteristic of either methyl group reorientation about the C(gamma)-C(delta) bond axis or by additional librational motion about the C(alpha)-C(beta) and C(beta)-C(gamma) bond axes. The (2)H NMR line shapes of all CD(3)-labeled leucines are very similar below 0 degrees C, indicating that all of the residues are located inside the lipid bilayer. At higher temperatures, all three labeled leucine residues undergo rapid reorientation about the C(alpha)-C(beta), C(beta)-C(gamma), and C(gamma)-C(delta) bond axes as indicated by (2)H line-shape simulations and reduced quadrupolar splittings. At all of the temperatures studied, the (2)H NMR spectra indicated that the Leu51 side chain has less motion than Leu39 or Leu28, which is attributed to its incorporation in the pentameric PLB leucine zipper motif. The (15)N powder spectra of Leu39 and Leu42 residues indicated no backbone motion, while Leu28 exhibited slight backbone motion. The chemical-shift anisotropy tensor values for (15)N-labeled Leu TM-PLB were sigma(11) = 50.5 ppm, sigma(22) = 80.5 ppm, and sigma(33) = 229 ppm within +/-3 ppm experimental error. The (15)N chemical-shift value from the mechanically aligned spectrum of (15)N-labeled Leu39 PLB in DOPC/DOPE phospholipid bilayers was 220 ppm and is characteristic of a TM peptide that is nearly parallel with the bilayer normal.     

The interaction of beta(2)-glycoprotein I domain V with chaperonin GroEL: the similarity with the domain V and membrane interaction

To clarify the mechanism of interaction between chaperonin GroEL and substrate proteins, we studied the conformational changes; of the fifth domain of human beta(2)-glycoprotein I upon binding to GroEL. The fifth domain has a large flexible loop, containing several hydrophobic residues surrounded by positively charged residues, which has been proposed to be responsible for the binding of beta(2)-glycoprotein I to negatively charged phospholipid membranes. The reduction by dithiothreitol of the three intramolecular disulfide bonds of the fifth domain was accelerated in the presence of stoichiometric amounts of GroEL, indicating that the fifth domain was destabilized upon interaction with GroEL. To clarify the GroEL-induced destabilization at the atomic level, we performed H/(2)H exchange of amide protons using heteronuclear NMR spectroscopy. The presence of GroEL promoted the H/(2)H exchange of most of the protected amide protons, suggesting that, although the flexible loop of the fifth domain is likely to be responsible for the initiation of binding to GroEL, the interaction with GroEL destabilizes the overall conformation of the fifth domain.     

Conformational dynamics of yeast calmodulin in the Ca(2+)-bound state probed using NMR relaxation dispersion

Most calmodulin (CaM) in apo and Ca(2+)-bound states show a dumb-bell-like structure, involving the N- and C-terminal domains, connected with a flexible linker. However, Ca(2+)-bound yeast calmodulin (yCaM) takes on a unique globular structure; the target-binding site of this protein is autoinhibited. We applied NMR relaxation dispersion experiments to yCaM in the Ca(2+)-bound state. The amide (15)N and (1)H(N) relaxation dispersion profiles indicated the presence of conformational dynamics for specific residues at the interface between the N- and C-terminal domains. We conclude that these conformational dynamics were derived from the mobility of the C-terminal domain.