The structure and dynamics of rat apo-cellular retinol-binding protein II in solution: comparison with the X-ray structure

The structure and dynamics of rat apo-cellular retinol binding protein II (apo-CRBP II) in solution has been determined by multidimensional NMR analysis of uniformly enriched recombinant rat 13C, 15N-apo-CRBP II and 15N-apo-CRBP II. The final ensemble of 24 NMR structures has been calculated from 3274 conformational restraints or 24.4 restraints/residue. The average root-mean-square deviation of the backbone atoms for the final 24 structures relative to their mean structure is 1.06 A. Although the average solution structure is very similar to the crystal structure, it differs at the putative entrance to the binding cavity, which is formed by the helix-turn-helix motif, the betaC-betaD turn and the betaE-betaF turn. The mean coordinates of the main-chain atoms of amino acid residues 28-38 are displaced in the solution structure relative to the crystal structure. The side-chain of F58, located on the betaC-betaD turn, is reoriented such that it interacts with L37 and no longer blocks entry into the ligand-binding pocket. Residues 28-35, which form the second helix of the helix-turn-helix motif in the crystal structure, do not exhibit a helical conformation in the solution structure. The solution structure of apo-CRBP II exhibits discrete regions of backbone disorder which are most pronounced at residues 28-32, 37-38 and 73-76 in the betaE-betaF turn as evaluated by the consensus chemical shift index, the root-mean-square deviation, amide 1H exchange rates and 15N relaxation studies. These studies indicate that fluctuations in protein conformation occur on the microseconds to ms time-scale in these regions of the protein. Some of these exchange processes can be directly observed in the three-dimensional 15N-resolved NOESY spectrum. These results suggest that in solution, apo-CRBP II undergoes conformational changes on the microseconds to ms time-scale which result in increased access to the binding cavity.     

Staphylococcal nuclease: sequential assignments and solution structure

Sequential assignments are reported for backbone 15N and 1H of nearly all residues of staphylococcal nuclease (Nase) complexed with thymidine 3',5'-diphosphate and Ca2+. Because of the relatively large size of the Nase ternary complex, Mr 18K, the crucial element of our assignment strategy was the use of isotope-edited two-dimensional NMR spectra, particularly 15N-edited nuclear Overhauser enhancement spectroscopy (NOESY), 15N-edited J-correlated spectroscopy (COSY), and 1H/15N or 1H/13C heteronuclear multiple quantum shift correlation spectroscopy (HMQC). These experiments, together with the more conventional NOESY, COSY, and homonuclear Hartmann-Hahn spectra of natural abundance or deuteriated samples, yielded backbone assignments of 127 of the 136 residues in the structured part of the protein. Using the NOESY data, we identified three helical domains and several beta-sheets which were in close correspondence with secondary structure identified in the crystal structure. Moreover, many long-range NOESY connectivities were identified that were in agreement with distances derived from the crystal structure. The region of the sequence in the neighborhood of residue 50 appears to be more flexible and disordered in solution than in the crystal. Very slowly exchanging amide protons are those found to be hydrogen bonded in the crystal structure; however, even hydrogen-bonded amides located within similar types of regular secondary structures, e.g., alpha-helices, exchange with greatly different rates.     

1H, 13C, and 15N resonance assignments of Fusarium solani pisi cutinase and preliminary features of the structure in solution.

Essentially complete (96%) sequence-specific assignments were made for the backbone and side-chain 1H, 13C, and 15N resonances of Fusarium solani pisi cutinase, produced as a 214-residue heterologous protein in Escherichia coli, using heteronuclear NMR techniques. Three structural features were noticed during the assignment. (1) The secondary structure in solution corresponds mostly with the structure from X-ray diffraction, suggesting that both structures are globally similar. (2) The HN of Ala32 has a strongly upfield-shifted resonance at 3.97 ppm, indicative of an amide-aromatic hydrogen bond to the indole ring of Trp69 that stabilizes the N-terminal side of the parallel beta-sheet. (3) The NMR data suggest that the residues constituting the oxyanion hole are quite mobile in the free enzyme in solution, in contrast to the existence of a preformed oxyanion hole as observed in the crystal structure. Apparently, cutinase forms its oxyanion hole upon binding of the substrate like true lipases.     

Spatial structure of cro-repressor in a solution. II. Effect of ionized groups

The technique of 1H NMR spectroscopy and absorption UV spectroscopy were used to study the ionization of the tyrosine phenol cycles and the effect of ionizable groups on the chemical shifts of signals from protons in the side chains of several amino acid residues. The microenvironment of these residues was established by analysing the titration curves. The mutual orientation of two functionally important adjacent alpha-helical protein regions was determined in solution. The signals from methionine residues belonging to different regions of the secondary structure were assigned in the NMR spectrum. The results indicate that the spatial structure of the repressor is similar in solution an in the crystal. They confirm the model proposed for the cro repressor interaction with DNA and based on the data of X-ray diffraction analysis.     

Monomeric solution structure of the prototypical 'C' chemokine lymphotactin

Lymphotactin, the sole identified member of the C class of chemokines, specifically attracts T lymphocytes and natural killer cells. This 93-residue protein lacks 2 of the 4 conserved cysteine residues characteristic of the other 3 classes of chemokines and possesses an extended carboxyl terminus, which is required for chemotactic activity. We have determined the three-dimensional solution structure of recombinant human lymphotactin by NMR spectroscopy. Under the conditions used for the structure determination, lymphotactin was predominantly monomeric; however, pulsed field gradient NMR self-diffusion measurements and analytical ultracentrifugation revealed evidence of dimer formation. Sequence-specific chemical shift assignments were determined through analysis of two- and three-dimensional NMR spectra of (15)N- and (13)C/(15)N-enriched protein samples. Input for the torsion angle dynamics calculations used in determining the structure included 1258 unique NOE-derived distance constraints and 60 dihedral angle constraints obtained from chemical-shift-based searching of a protein conformational database. The ensemble of 20 structures chosen to represent the structure had backbone and heavy atom rms deviations of 0.46 +/- 0.11 and 1.02 +/- 0.14 A, respectively. The results revealed that human lymphotactin adopts the conserved chemokine fold, which is characterized by a three-stranded antiparallel beta-sheet and a C-terminal alpha-helix. Two regions are dynamically disordered as evidenced by (1)H and (13)C chemical shifts and [(15)N]-(1)H NOEs: residues 1-9 of the amino terminus and residues 69-93 of the C-terminal extension. A functional role for the C-terminal extension, which is unique to lymphotactin, remains to be elucidated.     

An expectation/maximization nuclear vector replacement algorithm for automated NMR resonance assignments

We report an automated procedure for high-throughput NMR resonance assignment for a protein of known structure, or of an homologous structure. Our algorithm performs Nuclear Vector Replacement (NVR) by Expectation/Maximization (EM) to compute assignments. NVR correlates experimentally-measured NH residual dipolar couplings (RDCs) and chemical shifts to a given a priori whole-protein 3D structural model. The algorithm requires only uniform (15)N-labelling of the protein, and processes unassigned H(N)-(15)N HSQC spectra, H(N)-(15)N RDCs, and sparse H(N)-H(N) NOE's (d(NN)s). NVR runs in minutes and efficiently assigns the (H(N),(15)N) backbone resonances as well as the sparse d(NN)s from the 3D (15)N-NOESY spectrum, in O (n(3)) time. The algorithm is demonstrated on NMR data from a 76-residue protein, human ubiquitin, matched to four structures, including one mutant (homolog), determined either by X-ray crystallography or by different NMR experiments (without RDCs). NVR achieves an average assignment accuracy of over 99%. We further demonstrate the feasibility of our algorithm for different and larger proteins, using different combinations of real and simulated NMR data for hen lysozyme (129 residues) and streptococcal protein G (56 residues), matched to a variety of 3D structural models.     

A new electron transport mechanism in mitochondrial steroid hydroxylase systems based on structural changes upon the reduction of adrenodoxin

The adrenal ferredoxin (adrenodoxin, Adx) is an acidic 14.4-kDa [2Fe-2S] ferredoxin that belongs to the vertebrate ferredoxin family. It is involved in the electron transfer from the flavoenzyme NADPH-adrenodoxin-reductase to cytochromes P-450(scc) and P-450(11)(beta). The interaction between the redox partners during electron transport has not yet been fully established. Determining the tertiary structure of an electron-transfer protein may be very helpful in understanding the transport mechanism. In the present work, we report a structural study on the oxidized and reduced forms of bovine adrenodoxin (bAdx) in solution using high-resolution NMR spectroscopy. The protein was produced in Escherichia coli and singly or doubly labeled with (15)N or (13)C/(15)N, respectively. Approximately 70 and 75% of the (15)N, (13)C, and (1)H resonances could be assigned for the reduced and the oxidized bAdx, respectively. The secondary and tertiary structures of the reduced and oxidized states were determined using NOE distance information. (1)H(N)-T(1) relaxation times of certain residues were used to obtain additional distance constraints to the [2Fe-2S] cluster. The results suggest that the solution structure of oxidized Adx is quite similar to the X-ray structure. However, structural changes occur upon reduction of the [2Fe-2S] cluster, as indicated by NMR measurements. It could be shown that these conformational changes, especially in the C-terminal region, cause the dissociation of the Adx dimer upon reduction. A new electron transport mechanism proceeding via a modified shuttle mechanism, with both monomers and dimers acting as electron carriers, is proposed.     

Binding of retinol induces changes in rat cellular retinol-binding protein II conformation and backbone dynamics

The structure and backbone dynamics of rat holo cellular retinol-binding protein II (holo-CRBP II) in solution has been determined by multidimensional NMR. The final structure ensemble was based on 3980 distance and 30 dihedral angle restraints, and was calculated using metric matrix distance geometry with pairwise Gaussian metrization followed by simulated annealing. The average RMS deviation of the backbone atoms for the final 25 structures relative to their mean coordinates is 0.85(+/-0.09) A. Comparison of the solution structure of holo-CRBP II with apo-CRBP II indicates that the protein undergoes conformational changes not previously observed in crystalline CRBP II, affecting residues 28-35 of the helix-turn-helix, residues 37-38 of the subsequent linker, as well as the beta-hairpin C-D, E-F and G-H loops. The bound retinol is completely buried inside the binding cavity and oriented as in the crystal structure. The order parameters derived from the (15)N T(1), T(2) and steady-state NOE parameters show that the backbone dynamics of holo-CRBP II is restricted throughout the polypeptide. The T(2) derived apparent backbone exchange rate and amide (1)H exchange rate both indicate that the microsecond to second timescale conformational exchange occurring in the portal region of the apo form has been suppressed in the holo form.     

Global folds of proteins with low densities of NOEs using residual dipolar couplings: application to the 370-residue maltodextrin-binding protein

The global fold of maltose-binding protein in complex with the substrate beta-cyclodextrin was determined by solution NMR methods. The two-domain protein is comprised of a single polypeptide chain of 370 residues, with a molecular mass of 42 kDa. Distance information in the form of H(N)-H(N), H(N)-CH(3) and CH(3)-CH(3) NOEs was recorded on (15)N, (2)H and (15)N, (13)C, (2)H-labeled proteins with methyl protonation in Val, Leu, and Ile (C(delta1) only) residues. Distances to methyl protons, critical for the structure determination, comprised 77 % of the long-range restraints. Initial structures were calculated on the basis of 1943 NOEs, 48 hydrogen bond and 555 dihedral angle restraints. A global pair-wise backbone rmsd of 5.5 A was obtained for these initial structures with rmsd values for the N and C domains of 2.4 and 3.8 A, respectively. Direct refinement against one-bond (1)H(N)-(15)N, (13)C(alpha)-(13)CO, (15)N-(13)CO, two-bond (1)H(N)-(13)CO and three-bond (1)H(N)-(13)C(alpha) dipolar couplings resulted in structures with large numbers of dipolar restraint violations. As an alternative to direct refinement against measured dipolar couplings we have developed an approach where discrete orientations are calculated for each peptide plane on the basis of the dipolar couplings described above. The orientation which best matches that in initial NMR structures calculated from NOE and dihedral angle restraints exclusively is used to refine further the structures using a new module written for CNS. Modeling studies from four different proteins with diverse structural motifs establishes the utility of the methodology. When applied to experimental data recorded on MBP the precision of the family of structures generated improves from 5.5 to 2.2 A, while the rmsd with respect to the X-ray structure (1dmb) is reduced from 5.1 to 3.3 A.     

Structure and dynamics of the Pf1 filamentous bacteriophage coat protein in micelles

The major coat protein of filamentous bacteriophage adopts its membrane-bound conformation in detergent micelles. High-resolution 1H and 15N NMR experiments are used to characterize the structure and dynamics of residues 30-40 in the hydrophobic midsection of Pf1 coat protein in sodium dodecyl sulfate micelles. Uniform and specific-site 15N labels enable the immobile backbone sites to be identified by their 1H/15N heteronuclear nuclear Overhauser effect and allow the assignment of 1H and 15N resonances. About one-third of the amide N-H protons in the protein undergo very slow exchange with solvent deuterons, which is indicative of sites in highly structured environments. The combination of results from 1H/15N heteronuclear correlation, 1H homonuclear correlation, and 1H homonuclear Overhauser effect experiments assigns the resonances to specific residues and demonstrates that residues 30-40 of the coat protein have a helical secondary structure.     

The secondary structure of the colicin E3 immunity protein as studied by 1H-1H and 1H-15N two-dimensional NMR spectroscopy.

By performing 1H-1H and 1H-15N two-dimensional (2D) nuclear magnetic resonance (NMR) experiments, the complete sequence-specific resonance assignment was determined for the colicin E3 immunity protein (84 residues; ImmE3), which binds to colicin E3 and inhibits its RNase activity. First, the fingerprint region of the spectrum was analyzed by homonuclear 1H-1H HOHAHA and NOESY methods. For the identification of overlapping resonances, heteronuclear 1H-15N (HMQC-HOHAHA, HMQC-NOESY) experiments were performed, so that the complete 1H and 15N resonance assignments were provided. Then the secondary structure of ImmE3 was determined by examination of characteristic patterns of sequential backbone proton NOEs in combination with measurement of exchange rates of amide protons and 3JHN alpha coupling constants. From these results, it was concluded that ImmE3 contains a four-stranded antiparallel beta-sheet (residues 2-10, 19-22, 47-49, and 71-79) and a short alpha-helix (residues 31-36).     

NMR mapping of the recombinant mouse major urinary protein I binding site occupied by the pheromone 2-sec-butyl-4,5-dihydrothiazole.

The interactions between the mouse major urinary protein isoform MUP-I and the pheromone 2-sec-butyl-4,5-dihydrothiazole have been characterized in solution. (15)N-labeled and (15)N, (13)C-doubly-labeled recombinant MUP-I were produced in a bacterial expression system and purified to homogeneity. Racemic 2-sec-butyl-4, 5-dihydrothiazole was produced synthetically. An equilibrium diffusion assay and NMR titration revealed that both enantiomers of the pheromone bind to the recombinant protein with a stoichiometry of 1 equiv of protein to 1 equiv of racemic pheromone. A micromolar dissociation constant and slow-exchange regime dissociation kinetics were determined for the pheromone-protein complex. (1)H, (15)N, and (13)C chemical shifts of MUP-I were assigned using triple resonance and (15)N-correlated 3D NMR experiments. Changes in protein (1)H(N) and (15)N(H) chemical shifts upon addition of pheromone were used to identify the ligand binding site. Several amide signals, corresponding to residues on one side of the binding site, were split into two peaks in the saturated protein-ligand complex. Similarly, two overlapping ligand spin systems were present in isotope-filtered NMR spectra of labeled protein bound to unlabeled pheromone. The two sets of peaks were attributed to the two possible chiralities of the pheromone. Intermolecular NOEs indicated that the orientation of the pheromone in the MUP-I binding cavity is opposite to that modeled in a previous X-ray structure.     

Nuclear magnetic resonance solution structure of truncated human GRObeta [5-73] and its structural comparison with CXC chemokine family members GROalpha and IL-8

The three-dimensional structure of a novel four amino acid truncated form of the CXC chemokine GRObeta [5-73] isolated from bone marrow stromal cells with potent hematopoietic and anti-infective activities has been determined by two-dimensional (1)H nuclear magnetic resonance (NMR) spectroscopy in solution. On the basis of 1878 upper distance constraints derived from nuclear Overhauser effects (NOE) and 314 dihedral angle constraints, a group of 20 conformers representing the solution structure of the human GRObeta [5-73] was computed with the program DYANA. At the concentrations used for NMR study, GRObeta [5-73] forms a dimer in solution that is architectured by a six-stranded antiparallel beta-sheet (residues 25 to 29, 39 to 44, 49 to 52) and a pair of helices (residues 58 to 68) with 2-fold symmetry, while the C terminus of the protein is disordered. The average of the pairwise root-mean-square deviations of individual NMR conformers relative to the mean coordinates for the backbone atoms N, C(alpha) and C' of residues 5 to 68 is 0.47 A. Overall, the global fold of GRObeta [5-73] is similar to that of the previously reported NMR structure of GROalpha and the NMR and X-ray structures of interleukin-8. Among these three CXC chemokines, GRObeta [5-73] is most similar in structure to GROalpha. Significant differences between GRObeta [5-73], GROalpha and interleukin-8 are in the N-terminal loop comprising residues 12 to 19. The N-terminal arm containing the conserved ELR motif and the loop of residues 30 to 38 containing the GPH motif are different among these three CXC chemokines. The structural differences in these two regions may be responsible for the specificity of the receptor binding and biological activity of different chemokines.     

Validation of NMR side-chain conformations by packing calculations

The precision and accuracy of protein structures determined by nuclear magnetic resonance (NMR) spectroscopy depend on the completeness of input experimental data set. Typically, rather than a single structure, an ensemble of up to 20 equally representative conformers is generated and routinely deposited in the Protein Database. There are substantially more experimentally derived restraints available to define the main-chain coordinates than those of the side chains. Consequently, the side-chain conformations among the conformers are more variable and less well defined than those of the backbone. Even when a side chain is determined with high precision and is found to adopt very similar orientations among all the conformers in the ensemble, it is possible that its orientation might still be incorrect. Thus, it would be helpful if there were a method to assess independently the side-chain orientations determined by NMR. Recently, homology modeling by side-chain packing algorithms has been shown to be successful in predicting the side-chain conformations of the buried residues for a protein when the main-chain coordinates and sequence information are given. Since the main-chain coordinates determined by NMR are consistently more reliable than those of the side-chains, we have applied the side-chain packing algorithms to predict side-chain conformations that are compatible with the NMR-derived backbone. Using four test cases where the NMR solution structures and the X-ray crystal structure of the same protein are available, we demonstrate that the side-chain packing method can provide independent validation for the side-chain conformations of NMR structures. Comparison of the side-chain conformations derived by side-chain packing prediction and by NMR spectroscopy demonstrates that when there is agreement between the NMR model and the predicted model, on average 78% of the time the X-ray structure also concurs. While the side-chain packing method can confirm the reliable residue conformations in NMR models, more importantly, it can also identify the questionable residue conformations with an accuracy of 60%. This validation method can serve to increase the confidence level for potential users of structural models determined by NMR.     

Interaction of the E2 and E3 components of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus. Use of a truncated protein domain in NMR spectroscopy

A (15)N-labelled peripheral-subunit binding domain (PSBD) of the dihydrolipoyl acetyltransferase (E2p) and the dimer of a solubilized interface domain (E3int) derived from the dihydrolipoyl dehydrogenase (E3) were used to investigate the basis of the interaction of E2p with E3 in the assembly of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus. Thirteen of the 55 amino acids in the PSBD show significant changes in either or both of the (15)N and (1)H amide chemical shifts when the PSBD forms a 1 : 1 complex with E3int. All of the 13 amino acids reside near the N-terminus of helix I of PSBD or in the loop region between helix II and helix III. (15)N backbone dynamics experiments on PSBD indicate that the structured region extends from Val129 to Ala168, with limited structure present in residues Asn126 to Arg128. The presence of structure in the region before helix I was confirmed by a refinement of the NMR structure of uncomplexed PSBD. Comparison of the crystal structure of the PSBD bound to E3 with the solution structure of uncomplexed PSBD described here indicates that the PSBD undergoes almost no conformational change upon binding to E3. These studies exemplify and validate the novel use of a solubilized, truncated protein domain in overcoming the limitations of high molecular mass on NMR spectroscopy.     

Ubiquitin binding interface mapping on yeast ubiquitin hydrolase by NMR chemical shift perturbation.

The interaction between the 26 kDa yeast ubiquitin hydrolase (YUH1), involved in maintaining the monomeric ubiquitin pool in cells, and the 8.5 kDa yeast ubiquitin protein has been studied by heteronuclear multidimensional NMR spectroscopy. Chemical shift perturbation of backbone (1)H(N), (15)N, and (13)C(alpha) resonances of YUH1, in a YUH1-ubiquitin mixture and in a 35 kDa covalent complex with ubiquitin (a stable analogue of the tetrahedral reaction intermediate), was employed to identify the ubiquitin binding interface of YUH1. This interface mapped on the secondary structure of YUH1 suggests a wide area of contact for ubiquitin, encompassing the N-terminus, alpha1, alpha4, beta2, beta3, and beta6, coincident with the high specificity of YUH1 for ubiquitin. The presence of several hydrophobic clusters in the ubiquitin binding interface of YUH1 suggests that hydrophobic interactions are equally important as ionic interactions in contacting ubiquitin. The residues in the binding interface exhibit a high percentage of homology among the members of the ubiquitin C-terminal hydrolase family, indicating the well-conserved nature of the ubiquitin binding interface reported in this study. The secondary structure of YUH1, from our NMR studies, was similar to the recently determined structure of its human homologue ubiquitin C-terminal hydrolase L3 (UCH-L3), except for the absence of the helix H3 of UCH-L3. This region in YUH1 (helix H3 of UCH-L3) was least perturbed upon ubiquitin binding. Therefore, the binding interface was mapped onto the corresponding residues in the UCH-L3 crystal structure. A model for ubiquitin binding to YUH1 is proposed, in which a good correlation was observed for the lateral binding of ubiquitin to UCH-L3 (YUH1), stabilized by the electrostatic and hydrophobic interactions.     

Model for the catalytic domain of the proofreading epsilon subunit of Escherichia coli DNA polymerase III based on NMR structural data.

The DNA polymerase III holoenzyme (HE) is the primary replicative polymerase of Escherichia coli. The epsilon subunit of the HE complex provides the 3'-exonucleolytic proofreading activity for this enzyme complex. epsilon consists of two domains: an N-terminal domain containing the proofreading exonuclease activity (residues 1-186) and a C-terminal domain required for binding to the polymerase (alpha) subunit (residues 187-243). Multidimensional NMR studies of (2)H-, (13)C-, and (15)N-labeled N-terminal domains (epsilon186) were performed to assign the backbone resonances and measure H(N)-H(N) nuclear Overhauser effects (NOEs). NMR studies were also performed on triple-lableled [U-(2)H,(13)C,(15)N]epsilon186 containing Val, Leu, and Ile residues with protonated methyl groups, which allowed for the assignment of H(N)-CH(3) and CH(3)-CH(3) NOEs. Analysis of the (13)C(alpha), (13)C(beta), and (13)CO shifts, using chemical shift indexing and the TALOS program, allowed for the identification of regions of the secondary structure. H(N)-H(N) NOEs provided information on the assembly of the extended strands into a beta-sheet structure and confirmed the assignment of the alpha helices. Measurement of H(N)-CH(3) and CH(3)-CH(3) NOEs confirmed the beta-sheet structure and assisted in the positioning of the alpha helices. The resulting preliminary characterization of the three-dimensional structure of the protein indicated that significant structural homology exists with the active site of the Klenow proofreading exonuclease domain, despite the extremely limited sequence homology. On the basis of this analogy, molecular modeling studies of epsilon186 were performed using as templates the crystal structures of the exonuclease domains of the Klenow fragment and the T4 DNA polymerase and the recently determined structure of the E. coli Exonuclease I. A multiple sequence alignment was constructed, with the initial alignment taken from the previously published hidden Markov model and NMR constraints. Because several of the published structures included complexed ssDNA, we were also able to incorporate an A-C-G trinucleotide into the epsilon186 structure. Nearly all of the residues which have been identified as mutators are located in the portion of the molecule which binds the DNA, with most of these playing either a catalytic or structural role.     

Solution secondary structure of calcium-saturated troponin C monomer determined by multidimensional heteronuclear NMR spectroscopy.

The solution secondary structure of calcium-saturated skeletal troponin C (TnC) in the presence of 15% (v/v) trifluoroethanol (TFE), which has been shown to exist predominantly as a monomer (Slupsky CM, Kay CM, Reinach FC, Smillie LB, Sykes BD, 1995, Biochemistry 34, forthcoming), has been investigated using multidimensional heteronuclear nuclear magnetic resonance spectroscopy. The 1H, 15N, and 13C NMR chemical shift values for TnC in the presence of TFE are very similar to values obtained for calcium-saturated NTnC (residues 1-90 of skeletal TnC), calmodulin, and synthetic peptide homodimers. Moreover, the secondary structure elements of TnC are virtually identical to those obtained for calcium-saturated NTnC, calmodulin, and the synthetic peptide homodimers, suggesting that 15% (v/v) TFE minimally perturbs the secondary and tertiary structure of this stably folded protein. Comparison of the solution structure of calcium-saturated TnC with the X-ray crystal structure of half-saturated TnC reveals differences in the phi/psi angles of residue Glu 41 and in the linker between the two domains. Glu 41 has irregular phi/psi angles in the crystal structure, producing a kink in the B helix, whereas in calcium-saturated TnC, Glu 41 has helical phi/psi angles, resulting in a straight B helix. The linker between the N and C domains of calcium-saturated TnC is flexible in the solution structure.