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Bacterial MinD and MinE form a standing oscillatory wave which positions the cell division inhibitor MinC, that binds MinD, everywhere on the membrane except at the midpoint of the cell, ensuring midcell positioning of the cytokinetic septum. During this process MinE undergoes fold switching as it interacts with different partners. We explore the exchange dynamics between major and excited states of the MinE dimer in 3 forms using 15 N relaxation dispersion NMR: the full-length protein (6-stranded β-sheet sandwiched between 4 helices) representing the resting state; a 10-residue N-terminal deletion (Δ10) mimicking the membrane-binding competent state where the N-terminal helix is detached to interact with membrane; and N-terminal deletions of either 30 (Δ30) or 10 residues with an I24N mutation (Δ10/I24N), in which the β1-strands at the dimer interface are extruded and available to bind MinD, leaving behind a 4-stranded β-sheet. Full-length MinE samples 2 “excited” states: The first is similar to a full-length/Δ10 heterodimer; the second, also sampled by Δ10, is either similar to or well along the pathway toward the 4-stranded β-sheet form. Both Δ30 and Δ10/I24N sample 2 excited species: The first may involve destabilization of the β3- and β3′-strands at the dimer interface; changes in the second are more extensive, involving further disruption of secondary structure, possibly representing an ensemble of states on the pathway toward restoration of the resting state. The quantitative information on MinE conformational dynamics involving these excited states is crucial for understanding the oscillation pattern self-organization by MinD–MinE interaction dynamics on the membrane.

The solution structure of the 48-kDa IIA(Man)-HPr complex of the mannose branch of the Escherichia coli phosphotransferase system has been solved by NMR using conjoined rigid body/torsion angle-simulated annealing on the basis of intermolecular nuclear Overhauser enhancement data and residual dipolar couplings. IIA(Man) is dimeric and has two symmetrically related binding sites per dimer for HPr. A convex surface on HPr, formed primarily by helices 1 and 2, interacts with a deep groove at the interface of the two subunits of IIA(Man). The interaction surface on IIA(Man) is predominantly helical, comprising helix 3 from the subunit that bears the active site His-10 and helices 1, 4, and 5 from the other subunit. The total buried accessible surface area at the protein-protein interface is 1450 A(2). The binding sites on the two proteins are complementary in terms of shape and distribution of hydrophobic, hydrophilic, and charged residues. The active site histidines, His-10 of IIA(Man) and His-15 (italics indicate HPr residues) of HPr, are in close proximity. An associative transition state involving a pentacoordinate phosphoryl group with trigonal bipyramidal geometry bonded to the N-epsilon2 atom of His-10 and the N-delta1 atom of His-15 can be readily formed with negligible displacement in the backbone coordinates of the residues immediately adjacent to the active site histidines. Comparing the structures of complexes of HPr with three other structurally unrelated phosphotransferase system proteins, enzymes I, IIA(glucose), and IIA(mannitol), reveals a number of common features that provide a molecular basis for understanding how HPr specifically recognizes a wide range of diverse proteins.

The side chains of Arg 50 and Arg 52 at positions P6' and P8', respectively, anchor the binding loop to the protein scaffold by means of hydrogen bonds in Cucurbita maximatrypsin inhibitor-V (CMTI- V), a potato I family member. Here, we have investigated the relative contributions of Arg 50 and Arg 52 to the binding-loop flexibility and stability by determining changes in structure, dynamics, and proteolytic stability as a consequence of individually mutating them into an alanine. We have compared chemical shift assignments of main-chain hydrogens and nitrogens, and 1 H- 1 H interresidue nuclear Overhauser effects (NOEs) for the two mutants with those of the wild-type protein. We have also measured NMR longitudinal and transverse relaxation rates and 15 N- 1 H NOE enhancements for all backbone and side- chain NH groups and calculated the model-free parameters for R50A-rCMTI-V and R52A-rCMTI-V. The three-dimensional structures and backbone dynamics of the protein scaffold region remain very similar for both mutants, relative to the wild-type protein. The flexibility of the binding loop is increased in both R50A- and R52A-rCMTI-V. In R52A-rCMTI-V, the mean generalized order parameter ( )o f the P6-P1 residues of the binding loop (39-44) decreases to 0.68 ( 0.02 from 0.76 ( 0.04 observed for the wild-type protein. However, in R50A-rCMTI-V, the flexibility of the whole binding loop increases, especially that of the P1'-P3' residues (45-47), whose value drops dramatically to 0.35 ( 0.03 from 0.68 ( 0.03 determined for rCMTI-V. More strikingly, S 2 values of side-chain NHs reveal that, in the R50A mutant, removal of the R50 hydrogen bond results in the loss of the R52 hydrogen bond too, whereas in R52A, the R50 hydrogen bond remains unaffected. Kinetic data on trypsin-catalyzed hydrolysis of the reactive-site peptide bond (P 1-P1') suggest that the activation free energy barrier of the reaction at 25 °C is reduced by 2.1 kcal/mol for R50A-rCMTI-V and by 1.5 kcal/mol for R52A-rCMTI-V, relative to rCMTI-V. Collectively, the results suggest that although both the P 6' and P8' anchors are required for optimal inhibitor function and stability in the potato I family, the former is essential for the existence of the latter and has greater influence on the binding-loop structure, dynamics, and stability.

The solution structure of the N-terminal zinc binding domain (residues 1-55; IN1-55) of HIV-1 integrase has been solved by NMR spectroscopy. IN1-55 is dimeric, and each monomer comprises four helices with the zinc tetrahedrally coordinated to His 12, His 16, Cys 40 and Cys 43. IN1-55 exists in two interconverting conformational states that differ with regard to the coordination of the two histidine side chains to zinc. The different histidine arrangements are associated with large conformational differences in the polypeptide backbone (residues 9-18) around the coordinating histidines. The dimer interface is predominantly hydrophobic and is formed by the packing of the N-terminal end of helix 1, and helices 3 and 4. The monomer fold is remarkably similar to that of a number of helical DNA binding proteins containing a helix-turn-helix (HTH) motif with helices 2 and 3 of IN1-55 corresponding to the HTH motif. In contrast to the DNA binding proteins where the second helix of the HTH motif is employed for DNA recognition, IN1-55 uses this helix for dimerization.

The side chains of Arg50 and Arg52 iin Cucurbita maxima trypsin inhibitor-V (CMTI-V) anchor the binding loop to the scaffold region [Cai, M., Gong, Y., Kao, J.L-F.,Krishnamoorthi, R. (1995) Biochemistry 34, 5201-5211]. The consequences of these hydrogen-bonding and electrostatic interactions on the conformational flexibility and stability of the binding loop were evaluated by trypsin-catalyzed hydrolysis of CMTI-V mutants, in which each of the arginines was individually replaced with Ala, Lys, or Gln by genetic engineering methods. All mutants exhibited significantly increased vulnerability to the protease attack at many sites, including the reactive-site (Lys44-Asp45 peptide bond), with the R50 mutants showing much more pronounced effects than the R52 counterparts. For CmTI-V and the mutants studied, a qualitative correlation was inferred between binding loop flexibility and retention time on a reverse-phase high-pressure liquid chromatography C-18 column. The R50 mutants were found to be more flexible than the corresponding R52 versions. These results demonstrate that Arg50 contributes more to the stability and function of CMTI-V. The differing strengths of the hydrogen bonds made by Arg50 and Arg52 were characterized by determining the internal dynamics of their side chains at pH 5.0 and 2.5: 15N NMR longitudinal and transverse relaxation rates and 15N-1H nuclear Overhauser effect (NOE) enhancements were measured for the main-chain and side-chain NH groups in 15N-labeled recombinant CMTI-V (rCMTI-V) and the model-free parameters [Lipari, G.,Szabo, A.(1982) J. Am. Chem. Soc. 104, 4546-59; 4559-4570] were calculated. At both pH 5.0 and 2.5, the arginines at positions 26, 47, 58 and 66 are found to be highly mobile, as the caluculated general order parameters, S2 values, of their NepsilonH groups fall in the range 0.03-0.18. The corresponding values for Arg50 amd Arg52 are 0.73 and 0.63, respectively, at pH 5.0, thus confirming that the two arginines are rigid and hydrogen bonded. At pH 2.5, these hydrogen bonds are still retained with Arg50 appearing to be more restrained (S2 = 0.71) than Arg52 (S2 = 0.56). This is consistent a greater contribution by Arg50 to the conformational stability of the reactive-site loop in CMTI-V. The results also indicate that the Arg50 and Arg52 side chains are not hydrogen-bonded to carboxylate groups, which would be protonated at pH 2.5 and, hence, unavailable for hydrogen-bonding interactions. The overall folding of rCMTI-V appears not to be significantly affected by the pH change, as indicated by comparisons of 1H and 15N chemical shifts, sequential NOE cross-peaks, and S2 values of the backbone atoms, and the conserved side-chain dynamics of Trp9 and Trp54--residues that are involved in hydrophobic and hydrogen-bonding interactions with others in the protein core and the binding loop, respectively.

Reactive-site (Lys44-Asp45 peptide bond) hydrolyzed Cucurbita maxima trypsin inhibitor-V (CMTI-V*) was prepared and characterized: In comparison to the intact form, CMTI-V* exhibited markedly reduced inhibitory properties and binding affinities toward trypsin and human blood coagulation factor XIIa. The equilibrium constant of trypsin-catalyzed hydrolysis, Khyd, defined as [CMTI-V*]/[CMTI-V], was measured to be approximately 9.4 at 25 degrees C (delta G degrees = -1.3 kcal.mol-1). From the temperature dependence of delta G degrees, the following thermodynamic parameters were estimated: delta H degrees = 1.6 kcal.mol-1 and delta S degrees = 9.8 eu. In order to understand the functional and thermodynamic differences between the two forms, the three-dimensional solution structure of CMTI-V* was determined by a combined approach of NMR, distance geometry, and simulated annealing methods. Thus, following sequence-specific and stereospecific resonance assignments, including those of beta-, gamma-, delta-, and epsilon-hydrogens and valine methyl hydrogens, 809 interhydrogen distances and 123 dihedral angle constraints were determined, resulting in the computation and energy-minimization of 20 structures for CMTI-V*. The average root mean squared deviation in position for equivalent atoms between the 20 individual structures and the mean structure obtained by averaging their coordinates is 0.67 +/- 0.15 A for the main chain atoms and 1.19 +/- 0.23 A for all the non-hydrogen atoms of residues 5-40 and residues 48-67.(ABSTRACT TRUNCATED AT 250 WORDS)

Three different conformations of proline rings in a protein in solution, Up, Down and Twist, have been distinguished, and stereospecific assignments of the pyrrolidine beta-, gamma- and delta-hydrogens have been made on the basis of 1H-1H vicinal coupling constant patterns and intraresidue NOEs. For all three conformations, interhydrogen distances in the pairs alpha-beta 3, beta 3-gamma 3, beta 2-gamma 2, gamma 2-delta 2, and gamma 3-delta 3 (2.3 A) are shorter than those in the pairs alpha-beta 2, beta 2-gamma 3, beta 3-gamma 2, gamma 2-delta 3, and gamma 3-delta 2 (2.7-3.0 A), resulting in stronger NOESY cross peaks. For the Up conformation, the beta 3-gamma 2 and gamma 2-delta 3 spin-spin coupling constants are small (3 Hz), and weak cross peaks are obtained in a short-mixing-time (10 ms) TOCSY spectrum; all other vicinal coupling constants are in the range 5-12 Hz, and result in medium to strong TOCSY cross peaks. For the Down form, the alpha-beta 2, beta 2-gamma 3, and gamma 3-delta 2 vicinal coupling constants are small, leading to weak TOCSY cross peaks; all other couplings again are in the range 5-12 Hz, and result in medium to strong TOCSY cross peaks. In the case of a Twist conformation, dynamically averaged coupling constants are anticipated. The procedure has been applied to bovine pancreatic trypsin inhibitor and Cucurbita maxima trypsin inhibitor-V, and ring conformations of all prolines in the two proteins have been determined.

The solution structure of the IIA-IIB complex of the N,N′-diacetylchitobiose (Chb) transporter of the Escherichia coli phosphotransferase system has been solved by NMR. The active site His-89 of IIAChb was mutated to Glu to mimic the phosphorylated state and the active site Cys-10 of IIBChb was substituted by serine to prevent intermolecular disulfide bond formation. Binding is weak with a KD of ∼1.3 mm. The two complementary interaction surfaces are largely hydrophobic, with the protruding active site loop (residues 9–16) of IIBChb buried deep within the active site cleft formed at the interface of two adjacent subunits of the IIAChb trimer. The central hydrophobic portion of the interface is surrounded by a ring of polar and charged residues that provide a relatively small number of electrostatic intermolecular interactions that serve to correctly align the two proteins. The conformation of the active site loop in unphosphorylated IIBChb is inconsistent with the formation of a phosphoryl transition state intermediate because of steric hindrance, especially from the methyl group of Ala-12 of IIBChb. Phosphorylation of IIBChb is accompanied by a conformational change within the active site loop such that its path from residues 11–13 follows a mirror-like image relative to that in the unphosphorylated state. This involves a transition of the φ/ψ angles of Gly-13 from the right to left α-helical region, as well as smaller changes in the backbone torsion angles of Ala-12 and Met-14. The resulting active site conformation is fully compatible with the formation of the His-89-P-Cys-10 phosphoryl transition state without necessitating any change in relative translation or orientation of the two proteins within the complex.

A method for accurately measuring H(N)-H(alpha) residual dipolar couplings is described. Using this technique, both the sign and magnitude of the coupling can be determined easily. Residual dipolar coupling between H(N)(i)-H(alpha)(i) and H(N)(i)-H(alpha)(i-1) were measured for the FK506 binding protein complexed to FK506. The experimental values were in excellent agreement with predictions based on an X-ray crystal structure of the protein/ligand complex, suggesting that these residual dipolar couplings will provide accurate structural constraints for the refinement of protein structures determined by NMR.

The barrier-to-autointegration factor BAF binds to the LEM domain (Em(LEM)) of the nuclear envelope protein emerin and plays an essential role in the nuclear architecture of metazoan cells. In addition, the BAF(2) dimer bridges and compacts double-stranded DNA nonspecifically via two symmetry-related DNA binding sites. In this article we present biophysical and structural studies on a complex of BAF(2) and Em(LEM). Light scattering, analytical ultracentrifugation, and NMR indicate a stoichiometry of one molecule of Em(LEM) bound per BAF(2) dimer. The equilibrium dissociation constant (K(d)) for the interaction of the BAF(2) dimer and Em(LEM), determined by isothermal titration calorimetry, is 0.59 +/- 0.03 microm. Z-exchange spectroscopy between corresponding cross-peaks of the magnetically non-equivalent subunits of the BAF(2) dimer in the complex yields a dissociation rate constant of 78 +/- 2s(-1). The solution NMR structure of the BAF(2)-Em(LEM) complex reveals that the LEM and DNA binding sites on BAF(2) are non-overlapping and that both subunits of the BAF(2) dimer contribute approximately equally to the Em(LEM) binding site. The relevance of the implications of the structural and biophysical data on the complex in the context of the interaction between the BAF(2) dimer and Em(LEM) at the nuclear envelope is discussed.

The solution structure of a stably phosphorylated form of the cytoplasmic B domain of the mannitol-specific transporter (IIB(Mtl)) of the Escherichia coli phosphotransferase system, containing a mutation of the active site Cys384 to Ser, has been solved by NMR. The strategy employed relies principally on backbone residual dipolar couplings recorded in three different alignment media, supplemented by nuclear Overhauser enhancement data and torsion angle restraints related specifically to the active site loop (residues 383-393). As judged from the dipolar coupling data, the remainder of the structure is unchanged upon phosphorylation within the errors of the coordinates of the previously determined solution structure of unphosphorylated wild-type IIB(Mtl). Thus, only the active site loop was refined. Phosphorylation results in a backbone atomic rms shift of approximately 0.7 angstroms in the active site loop. The resulting conformation is less than 0.5 angstroms away from the equivalent P-loop in both the low and high molecular mass eukaryotic tyrosine phosphatases. 3J(NP) coupling constant measurements using quantitative J-correlation spectroscopy provide a direct demonstration of a hydrogen bond between the phosphoryl group and the backbone amide of Ser391 at position i + 7 from phospho-Ser384, with an approximately linear P-O-H(N) bond angle. The structure also reveals additional hydrogen bonding interactions involving the backbone amides of residues at positions i + 4 and i + 5, and the hydroxyl groups of two serine residues at positions i + 6 and i + 7 that stabilize the phosphoryl group.

The solution structure of the cytoplasmic B domain of the mannitol (Mtl) transporter (II(Mtl)) from the mannitol branch of the Escherichia coli phosphotransferase system has been solved by multidimensional NMR spectroscopy with extensive use of residual dipolar couplings. The ordered IIB(Mtl) domain (residues 375-471 of II(Mtl)) consists of a four-stranded parallel beta-sheet flanked by two helices (alpha(1) and alpha(3)) on one face and helix alpha(2) on the opposite face with a characteristic Rossmann fold comprising two right-handed beta(1)alpha(1)beta(2) and beta(3)alpha(2)beta(4) motifs. The active site loop is structurally very similar to that of the eukaryotic protein tyrosine phosphatases, with the active site cysteine (Cys-384) primed in the thiolate state (pK(a)5.6) for nucleophilic attack at the phosphorylated histidine (His-554) of the IIA(Mtl) domain through stabilization by hydrogen bonding interactions with neighboring backbone amide groups at positions i + 2/3/4 from Cys-384 and with the hydroxyl group of Ser-391 at position i + 7. Modeling of the phosphorylated state of IIB(Mtl) suggests that the phosphoryl group can be readily stabilized by hydrogen bonding interactions with backbone amides in the i + 2/4/5/6/7 positions as well as with the hydroxyl group of Ser390 at position i + 6. Despite the absence of any significant sequence identity, the structure of IIB(Mtl) is remarkably similar to the structures of bovine protein tyrosine phosphatase (which contains two long insertions relative to IIB(Mtl)) and the cytoplasmic B component of enzyme II(Chb), which fulfills an analogous role to IIB(Mtl) in the N,N'-diacetylchitobiose branch of the phosphotransferase system. All three proteins utilize a cysteine residue in the nucleophilic attack of a phosphoryl group covalently bound to another protein.

The solution structure of His12 --> Cys mutant of the N-terminal zinc binding domain (residues 1-55; IN(1-55)) of HIV-1 integrase complexed to cadmium has been solved by multidimensional heteronuclear NMR spectroscopy. The overall structure is very similar to that of the wild-type N-terminal domain complexed to zinc. In contrast to the wild-type domain, however, which exists in two interconverting conformational states arising from different modes of coordination of the two histidine side chains to the metal, the cadmium complex of the His12 --> Cys mutant exists in only a single form at low pH. The conformation of the polypeptide chain encompassing residues 10-18 is intermediate between the two forms of the wild-type complex.

The solution structure of the ectodomain of simian immunodeficiency virus (SIV) gp41 (e-gp41), consisting of residues 27‐149, has been determined by multidimensional heteronuclear NMR spectroscopy. SIV e-gp41 is a symmetric 44 kDa trimer with each subunit consisting of antiparallel N-terminal (residues 30‐80) and C-terminal (residues 107‐147) helices connected by a 26 residue loop (residues 81‐106). The N-terminal helices of each subunit form a parallel coiled-coil structure in the interior of the complex which is surrounded by the C-terminal helices located on the exterior of the complex. The loop region is ordered and displays numerous intermolecular and non-sequential intramolecular contacts. The helical core of SIV e-gp41 is similar to recent X-ray structures of truncated constructs of the helical core of HIV-1 e-gp41. The present structure establishes unambiguously the connectivity of the N- and C-terminal helices in the trimer, and characterizes the conformation of the intervening loop, which has been implicated by mutagenesis and antibody epitope mapping to play a key role in gp120 association. In conjunction with previous studies, the solution structure of the SIV e-gp41 ectodomain provides insight into the binding site of gp120 and the mechanism of cell fusion. The present structure of SIV e-gp41 represents one of the largest protein structures determined by NMR to date.

The gp41 protein of the human (HIV) and simian (SIV) immunodeficiency viruses is part of the envelope glycoprotein complex gp41/gp120 which plays an essential role in viral infection. We present a multidimensional NMR study on the trimeric 44 kDa soluble ectodomain of SIV gp41 (e-gp41), comprising residues 27 to 149. Despite the large molecular weight and very limited spectral dispersion, complete backbone 1H, 13C, 13CO and 15N assignments have been made using a combination of triple resonance experiments on uniformly 13C/15N and 2H/13C/15N-labeled samples. The secondary structure of SIV e-gp41, derived on the basis of 13C chemical shifts, NH exchange rates, medium range nuclear Overhauser enhancements (NOE), and 3JHNα coupling constants, consists of a 49 residue helix at the N terminus (residues 29 to 77) and a 40 residue helix at the C terminus (residues 108 to 147), connected by a 30 residue loop which does not display any of the characteristics of regular secondary structure. The cross-peak intensities of the loop region in scalar correlation experiments suggests that it is more mobile than the core helical regions. The presence, however, of numerous long range NOEs, both intra and inter-subunit, within the loop indicates that it adopts a well-defined structure in which the loops from the three subunits interact with each other. Based on a number of long range intra and inter-subunit NOEs, a topological model is presented for the symmetric SIV e-gp41 trimer in which the N-terminal helices are packed within the protein interior in a parallel trimeric coiled-coil arrangement, while the C-terminal helices are located on the protein exterior, oriented antiparallel to the N-terminal helices.

The solution structure of recombinant Cucurbita maxima trypsin inhibitor-V (rCMTI-V), whose N-terminal is unacetylated and carries an extra glycine residue, was determined by means of two-dimensional (2D) homo and 3D hetero NMR experiments in combination with a distance geometry and simulated annealing algorithm. A total of 927 interproton distances and 123 torsion angle constraints were utilized to generate 18 structures. The root mean squared deviation (RMSD) of the mean structure is 0.53 A for main-chain atoms and 0.95 A for all the non-hydrogen atoms of residues 3-40 and 49-67. The average structure of rCMTI-V is found to be almost the same as that of the native protein [Cai, M., Gong, Y., Kao, J.-L.,Krishnamoorthi, R. (1995) Biochemistry 34, 5201-5211]. The backbone dynamics of uniformly 15N-labeled rCMTI-V were characterized by 2D 1H-15N NMR methods. 15N spin-lattice and spin-spin relaxation rate constants (R1 and R2, respectively) and [1H]-15N steady-state heteronuclear Overhauser effect enhancements were measured for the peptide NH units and, using the model-free formalism [Lipari, G.,Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4559, 4559-4570], the following parameters were determined: overall tumbling correlation time for the protein molecule (tau m), generalized order parameters for the individual N-H vectors (S2), effective correlation times for their internal motions (tau e), and terms to account for motions on a slower time scale (second) due to chemical exchange and/or conformational averaging (R(ex)). Most of the backbone NH groups of rCMTI-V are found to be highly constrained ((S2) = 0.83) with the exception of those in the binding loop (residues 41-48, (S2) = 0.71) and the N-terminal region ((S2) = 0.73). Main-chain atoms in these regions show large RMSD values in the average NMR structure. Residues involved in turns also appear to have more mobility ((S2) = 0.80). Dynamical properties of rCMTI-V were compared with those of two other inhibitors of the potato I family--eglin c [Peng, J. W.,Wagner, G. (1992) Biochemistry 31, 8571-8586] and barley chymotrypsin inhibitor 2 [CI-2; Shaw, G. L., Davis, B., Keeler, J.,Fersht, A. R. (1995) Biochemistry 34, 2225-2233]. The Cys3-Cys48 linkage found only in rCMTI-V appears to somewhat reduce the N-terminal flexibility; likewise, the C-terminal of rCMTI-V, being part of a beta-sheet, appears to be more rigid.

The solution structure of Cucurbita maxima trypsin inhibitor-V (CMTI-V), which is also a specific inhibitor of the blood coagulation protein, factor XIIa, was determined by 1H NMR spectroscopy in combination with a distance-geometry and simulated annealing algorithm. Sequence-specific resonance assignments were made for all the main-chain and most of the side-chain hydrogens. Stereospecific assignments were also made for some of the beta-, gamma-, delta-, and epsilon-hydrogens and valine methyl hydrogens. The ring conformations of all six prolines in the inhibitor were determined on the basis of 1H-1H vicinal coupling constant patterns; most of the proline ring hydrogens were stereospecifically assigned on the basis of vicinal coupling constant and intraresidue nuclear Overhauser effect (NOE) patterns. Distance constraints were determined on the basis of NOEs between pairs of hydrogens. Dihedral angle constraints were determined from estimates of scalar coupling constants and intraresidue NOEs. On the basis of 727 interproton distance and 111 torsion angle constraints, which included backbone phi angles and side-chain chi 1, chi 2, chi 3, and chi 4 angles, 22 structures were calculated by a distance geometry algorithm and refined by energy minimization and simulated annealing methods. Both main-chain and side-chain atoms are well-defined, except for a loop region, two terminal residues, and some side-chain atoms located on the molecular surface. The average root mean squared deviation in the position for equivalent atoms between the 22 individual structures and the mean structure obtained by averaging their coordinates is 0.58 +/- 0.06 A for the main-chain atoms and 1.01 +/- 0.07 A for all the non-hydrogen atoms of residues 3-40 and 49-67. These structures were compared to the X-ray crystallographic structure of another protein of the same inhibitor family-chymotrypsin inhibitor-2 from barley seeds [CI-2; McPhalen, C. A., & James, M. N. G. (1987) Biochemistry 26, 261-269]. The main-chain folding patterns are highly similar for the two proteins, which possess 62% sequence differences. However, major differences are noted in the N- and C-terminal segments, which may be due to the presence of a disulfide bridge in CMTI-V, but not in CI-2.

1H NMR spectroscopy and solution structure computations have been used to examine ferrocytochrome c-551 from Pseudomonas stutzeri ZoBell (ATCC 14405). Resonance assignments are proposed for all main-chain and most side-chain protons. Stereospecific assignments were also made for some of the beta-methylene protons and valine methyl protons. Distance constraints were determined based upon nuclear Overhauser enhancements between pairs of protons. Dihedral angle constraints were determined from estimates of scalar coupling constants and intra-residue NOEs. Twenty structures were calculated by distance geometry and refined by energy minimization and simulated annealing on the basis of 1012 interproton distance and 74 torsion angle constraints. Both the main-chain and side-chain atoms are well defined except for two terminal residues, and some side-chain atoms located on the molecular surface. The average root mean squared deviation in the position for equivalent atoms between the 20 individual structures and the mean structure obtained by averaging their coordinates is 0.56 +/- 0.10 A for the main-chain atoms, and 0.95 +/- 0.09 A for all nonhydrogen atoms of residue 3 to 80 plus the heme group. The average structure was compared with an analogous protein, cytochrome c-551 from pseudomonas stutzeri. The main-chain folding patterns are very consistent, but there are some differences, some of which can be attributed to the loss of normally conserved aromatic residues in the ZoBell c-551.