Solution conformation of salmon calcitonin in sodium dodecyl sulfate micelles as determined by two-dimensional NMR and distance geometry calculations

The 32 amino acid hormone salmon calcitonin was studied at pH 3.7 and 7.4 by two-dimensional NMR in sodium dodecyl sulfate (SDS) micelles at 310 K. The spectrum was fully assigned, and the secondary structure was obtained from nuclear Overhauser enhancement spectroscopy (NOESY), 3JHN alpha coupling constants, and slowly exchanging amide data. Three-dimensional structures consistent with NMR data were generated by using distance geometry calculations. A set of 260 interproton distances, derived from NOESY, and hydrogen-bond constraints, obtained from analysis of the amide exchange, were used. From the initial random conformations, 13 distance geometry structures with minimal violations were selected for further refinement with restrained energy minimization. In SDS, at both pHs, the main conformational feature of the hormone is an alpha-helix from Thr6 through Tyr22, thus including the amphipathic 8-22 segment and two residues of the Cys1-Cys7 N-terminal loop. The C-terminal decapeptide forms a loop folded back toward the helix. The biological significance of this conformation is discussed.     

Solution structure of human calcitonin in membrane-mimetic environment: The role of the amphipathic helix

The 32 amino acid hormone human calcitonin was studied at pH 3.7 and 7.4 by multidimensional NMR spectroscopy in sodium dodecyl sulfate micelles at 310K. The secondary structure was obtained from nuclear Overhauser enhancement spectroscopy (NOESY), ³J_HNα coupling constants, and slowly exchanging amide data. Three-dimensional structures consistent with NMR data were generated by using distance geometry calculations. A set of 265 interproton distances derived from NOESY experiments, hydrogen-bond constraints obtained from amide exchange, and coupling constants were used. From the initial random conformations, 30 distance geometry structures with minimal violations were selected for further refinement with restrained energy minimization. In micelles, at both pHs, the hormone assumes an amphipathic α-helix from Leu9 to Phe16, followed by a type-I β-turn between residues Phe16 and Phe19. From His20 onward the molecule is extended and no interaction with the helix was observed. The relevance of the amphipathic helix for the structure–activity relationship, the possible mechanisms of interaction with the receptor, as well as the formation of fibrillar aggregates, is discussed. Proteins 32:314–323, 1998. © 1998 Wiley-Liss, Inc.     

Conformational analysis of biologically active truncated linear analogs of endothelin-1 using NMR and molecular modeling.

Some linear truncated analogs of endothelin-1 display potent agonistic activity at the ET(B) receptor, especially when the side chain of Trp21 is N-formylated. Then, the three-dimensional arrangements of six structurally reduced linear analogs, three formylated and three nonformylated, have been investigated by high resolution NMR spectroscopy and molecular modeling, in order to pinpoint the conformational features related to the biological activity. Two-dimensional double-quantum-filtered correlation spectroscopy (DQFCOSY), total correlation spectroscopy (TOCSY) and nuclear Overhauser enhancement spectroscopy (NOESY) were recorded and analyzed for each molecule. Interspatial distance constraints were derived from the intensity of the NOESY connectivities. The formation of hydrogen bonding was monitored from the temperature dependence of the NH chemical shifts. Molecular models calculated by means of distance geometry, simulated annealing and energy minimization, using the NMR constraints, strongly suggested a global elongated structure for the formylated analogs exhibiting biological activity, and a folded arrangement for the unformylated derivatives. Homology comparisons allowed the identification of a beta-turn-like folding of the C-terminal segment Asp18-Trp21 as a probable key-factor for activity.     

Simulated annealing with restrained molecular dynamics using CONGEN: energy refinement of the NMR solution structures of epidermal and type-alpha transforming growth factors.

The new functionality of the program CONGEN (Bruccoleri RE, Karplus M, 1987, Biopolymers 26:137-168; Bassolino-Klimas D et al., 1996, Protein Sci 5:593-603) has been applied for energy refinement of two previously determined solution NMR structures, murine epidermal growth factor (mEGF) and human type-alpha transforming growth factor (hTGF alpha). A summary of considerations used in converting experimental NMR data into distance constraints for CONGEN is presented. A general protocol for simulated annealing with restrained molecular dynamics is applied to generate NMR solution structures using CONGEN together with real experimental NMR data. A total of 730 NMR-derived constraints for mEGF and 424 NMR-derived constraints for hTGF alpha were used in these energy-refinement calculations. Different weighting schemes and starting conformations were studied to check and/or improve the sampling of the low-energy conformational space that is consistent with all constraints. The results demonstrate that loosened (i.e., "relaxed") sets of the EGF and hTGF alpha internuclear distance constraints allow molecules to overcome local minima in the search for a global minimum with respect to both distance restraints and conformational energy. The resulting energy-refined structures of mEGF and hTGF alpha are compared with structures determined previously and with structures of homologous proteins determined by NMR and X-ray crystallography.     

Conformation of recombinant desulfatohirudin in aqueous solution determined by nuclear magnetic resonance

The three-dimensional structure of recombinant desulfatohirudin in aqueous solution was determined by 1H nuclear magnetic resonance at 600 MHz and distance geometry calculations with the program DISMAN. The input for the structure calculations was prepared on the basis of complete sequence-specific resonance assignments at pH 4.5 and 22 degrees C and consisted of 425 distance constraints from nuclear Overhauser enhancements and 159 supplementary constraints from spin-spin coupling constants and from the identification of intramolecular hydrogen bonds. Residues 3-30 and 37-48 form a molecular core with two antiparallel beta-sheets and several well-defined turns. The three disulfide bonds 6-14, 16-28, and 22-39 were identified by NMR. In contrast to this well-defined molecular core, with an average root mean square distance for the polypeptide backbone of 0.8 A for a group of nine DISMAN solutions, no preferred conformation was found for the C-terminal segment 49-65, and a loop consisting of residues 31-36 is not uniquely constrained by the NMR data either. These structural properties of recombinant desulfatohirudin coincide closely with the previously described solution conformation of natural hirudin, but the presence of localized differences is indicated by chemical shift differences for residues Asp 5, Ser 9, Leu 15, Asp 53, Gly 54, and Asp 55.     

Structural Characterization of Neurokinin-3 Receptor Selective Peptide Agonist Scyliorhinin II Bound to DPC Micelles

Abstract

Scyliorhinin II, a cyclic Tachykinin peptide, is a potent NK3 receptor agonist. The pharmacology of NK3 receptor is least characterized out of the three tachykinin receptor subtypes cloned and characterized for Tachykinins. To understand the structural basis of peptide-receptor interaction, the three-dimensional structure of the Scyliorhinin II in aqueous and micellar environments has been studied by two-dimensional proton nuclear magnetic resonance (2D ¹H-NMR spectroscopy) and distance geometry calculations. Proton NMR assignments have been carried out with the aid of correlation spectroscopy (gradient-COSY and TOCSY) and nuclear Overhauser effect spectroscopy (NOESY and ROESY) experiments. The inter proton distances and dihedral angle constraints obtained from the NMR data have been used in torsion angle dynamics algorithm for NMR applications (DYANA) to generate a family of structures, which have been refined using restrained energy minimization and dynamics. The results show that in an aqueous environment, Scyliorhinin II lacks a definite secondary structure. The structure is well-defined in presence of dodecyl phosphocholine micelles. The global fold of Scyliorhinin II bound to DPC micelles consists of a well-defined helix in the C-terminal region from residue 12–18 and a series of turns towards N-terminus. The structure is further stabilized by disulfide bond between Cys7 and Cys13. The conformational range of the peptide revealed by NMR and CD studies has been analyzed in terms of characteristic secondary features. Observed conformational features have been compared with those of Substance P, Neurokinin A and Neurokinin B, potent NK1, NK2, and NK3 agonists, respectively.     

Tertiary structure of mouse epidermal growth factor determined by two-dimensional 1H NMR

The tertiary structure of mouse epidermal growth factor (EGF) in solution (28 degrees C, pH 2.0) was studied by two-dimensional NMR spectroscopy. Proton-proton distance constraints derived from NOESY spectra were used to construct a mechanical molecular model of mouse EGF, which was subsequently checked by means of a preliminary distance geometry calculation. The chain-folds in the two structural domains of mouse EGF were very similar to those previously reported (Montelione et al. (1987) Proc. Natl. Acad. Sci. U.S. 84, 5226-5230). However, the relative orientations of the two domains were different. Because we could assign much more inter-domain NOEs, the relative orientations of the two domains were well determined in our model. The hollow between the two domains may function as a binding site for the EGF receptor.     

NMR snapshots of a fluctuating protein structure: ubiquitin at 30 bar-3 kbar

Conformational fluctuation plays a key role in protein function, but we know little about the associated structural changes. Here we present a general method for elucidating, at the atomic level, a large-scale shape change of a protein molecule in solution undergoing conformational fluctuation. The method utilizes the intimate relationship between conformation and partial molar volume and determines three-dimensional structures of a protein at different pressures using variable pressure NMR technique, whereby NOE distance and torsion angle constraints are used to create average coordinates. Ubiquitin (pH 4.6 at 20 degrees C) was chosen as the first target, for which structures were determined at 30 bar and at 3 kbar, giving "NMR snapshots" of a fluctuating protein structure at atomic resolution. The result reveals that the helix swings in and out by >3 angstroms with a simultaneous reorientation of the C-terminal segment, providing an "open" conformer suitable for enzyme recognition. Spin relaxation analysis indicates that this fluctuation occurs in the ten microsecond time range with activation volumes -4.2(+/-3.2) and 18.5(+/-3.0) ml/mol for the "closed-to-open" and the "open-to-closed" transitions, respectively.     

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.     

Structural characterization of neurokinin-3 receptor selective peptide agonist scyliorhinin II bound to DPC micelles

Scyliorhinin II, a cyclic Tachykinin peptide, is a potent NK3 receptor agonist. The pharmacology of NK3 receptor is least characterized out of the three tachykinin receptor subtypes cloned and characterized for Tachykinins. To understand the structural basis of peptide-receptor interaction, the three-dimensional structure of the Scyliorhinin II in aqueous and micellar environments has been studied by two-dimensional proton nuclear magnetic resonance (2D 1H-NMR spectroscopy) and distance geometry calculations. Proton NMR assignments have been carried out with the aid of correlation spectroscopy (gradient-COSY and TOCSY) and nuclear Overhauser effect spectroscopy (NOESY and ROESY) experiments. The inter proton distances and dihedral angle constraints obtained from the NMR data have been used in torsion angle dynamics algorithm for NMR applications (DYANA) to generate a family of structures, which have been refined using restrained energy minimization and dynamics. The results show that in an aqueous environment, Scyliorhinin II lacks a definite secondary structure. The structure is well-defined in presence of dodecyl phosphocholine micelles. The global fold of Scyliorhinin II bound to DPC micelles consists of a well-defined helix in the C-terminal region from residue 12-18 and a series of turns towards N-terminus. The structure is further stabilized by disulfide bond between Cys7 and Cys13. The conformational range of the peptide revealed by NMR and CD studies has been analyzed in terms of characteristic secondary features. Observed conformational features have been compared with those of Substance P, Neurokinin A and Neurokinin B, potent NK1, NK2, and NK3 agonists, respectively.     

Solution conformation of endothelin, a potent vaso-constricting bicyclic peptide. A combined use of 1H NMR spectroscopy and distance geometry calculations

The solution structure of endothelin-1, a newly discovered potent bicyclic peptide vaso-constrictor agent, has been investigated using 1H NMR conformational constraints and distance geometry calculations. The conformation is constrained by two disulphide bridges between Cys1-Cys15 and Cys3-Cys11 but the NMR data and computed conformers show additional helical structure between residues Leu6 and Cys11. Our results are compared with previous conflicting reports on the solution conformation of this peptide.     

Solution structure of tertiapin determined using nuclear magnetic resonance and distance geometry

The solution structure of tertiapin, a 21-residue bee venom peptide, has been characterized by circular dichroism (CD), two-dimensional nuclear magnetic resonance (NMR) spectroscopy, and distance geometry. A total of 21 lowest error structures were obtained from distance geometry calculations. Superimposition of these structures shows that the backbone of tertiapin is very well defined. One type-I reverse turn from residue 4 to 7 and an α-helix from residue 12 to 19 exist in the structure of tertiapin. The α-helical region is best defined from both conformational analysis and structural superimposition. The overall three-dimensional structure of tertiapin is highly compact resulting from side chain interactions. The structural information obtained from CD and NMR are compared for both tertiapin and apamin (ref. 3), another bee venom peptide. Tertiapin and apamin have some similar secondary structure, but display different tertiary structures. © 1993 Wiley-Liss, Inc.     

pH-dependent aggregate forms and conformation of Alzheimer amyloid beta-peptide (12-24)

The conformational transition to a beta-structure and the aggregation process of Alzheimer amyloid beta-peptide (12-24) [abbreviated as A beta(12-24)] were studied. The influence of sample dissolution methods for the aggregate structure was examined by electron microscopy (EM). The difference in the width of the aggregate of A beta(12-24) depended on the pH immediately after sample dissolution. Two types of sample dissolution methods, F and R, were employed. For dissolution method F, the peptide sample was immediately dissolved in water and then adjusted to pH 2.2 by adding buffer, while for dissolution method R, the peptide was directly dissolved in the buffer solution. In the latter case, the starting pH was 3.0. Slight fibrils (10-12 nm in diameter) were observed with method F, and wider ribbon-like aggregates (17-20 nm in diameter) with method R, despite the same pH range. A difference between methods F and R was also detected in the CD spectra, especially at pHs near 5.0. The CD intensity of the 214 nm band with method R changed with pH, with the highest value at pH 3.7, whereas that with method F was unchanged at pHs below 5.0. The temperature-dependent CD results showed that a thermostable aggregate of A beta(12-24) occurs at higher pHs than 3.0. NMR analysis showed that deprotonation of the C-terminal carboxylate group in A beta(12-24) triggered the aggregate formation, and the transition from a random coil to a beta-conformation in the C-terminal region of V18-V24 was detected on analysis of the (3)Ja(N) coupling constant in the pH range of 2.2 to 3.0.     

Solution structure of endothelin-3 determined using NMR spectroscopy.

The aqueous solution structure of the 21-residue vasoactive peptide hormone endothelin-3 has been determined using high-resolution NMR spectroscopy. A total of 177 proton-proton distance measurements and 5 chi 1 dihedral angle constraints derived from NMR spectra were used to calculate the structure using a combination of distance geometry and dynamical simulated annealing calculations. The calculations reveal a highly ordered, compact conformation in which a helical region extending from K9 to C15 lies in close apposition with the C-terminal hexapeptide; this interaction seems to be largely driven by hydrophobic interactions. Structure-activity studies are interpreted in terms of the conformational features of the calculated endothelin-3 structure.     

NMR conformational analyses on (des-bromo) neuropeptide B [1-23] and neuropeptide W [1-23]: the importance of alpha-helices, a cation-pi interaction and a beta-turn

The preferred conformations of the orphan G-protein coupled receptor agonists (des-bromo) neuropeptide B [1-23] and neuropeptide W [1-23], referred to as NPB and NPW, have been determined by (1)H NMR, CD, and molecular modeling. The sequences of NPB and NPW are WYKPAAGHSSYSVGRAAGLLSGL and WYKHVASPRYHTVGRAAGLLMGL, respectively. These are hypothalamic peptides that exert their biological actions on GPR7 and GPR8 receptors. Micellar solutions using the membrane mimetic, sodium dodecylsulphate-d(25) (SDS), were used to mimic a physiological environment for the peptides. The secondary structure of NPB consists of a type II beta-turn involving residues Lys(3) to Ala(6). The C-terminal region of NPB exists in a conformational equilibrium between different secondary structures, including an alpha-helix from residues Arg(15) to Ser(21), and a 3(10)-helix from residues Ser(12) to Ser(21). The N-terminus of NPW exhibits a cation-pi interaction between the Lys(3) side chain and the quadrupole moment of the Trp(1) indole group. At the C-terminus of NPW, a well-defined alpha-helical conformation exists from Arg(15) to Met(21). As NPB and NPW have 91% sequence homology from residues Val(13) to Leu(23), with only residue 21 differing between the two peptides, the similar C-terminal secondary structures of these two peptides are consistent with the sequences. This is supported by the similar CD spectra. The different secondary structures at the N-termini for NPB and NPW point to the importance of the N-terminus in receptor binding. This is consistent with the work of Fujii et al. [J. Biol. Chem. 277, 34010-34016 (2002)] who observed that iodination of the NPB Tyr(2) resulted in decreased agonistic activity at GPR7. In addition, Tanaka et al. [Proc. Natl. Acad. Sci. USA 100, 6251-6256 (2003)] showed that deletion of Trp(1) from NPB or NPW drastically decreased activity at GPR7 for NPB and GPR7 and GPR8 for NPW. Therefore, we postulate that the N-terminus is involved in membrane recognition and receptor binding.     

Three-dimensional structure of neuropeptide k bound to dodecylphosphocholine micelles

Neuropeptide K (NPK), an N-terminally extended form of neurokinin A (NKA), represents the most potent and longest lasting vasodepressor and cardiomodulatory tachykinin reported thus far. NPK has been shown to have high selectivity for the NK2 receptor. Because the micelle-associated structure may be relevant to the NPK-receptor interaction, the three-dimensional structure of the NPK in aqueous and micellar environments has been studied by two-dimensional proton nuclear magnetic resonance (2D (1)H NMR spectroscopy) and distance geometry calculations. Proton NMR assignments have been carried out with the aid of correlation spectroscopy (DQF-COSY and TOCSY) and nuclear Overhauser effect spectroscopy (NOESY and ROESY) experiments. The interproton distances and dihedral angle constraints obtained from the NMR data have been used in torsion angle dynamics algorithm for NMR applications (DYANA) to generate a family of structures, which have been refined using restrained energy minimization and dynamics. The results show that in an aqueous environment NPK lacks a definite secondary structure, although some turn-like elements are present in the N terminus. The structure is well-defined in the presence of dodecylphosphocholine micelles. The global fold of NPK bound to DPC micelles consists of two well-defined helices from residues 9 to 18 and residues 27 to 33 connected by a noncanonical beta turn. The N terminus of the peptide is characterized by a 3(10) helix or a series of dynamic beta turns. The conformational range of the peptide revealed by NMR and circular dichroism (CD) studies has been analyzed in terms of characteristic secondary features. The observed conformational features have been further compared to a NKA and neuropeptide gamma (NPgamma) potent endogenous agonist for the NK2 receptor.     

Structure refinement of a cyclic peptide from two-dimensional NMR data and molecular modeling

The conformational and dynamic properties of a cyclic peptide designed to inhibit human renin have been examined by using NMR and molecular modeling. From a quantitative analysis of a series of two-dimensional NOE data sets, proton-proton distances were calculated. Several different methods were explored and compared to incorporate these distance constraints as well as those derived from vicinal spin-spin coupling constants into computer-generated three-dimensional structures. These methods included interactive manual manipulation of the structures to fit the NMR-determined distance constraints, distance geometry, constrained energy minimizations, and constrained molecular dynamics. The advantages and disadvantages of the methods are discussed. In addition, to gain insight into the conformations accessible to the cyclic peptide and the relative flexibility of the different parts of the molecule, molecular dynamics calculations were performed at three different temperatures. Average interproton distances and dihedral angles were obtained from the structures generated in the dynamics trajectories and compared to those obtained from the NMR experiments. Despite the four methylene groups and ether linkage contained in the cyclic portion of the peptide, our NMR results indicated a preferred conformation for the macrocyclic ring of the peptide and supported the presence of a cis Phe-Ala peptide bond. In contrast, both the molecular dynamics and NMR data indicated a considerable amount of flexibility for the remaining noncyclic portion of the molecule. These results are used to propose an explanation for the cyclic peptide's inability to inhibit human renin.     

Conformational comparison of mu-selective endomorphin-2 with its C-terminal free acid in DMSO solution, by 1H NMR spectroscopy and molecular modeling calculation.

In order to make clear the structural role of the C-terminal amide group of endomorphin-2 (EM2, H-Tyr-Pro-Phe-Phe-NH2), an endogenous mu-receptor ligand, in the biological function, the solution conformations of endomorphin-2 and its C-terminal free acid (EM2OH, H-Tyr-Pro-Phe-Phe-OH), studied using two-dimensional 1H NMR measurements and molecular modeling calculations, were compared. Both peptides were in equilibrium between the cis and trans isomers around the Tyr-Pro omega bond in a population ratio of approximately/= 1:2. The lack of significant temperature and concentration dependence of NH protons suggested that the NMR spectra reflected the conformational features of the respective molecules themselves. Fifty possible 3D structures for the each isomer were generated by the dynamical simulated annealing method under the proton-proton distance constraints derived from the ROE cross-peaks. These energy-minimized conformers, which were all in the phi torsion angles estimated from J(NHCalphaH) coupling constants within +/- 30 degrees, were then classified in groups one or two according to the folding backbone structures. All trans and cis EM2 conformers adopt an open conformation in which their extended backbone structures are twisted at the Pro2-Phe3 moiety. In contrast, the trans and cis conformers of EM2OH show conformational variation between the 'bow'-shaped extended and folded backbone structures, although the cis conformers of its zwitterionic form are refined into the folded structure of the close disposition of C- and N-terminal groups. These results indicate clearly that the substitution of carboxyl group for C-terminal amide group makes the peptide flexible. The conformational requirement for mu-receptor activation has been discussed based on the active form proposed for endomorphin-1 and by comparing conformational features of EM2 and EM2OH.     

Fluoroalcohols as structure modifiers in peptides and proteins: hexafluoroacetone hydrate stabilizes a helical conformation of melittin at low pH.

The effect of hexafluoroacetone hydrate (HFA) on the structure of the honey bee venom peptide melittin has been investigated. In aqueous solution at low pH melittin is predominantly unstructured. Addition of HFA at pH approximately 2.0 induces a structural transition from the unstructured state to a predominantly helical conformation as suggested by intense diagnostic far UV CD bands. The structural transition is highly cooperative and complete at 3.6 M (50% v/v) HFA. A similar structural transition is also observed in 2,2,2 trifluoroethanol which is complete only at a cosolvent concentration of approximately 8 M. Temperature dependent CD experiments support a 'cold denaturation' of melittin at low concentrations of HFA, suggesting that selective solvation of peptide by HFA is mediated by hydrophobic interactions. NMR studies in 3.6 M HFA establish a well-defined helical structure of melittin at low pH, as suggested by the presence of strong NH/NHi+1 NOEs throughout the sequence, along with many medium range helical NOEs. Structure calculations using NOE-driven distance constraints reveal a well-ordered helical fold with a relatively flexible segment around residues T10-G11-T12. The helical structure of melittin obtained at 3.6 M HFA at low pH is similar to those determined in methanolic solution and perdeuterated dodecylphosphocholine micelles. HFA as a cosolvent facilitates helix formation even in the highly charged C-terminal segment.     

Solution structure of murine epidermal growth factor determined by NMR spectroscopy and refined by energy minimization with restraints.

The solution structure of murine epidermal growth factor (mEGF) at pH 3.1 and a temperature of 28 degrees C has been determined from NMR data, using distance geometry calculations and restrained energy minimization. The structure determination is based on 730 conformational constraints derived from NMR data, including 644 NOE-derived upper bound distance constraints, constraints on the ranges of 32 dihedral angles based on measurements of vicinal coupling constants, and 54 upper and lower bound constraints associated with nine hydrogen bonds and the three disulfide bonds. The distance geometry interpretation of the NMR data is based on previously published sequence-specific 1H resonance assignments [Montelione et al. (1988) Biochemistry 27, 2235-2243], supplemented here with individual assignments for some side-chain amide, methylene, and isopropyl methyl protons. The molecular architecture of mEGF is the same as that described previously [Montelione et al. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 5226-5230], but the structure is overall more precisely determined by a more extensive set of NMR constraints. Analysis of proton NMR line widths, amide proton exchange rates, and side-chain 3J(H alpha-H beta) coupling constants provides evidence for internal motion in several regions of the mEGF molecule. Because mEGF is one member of a large family of homologous growth factors and protein domains for which X-ray crystal structures are not yet available, the atomic coordinates resulting from the present structure refinement (which we have deposited in the Brookhaven Protein Data Bank) are important data for understanding the structures of EGF-like proteins and for further detailed comparisons of these structures with mEGF.