Structure of epidermal growth factor bound to perdeuterated dodecylphosphocholine micelles determined by two-dimensional NMR and simulated annealing calculations.

The interaction of mouse epidermal growth factor (mEGF) with micelles of a phospholipid analogue, perdeuterated dodecylphosphocholine (DPC), was investigated by two-dimensional 1H NMR. Sequence-specific resonance assignments of the micelle-bound mEGF have been made, and the chemical shifts were compared with those in the absence of DPC. DPC induced large chemical shift changes of the resonances from the residues in the C-terminal tail (residues 46-53) but little perturbation on the residues in the main core (residues 1-45). Starting from the three-dimensional structure in the absence of DPC, micelle-bound structures were calculated using the program XPLOR with interproton distance data obtained from NOESY spectra recorded in the presence of DPC. The C-terminal tail of mEGF was found to change conformation to form an amphiphilic structure when bound to the micelles. It is possible that induced fit in the C-terminal tail of mEGF occurs upon binding to a putative hydrophobic pocket of the EGF receptor.     

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.     

Sequence-specific 1H NMR assignment and secondary structure of the Arc repressor of bacteriophage P22, as determined by two-dimensional 1H NMR spectroscopy

The Arc repressor of bacteriophage P22 is a DNA binding protein that does not belong to any of the known classes of such proteins. We have undertaken a 1H NMR study of the protein with the aim of elucidating its three-dimensional structure in solution and its mode of binding of operator DNA. Here we present the 1H nuclear magnetic resonance (NMR) assignments of all backbone protons and most of the side-chain protons of Arc repressor. Elements of secondary structure have been identified on the basis of networks of characteristic sequential and medium-range nuclear Overhauser enhancements (NOEs). Two alpha-helical regions have been found in the peptide regions 16-29 and 35-45. The ends of the helices could not yet be firmly established and could extend to residue 31 for the first helix and to residue 49 for the second. Immediately before the first helix, between residues 8 and 14, a region is present with beta-sheet characteristics dominated by a close proximity of the alpha-protons of residues 9 and 13. Because of the dimeric nature of the protein there are still two possible ways in which the NOEs in the beta-sheet region can be interpreted. If the NOEs are intramonomer, this requires a tight turn involving residues 10-12. Alternatively, if the NOEs are intermonomer, then and antiparallel beta-sheet would be implicated comprising two strands of different Arc monomers. While the data presently do not allow an unambiguous choice between these two possibilities, some evidence is discussed that favors the latter (beta-sheet between monomers).(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure-function relationships in human epidermal growth factor studied by site-directed mutagenesis and 1H NMR

In order to elucidate the mechanism of interaction between human epidermal growth factor (EGF) and its receptor, selected variants of EGF, differing by single amino acid substitutions, have been made by site-directed mutagenesis. The receptor affinity of these mutants was determined by a receptor binding competition assay, and the effects of the substitution on the structure of the protein were assessed by 1H nuclear magnetic resonance techniques. Various substitutions of Arg-41 resulted in substantial reduction in receptor affinity of EGF whereas change of Tyr-13 did not affect binding to the receptor. The 1H resonances of all nonexchangeable protons of the Tyr-13----Leu, Arg-41----His, and Leu-47----Glu variants were assigned and compared in order to assess the structural integrity of these mutants, which possess very different spectral and biological properties. In the case of the Leu-47----Glu mutant, only minor localized spectral changes were observed, confirming that the tertiary structure of the protein is preserved upon mutation. In contrast, for both the Arg-41----His and Tyr-13----Leu variants, significant and strikingly similar spectra changes were observed for many residues located far away from the mutated residues. This implies that similar structural alterations have taken place in both proteins, an idea further supported by hydrogen-exchange experiments where the exchange rates of hydrogen-bonded amide protons for both the Tyr-13----Leu and the Arg-41----His mutants were found to be about 4 times faster than in the wild-type protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Complete sequence-specific 1H nuclear magnetic resonance assignments for mouse epidermal growth factor

The 1H NMR spectrum of the mouse epidermal growth factor (53 residues) was analyzed with the use of two-dimensional NMR techniques. All the observable 296 proton resonances were completely assigned in a sequential manner. For the spin system identification, two-dimensional homonuclear Hartmann-Hahn spectrum was useful, especially for arginine and proline residues. The easy spin system identification of these long-side-chain-bearing amino acid residues greatly facilitated the sequence-specific resonance assignment of the epidermal growth factor.     

Solution structure of alpha-conotoxin ImI determined by two-dimensional NMR spectroscopy.

The three-dimensional structure of alpha-conotoxin ImI, a potent antagonist targeting the neuronal alpha7 subtype of nicotinic acetylcholine receptor (nAChR), has been investigated by NMR spectroscopy. On the basis of 181 experimental constraints, a total of 25 converged structures were obtained. The average pairwise atomic root mean square difference is 0.40+/-0.11 A for the backbone atoms. The resulting structure indicates the presence of two successive type I beta-turns and a 310 helix for residues Cys2-Cys8 and Ala9-Arg11, respectively, and shows a significant structural similarity to that of alpha-conotoxin PnIA, which is also selective for the neuronal nAChR.     

Secondary and tertiary structure characteristics of Megasphaera elsdenii flavodoxin in the reduced state as determined by two-dimensional 1H NMR

The secondary structure of two-electron-reduced Megasphaera elsdenii flavodoxin has been determined by visual, qualitative inspection of the sequential connectivities involving C alpha H, C beta H and NH protons observed in NOESY (two-dimensional nuclear Overhauser enhancement spectroscopy) spectra. Results from an amide proton exchange experiment were used to confirm the secondary structure assignment and to demonstrate the compactness and stability of the protein. After the secondary structure elements were established, the global fold of the protein and the flavin binding site have been determined using nonsequential interresidual NOE connectivities as primary source of information. The secondary structure and the global fold of M. elsdenii and Clostridium MP flavodoxin appeared to be very similar, differences are observed however. M. elsdenii flavodoxin consists of a central parallel beta-sheet including five strands surrounded on both sides by a pair of alpha-helices.     

Interaction of epidermal growth factor with micelles monitored by photochemically induced dynamic nuclear polarization-1H NMR spectroscopy

Photochemically induced dynamic nuclear polarization (CIDNP)-1H-NMR spectroscopy has been used to study the interaction of the protein hormone epidermal growth factor (EGF) with micelles of sodium dodecyl sulfate (SDS) and dodecylphosphorylcholine (DPC). Conventional 1H-NMR spectra show that most protein resonances remain unperturbed when micelles are added to solution, which argues that the overall protein conformation is maintained in the presence of SDS or DPC at the concentrations used. Photo-CIDNP enhancements of resonances assigned to aromatic side chains of residues at the COOH terminus and beta-sheet regions of murine EGF (i.e. Trp-49, Trp-50, and Tyr-37) are considerably reduced in the presence of micelles, while resonances of aromatic side chains of residues found elsewhere on the protein surface are mostly unaffected. This suggests that the primary interaction between murine EGF and the micelle occurs at the micelle-bulk solvent interface. The overall negatively charged surface of SDS micelles tends to induce a stronger interaction with the protein compared to the zwitterionic DPC micelles, probably due to electrostatic interactions. Cleavage of the COOH-terminal pentapeptide containing both tryptophan residues enhances the already present, but weak, interaction with Tyr-10 and attenuates it with Tyr-37. A similar interaction pattern is found with rat EGF suggesting that at least concerning these two species of EGF the interaction is somewhat specific and conserved. A simple mass-action model for protein-micelle interaction is also presented.     

Secondary structure of a complement control protein module by two-dimensional 1H NMR

The complement control protein (CCP) module (also known as the short consensus repeat) is a consensus sequence of about 60 amino acid residues which is thought to fold independently. It occurs over 140 times in more than 20 extracellular mosaic proteins including 12 proteins of the complement cascade. An isolated CCP module, the 16th repeat from human complement factor H, has been expressed in a yeast vector and shown to fold with the same pattern of disulfide bond formation as is seen in the native protein. Two-dimensional 600-MHz 1H NMR spectra of this module have been recorded at pH 3.3 and 6.0 and analyzed to permit determination of secondary structure in solution. The CCP module comprises two predominantly extended segments (Glu1-His13 and Ala17-Glu27), two segments of double-stranded antiparallel beta-sheet (Gly14-Val16 paired with Tyr31-Cys33 and Gly38-Asp40 paired with Ser57-Ile59), and a short piece of triple-stranded beta-sheet (Glu27-Thr30, Ile44-Leu48, and Lys51-Ser53). Turns occur at Asp22, Gly36, and Glu50, while Gly41-Ala43 appear to form a looped-out segment or bulge. This structure is compared with a secondary structure prediction made on the basis of an alignment scheme of 101 sequences for CCP modules [Perkins, S. J., Haris, P. I., Sim, R. B., & Chapman, D. (1988) Biochemistry 27, 4004-4012]--the experimentally determined secondary structure bears an overall resemblance to the predicted one but differs in the number and position of turns. Some of those amino acid residues which are highly conserved throughout the range of CCP modules appear to play a role in stabilizing the global fold.     

Solution structure of Fe(II) cytochrome c551 from Pseudomonas aeruginosa as determined by two-dimensional 1H NMR.

The solution structure of Fe(II) cytochrome c551 from Pseudomonas aeruginosa based on 2D 1H NMR data is reported. Two sets of structure calculations were completed with a combination of simulated annealing and distance geometry calculations: one set of 20 structures included the heme-peptide covalent linkages, and one set of 10 structures excluded them. The main-chain atoms were well constrained within the two structural ensembles (1.30 and 1.35 A average RMSD, respectively) except for two regions spanning residues 30-40 and 60-70. The results were essentially the same when global fold comparisons were made between the ensembles with an average RMSD of 1.33 A. In total, 556 constraints were used, including 479 NOEs, 53 volume constraints, and 24 other distances. This report represents the first solution structure determination of a heme protein by 2D 1H NMR and should provide a basis for the application of these techniques to other proteins containing large prosthetic groups or cofactors.     

Solution structures of alpha-conotoxin G1 determined by two-dimensional NMR spectroscopy

Two-dimensional NMR data have been used to generate solution structures of alpha-conotoxin G1, a potent peptide antagonist of the acetylcholine receptor. Structural information was obtained in the form of proton-proton internuclear distance constraints, and initial structures were produced with a distance geometry algorithm. Energetically more favorable structures were generated by using the distance geometry structures as input for a constrained energy minimization program. The results of both of these calculations indicate that the overall backbone conformation of the molecule is well-defined by the NMR data whereas the side-chain conformations are generally less well-defined. The main structural features derived from the NMR data were the presence of tight turns centered on residues Pro5 and Arg9. The solution structures are compared with previous proposed models of conotoxin G1, and the NMR data are interpreted in conjunction with chemical modification studies and structural properties of other antagonists of the acetylcholine receptor to gain insight into structure-activity relationships in these peptide toxins.     

Sequence-specific 1H NMR assignments and identification of slowly exchanging amide protons in murine epidermal growth factor

Proton nuclear magnetic resonance (1H NMR) assignments for the murine epidermal growth factor (mEGF) in aqueous solution were determined by using two-dimensional NMR at pH 3.1 and 28 degrees C. The assignments are complete for all backbone hydrogen atoms, with the exception of the N-terminal amino group, and for 46 of the 53 side chains. Among the additional seven amino acid residues, three have complete assignments for all but one side-chain proton, and between two and four protons are missing for the remaining four residues. The sequential assignments by nuclear Overhauser effect spectroscopy are consistent with the chemically determined amino acid sequence. The NMR data show that the conformations of both the Tyr3-Pro4 and Cys6-Pro7 peptide bonds are trans in the predominant solution structure. Proton-deuterium exchange rate constants were also measured for 13 slowly exchanging amide protons. The information presented here has been used elsewhere to determine the three-dimensional structure of mEGF in aqueous solution.     

Structure of the oligosaccharide of hen phosvitin as determined by two-dimensional 1H NMR of the intact glycoprotein

The major form of the oligosaccharide of hen phosvitin was studied with two-dimensional 1H NMR of the intact glycoprotein. Its structure was determined from an analysis of the chemical shifts of the structural reporter groups, and it was further confirmed by comparison to several related model oligosaccharides. The oligosaccharide is N-linked and is present in a 1:1 stoichiometry to the protein. It has a complex type 1 triantennary structure with two NeuAc alpha 2,6Gal beta 1,4GlcNAc beta 1,2 arms linked to the Man-4 and Man-4' and a third Gal beta 1, 4GlcNAc beta 1,4 arm attached to the Man-4. The oligosaccharide contains the common core sequence which is present in all N-linked glycoproteins [Man alpha 1,3(Man alpha 1,6)-Man beta 1,4GlcNAc beta 1,4GlcNAc beta 1,N]. In the course of this study, we have found that unique spin systems for the GlcNAc and NeuAc are obtained for spectra recorded in 90% H2O. Their NH peaks were assigned at low pH, and these assignments proved useful for confirming the identity of cross-peaks in the anomeric region. In addition, the protons of GlcNAc-1 could be correlated to the NH of the asparagine link. The cross-peak patterns determined in phase-sensitive 2D experiments for the H1,H2 protons have a different appearance for each type of monosaccharide, and this information was also used for making first-order assignments. A comparison with model compounds suggests that the solution conformation of the oligosaccharide is not affected by its attachment to the protein.     

1H NMR assignment and secondary structure of the Ca2(+)-free form of the amino-terminal epidermal growth factor like domain in coagulation factor X

Blood coagulation factor X is composed of discrete domains, two of which are homologous to the epidermal growth factor (EGF). The N-terminal EGF like domain in factor X (fX-EGFN), residues 45-86 of the intact protein, contains a beta-hydroxylated aspartic acid and has one Ca2(+)-binding site. Using 2D NMR techniques, we have made a full assignment of the 500-MHz 1H NMR spectrum of Ca2(+)-free fX-EGFN. On the basis of this assignment and complementary NOESY experiments, we have also determined the secondary structure of Ca2(+)-free fX-EGFN in water solution. Residues 45-49 are comparatively mobile, whereas residues 50-56 are constrained by two disulfide bonds to one side of an antiparallel beta-sheet involving residues 59-64 and 67-72. Another antiparallel beta-sheet involves residues 76-77 and 83-84. A small, parallel beta-sheet connects residues 80-81 and 55-56 and thereby orients the two antiparallel beta-sheets relative to each other. Four beta-turns are identified, involving residues 50-53, 56-59, 64-67, and 73-76. Residues 78-82 adopt an extended bend structure. On the basis of secondary structure and the location of the three disulfide bonds, we find that Asp 46, Asp 48, and Hya 63 are sufficiently close to each other to form a Ca2(+)-binding site. However, the amino terminus of the Ca2(+)-free form of fX-EGFN is not part of a triple-stranded beta-sheet as in other EGF like peptides. Differences and similarities between fX-EFGN and murine EGF with respect to secondary structure and conformational shifts are discussed.     

Sequential resonance assignments in 1H NMR spectra of oligonucleotides by two-dimensional NMR spectroscopy

A sequential assignment procedure is outlined, based on two-dimensional NOE ( NOESY ) and two-dimensional J-correlated spectroscopy ( COSY ), for assigning the nonexchangeable proton resonances in NMR spectra of oligonucleotides. As presented here the method is generally applicable to right-handed helical oligonucleotides of intermediate size. We applied it to a lac operator DNA fragment consisting of d( TGAGCGG ) and d( CCGCTCA ) and obtained complete assignments for the adenine H8, guanine H8, cytosine H6 and H5, thymine H6 and 5-methyl, and the deoxyribose H1', H2', H2", H3', and H4' resonances, as well as some H5', H5" (pairwise) assignments. These assignments are required for the analysis of two-dimensional NOE and J-coupling data in terms of the solution structure of oligonucleotides.     

Secondary structure of acyl carrier protein as derived from two-dimensional 1H NMR spectroscopy

Sequence-specific assignments of 1H NMR resonances were obtained for the backbone protons in acyl carrier protein (ACP) from Escherichia coli, a protein of 77 residues. The observations, in the NOESY spectra, of 1H-1H sequential and medium-range connectivities indicate the presence of three or four alpha-helical segments joined by short sequences of mixed conformations. The observations are used to refine a secondary structure model previously proposed on the basis of a Chou-Fasman algorithm [Rock, C. O., & Cronan, J. E., Jr. (1979) J. Biol. Chem. 254, 9778-9785].