Controlled proteolysis of mouse epidermal growth factor. An RP-HPLC and 1H-n.m.r. study

Tryptic digestion of the mouse epidermal growth factor (mEGF) and the chromatographic separation of its proteolytic fragments by RP-HPLC affords the isolation of the pure hormone, of its 1-48 (Des(49-53)mEGF) and 1-45 (Des(46-53)mEGF) derivatives, and of the carboxyl-terminal pentapeptide W49-W50-E51-L52-R53. Kinetics of mEGF proteolytic degradation follows a two-state time-course: native mEGF being converted into Des(49-53)mEGF with an apparent half-time of 10 min; and Des(49-53)mEGF subsequently hydrolyzed to Des(46-53)mEGF with an apparent half-time of 7 h. Native mEGF and its proteolytic fragments have been characterized by 1H-n.m.r. spectroscopy. In the aromatic and aliphatic regions, the 1H-n.m.r. spectrum proved to be a sufficiently sensitive probe for following controlled proteolysis, and for analyzing the influence of the carboxyl-terminal sequence on the hormone conformation and stability.     

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.     

Isolation, purification and 13C- and 1H-n.m.r. assignments of peptide [1-24] of human serum albumin

Isolation, purification and 360 MHz 1H- and 13C-n.m.r. spectra of the residue corresponding to the NH2-terminal peptide fragment [1-24] of human serum albumin are reported. The various resonances have been assigned to individual amino acid residues and their spatial microenvironment has been determined in a straightforward manner on the basis of (i) pH dependent chemical shifts; (ii) combined use of multiple and selective proton-decoupled 1H- and 13C-n.m.r. spectra; (iii) the characteristic pK values exhibited by protons adjacent to sites of ionization in the molecule; and (iv) comparison of the spectra with the NH2-terminal tripeptide segment of human albumin. The pK values of different ionizable groups all fall in the normal range expected for each titrating sites and support a model of peptide fragment [1-24] in which there is no special structure-forming strong associations. These results are in agreement with those obtained by CD spectroscopy.     

Sequence-specific 1H n.m.r. assignments and determination of the three-dimensional structure of reduced Escherichia coli glutaredoxin

The determination of the nuclear magnetic resonance structure of reduced E. coli glutaredoxin in aqueous solution is described. Based on nearly complete, sequence-specific resonance assignments, 813 nuclear Overhauser effect distance constraints and 191 dihedral angle constraints were employed as the input for the structure calculations, for which the distance geometry program DIANA was used followed by simulated annealing with the program X-PLOR. The molecular architecture of reduced glutaredoxin is made up of three helices and four-stranded beta-sheet. The first strand of the beta-sheet (residues 2 to 7) runs parallel to the second strand (32 to 37) and antiparallel to the third strand (61 to 64), and the sheet is extended in an antiparallel fashion with a fourth strand (67 to 69). The first helix with residues 13 to 28 and the last helix (71 to 83) run parallel to each other on one side of the beta-sheet, with their direction opposite to that of the two parallel beta-strands, and the helix formed by residues 44 to 53 fills space available due to the twist of the beta-sheet and the reduced length of the last two beta-strands. The active site Cys11-Pro-Tyr-Cys14 is located after the first beta-strand and occupies the latter part of the loop connecting this strand with the first helix.     

1H-n.m.r. studies of squash seed trypsin inhibitor

1H-n.m.r. studies at 500 MHz have been performed on a trypsin inhibitor (CMTI-III) found in squash seed (Cucurbita maxima). The sequential resonance assignments have been made using two-dimensional techniques. The chemical shifts for the assigned protons are reported at 30 degrees, pH 2.8 and form a basis for the determination of the solution structure of CMTI-III. Analysis of the NOE data, NH-alpha CH vicinal coupling constants and pattern of slowly exchanging amide protons indicates that the predominant feature of the solution conformation is a triple stranded beta sheet consisting of residues 8-10, 21-23, and 26-29. Residues 12-15 appear to form a beta turn.     

High resolution 1H-n.m.r. study of the interaction between initiation factor IF1 and 30S ribosomal subunits

Interaction between Escherichia coli translational initiation factor IF1 (mol. wt. 8119) and 30S ribosomal subunits was followed by high resolution 1H-n.m.r. spectroscopy. Upon gradual addition of increasing yet largely sub-stoichiometric amounts of biologically active deuterated 30S ribosomal subunits, selective line broadenings and chemical shift changes were observed against the background of the gradual disappearance of the whole spectrum. At the highest 30S:IF1 ratio attained (0.25), all the resonance lines were broadened beyond meaningful detection. This behaviour, which can be partly reversed by increasing the ionic strength and/or the temperature, is due to the interaction between IF1 and the 30S ribosomal subunits, and can be explained by the existence of a medium-fast exchange dynamics between free and bound factor. The selective effects observed during titration with 30S ribosomal subunits shed some light on the mode of interaction of IF1 with 30S ribosomal subunits. At least one of the two His residues of the factor appears to be involved in the binding, since it undergoes a low-field change of chemical shift and becomes totally immobilized in the IF1-30S complex. Also strongly implicated in the interaction with 30S are more than one Ser and Arg residue and probably one lysine. Additional effects of the interaction of IF1 with ribosomes are a drastic reduction in the intensity of the ring current upfield shifted methyl resonances and mobilization of a previously rotationally hindered phenylalanine ring.     

Conformational studies on gastrin related peptides by high resolution 1H-n.m.r

The secondary structures of three gastrin analogs, HC1 X H-Trp-Nle-Asp(O-tBu)-Phe-NH2 (tetragastrin), pGlu-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2 (octagastrin), and H-Leu-(Glu)5-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2 (minigastrin) were studied by 1H-n.m.r. in dimethylsulfoxide and in trifluoroethanol. All three compounds were found to assume a random conformation in the former solvent, while some ordered secondary structure is present in trifluoroethanol even at the tetrapeptide level. This was shown by temperature studies and solvent titrations. At least four amide protons were found to be solvent shielded in the longer hormone.     

Sequence-specific 1H NMR assignments, secondary structure, and location of the calcium binding site in the first epidermal growth factor like domain of blood coagulation factor IX.

Factor IX is a blood clotting protein that contains three regions, including a gamma-carboxyglutamic acid (Gla) domain, two tandemly connected epidermal growth factor like (EGF-like) domains, and a serine protease region. The protein exhibits a high-affinity calcium binding site in the first EGF-like domain, in addition to calcium binding in the Gla domain. The first EGF-like domain, factor IX (45-87), has been synthesized. Sequence-specific resonance assignment of the peptide has been made by using 2D NMR techniques, and its secondary structure has been determined. The protein is found to have two antiparallel beta-sheets, and preliminary distance geometry calculations indicate that the protein has two domains, separated by Trp28, with the overall structure being similar to that of EGF. An NMR investigation of the calcium-bound first EGF-like domain indicates the presence and location of a calcium binding site involving residues on both strands of one of the beta-sheets as well as the N-terminal region of the peptide. These results suggest that calcium binding in the first EGF-like domain could induce long-range (possibly interdomain) conformational changes in factor IX, rather than causing structural alterations in the EGF-like domain itself.     

Polydepsipeptides. 13. Synthesis and 1H-n.m.r. analysis of collagen model structures

The synthesis of three collagen model analogs is described: Ac-Ala-Gly-Pro-Ala-Gly-Pro-NHMe, Ac-Ala-Gly-Pro-Ala-Glc-Pro-NHMe, and Ac-Ala-Glc-Pro-Ala-Gly-Pro-NHMe, where Glc stands for glycolic acid. The 1H-n.m.r. properties of these compounds in dimethylsulfoxide-d6 and trifluoroethanol are described. While in DMSO-d6 the compounds are random, in TFE the glycine amide protons seem to be less solvent exposed than the other amide protons. Little difference was found in the behavior of the three compounds.     

1H- and 2H-n.m.r. studies of water in gamma-irradiated phosphatidylcholine multilamellar liposomes

1H- and 2H-n.m.r. studies of gamma-irradiation-induced variations in the dynamic structure and proportional amounts of free, trapped and bound water species in multilamellar liposomes are reported and discussed. Bound water is shown to increase with dose and to be present in two different structural states. A dose-dependent decrease in the 1H-n.m.r. relaxation times of bound water following gamma-irradiation is reported. Variations are suggested as being due to large scale changes at the bilayer surface.     

A 2D-1H-n.m.r. study of some Shigella flexneri O-polysaccharides

A 1H-n.m.r. study of the O-polysaccharides from different types of Shigella flexneri has been performed. With the aid of 2D-n.m.r. techniques, namely, J-resolved, spin-spin correlation, and NOESY experiments, most of the structural features of these polysaccharides could be deduced. Sequences could generally be obtained from the NOESY experiments. When using a prolonged mixing time in these experiments, cross-peaks due to spin diffusion from one anomeric proton to the anomeric proton of an adjacent residue could be obtained, thereby giving complementary sequence information.     

1H-n.m.r. investigation of the interaction between cytochrome c and cytochrome b5.

The interaction between eukaryotic cytochrome c and the tryptic fragment of bovine liver microsomal cytochrome b5 was studied by 1H-n.m.r. spectroscopy, and a procedure was developed that may be generally applicable to the study of macromolecular interactions by n.m.r. At pH6.3 (27 degrees C, I approx. 0.04) the two ferricytochromes were found to form a 1:1 complex with an association constant of approx. 10(3) M -1. The protein--protein-interaction region was found to encompass the region of the surface of horse cytochrome c that includes Ile-81, Phe-82, Ala-83 and Ile-85, and Lys-13 and Lys-72 of horse cytochrome c were suggested to be involved in two important intermolecular interactions. Me3Lys-72 of Candida krusei cytochrome c was shown to be involved in the interaction.     

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.     

NMR backbone assignments of the tyrosine kinase domain of human fibroblast growth factor receptor 1

Members of the fibroblast growth factor receptor tyrosine kinase family (FGFR1–4) play an important role in many signalling cascades. Although tightly regulated, aberrant activity of these enzymes may lead to, or become features of, disease pathologies including cancer. FGFR isoforms have been the subject of drug discovery programmes, with a number of kinase-domain inhibitors in pre-clinical and clinical development. Here, we present the first (83 % complete) backbone resonance assignments of apo-FGFR1 kinase.     

1H- and 13C-n.m.r. studies of the antitumour antibiotic luzopeptin. Resonance assignments, conformation and flexibility in solution.

The depsipeptide DNA-intercalating antibiotic luzopeptin was studied in solution by n.m.r. methods. Two-dimensional 1H double-quantum-filtered correlation spectroscopy (DQF-COSY) and nuclear-Overhauser-effect spectroscopy (NOESY) confirm the primary structure and twofold symmetry of luzopeptin and provide details of its three-dimensional conformation in solution. Trans-annular hydrogen bonds between the glycine NH groups and carbonyl oxygen atoms have been identified in the crystalline state [Arnold & Clardy (1981) J. Am. Chem. Soc. 103, 1243-1244], and are important in maintaining an antiparallel beta-sheet conformation. The n.m.r. data indicate that the glycine NH protons are appreciably shielded from the solvent molecules, which suggests that these hydrogen bonds are maintained in solution. The orientation of the quinoline chromophores is defined by two-dimensional NOE cross-peaks that position the N-methyl group of the L-beta-hydroxyvaline residue close in space to both the quinoline H-8 and serine NH proton. This pattern of NOEs is in accord both with the chromophore configuration found in the crystal and one where the quinoline rings are aligned in a parallel manner at right-angles to the depsipeptide ring. The n.m.r. data are consistent with a hydrogen bond between the quinoline hydroxy groups and the quinoline carbonyl oxygen atoms. The pyridazine acetylmethyl groups give NOEs to the C(alpha)H groups of the beta-hydroxy-N-methylvaline residues, showing that the acetyl groups, for at least some of the time, stretch over the depsipeptide ring, occluding one face of the molecule. Both of the latter features are also found in the crystal structure. Resonances in the 13C-n.m.r. spectrum of luzopeptin have been assigned by transferring 1H assignments to their covalently bonded carbon atoms via a heteronuclear shift-correlation experiment (HETCOR). The measurement of spin-lattice relaxation times and 1H-13C NOEs at specific sites in the molecule has led us to conclude that segmental motions within the depsipeptide ring are restricted and that the 13C relaxation data for luzopeptin's protonated carbon atoms are adequately described by isotropic tumbling in solution. Furthermore, relaxation data for the carbon atoms of the quinoline chromophores show that these rings exhibit similar motion to the depsipeptide ring and are not rotating rapidly with respect to it. Taken together all the data imply that luzopeptin is fairly rigid in solution, on the time scale of molecular tumbling, and has, or can readily attain, a staple-like structure suitable for bisintercalation.(ABSTRACT TRUNCATED AT 400 WORDS)