Nuclear magnetic resonance investigation of the conformation of δ-haemolysin bound to dodecylphosphocholine micelles

δ-Haemolysin in mixed micelles with perdeuterated dodecylphosphocholine was investigated with two-dimensional proton nuclear magnetic resonance experiments at 500 MHz. A single set of resonance lines was observed for the micelle-bound polypeptide, indicating that δ-haemolysin adopts a single conformation in this environment. Nearly complete, sequence-specific assignments were obtained for the segment 5–23 of this 26 residue polypeptide chain. From the sequential connectivities and numerous medium-range nuclear Overhauser effects this central portion of the molecule was found to form an extended helix with pronounced amphipathic distribution of polar and nonpolar amino acid side-chains.     

Sequence-specific 1H NMR assignments and secondary structure of eglin c

Sequence-specific nuclear magnetic resonance assignments were obtained for eglin c, a polypeptide inhibitor of the granulocytic proteinases elastase and cathepsin G and some other proteinases. The protein consists of a single polypeptide chain of 70 residues. All proton resonances were assigned except for some labile protons of arginine side chains. The patterns of nuclear Overhauser enhancements and coupling constants and the observation of slow hydrogen exchange were used to characterize the secondary structure of the protein. The results indicate that the solution structure of the free inhibitor is very similar to the crystal structure reported for the same protein in the complex with subtilisin Carlsberg. However, a part of the binding loop seems to have a significantly different conformation in the free protein.     

N-terminal amino acid sequencing of EDP208 conjugative pili.

EDP208 conjugative pili contain a single polypeptide subunit of 11,500 daltons with a blocked N-terminus. This N-terminal blocking moiety was identified as an N-acetyl group by 1H nuclear magnetic resonance analysis of an N-terminal tripeptide isolated from pronase digests of EDP208 pilin. Limited acid hydrolysis of the tripeptide allowed its sequence to be determined as acetyl-NH-Thr-Asp-Leu. Trypsin digestion of EDP208 pilin resulted in the quantitative release of a fragment containing 12 residues from the N-terminus of the protein. The sequence of this dodecapeptide was determined to be acetyl-NH-Thr-Asp-Leu-Leu-Ala-Gly-Gly-Lys-Asp-Val-Asp-Lys.     

Systematic application of two-dimensional 1H nuclear-magnetic-resonance techniques for studies of proteins. 2. Combined use of correlated spectroscopy and nuclear Overhauser spectroscopy for sequential assignments of backbone resonances and elucidation of polypeptide secondary structures

This paper describes a new nuclear magnetic resonance approach for the determination of secondary structure in globular proteins. To illustrate the practical application of the new procedure, two-dimensional correlated spectroscopy and two-dimensional nuclear Overhauser enhancement spectroscopy were used to obtain individual assignments for all the backbone protons of the beta-sheet secondary structures in the basic pancreatic trypsin inhibitor. First, combined connectivity diagrams of these two methods recorded in both 2H2O solution and H2O solution of the inhibitor were employed to obtain sequential, individual resonance assignments for the separate strands in the beta sheet. Second, a 2D nuclear Overhauser enhancement spectrum recorded with a long mixing time was used to determine how the separate, extended polypeptide strands are linked by hydrogen bonds in the sheet structures. By combination of these results with the identifications of the amino acid side-chain resonances described in the preceding paper, the beta-sheet structures can, without reference to data on the spatial structure obtained with other techniques, be localized in the amino acid sequence. This investigation confirms results on limited regions of the beta sheet in the inhibitor obtained previously with one-dimensional nuclear magnetic resonance experiments and demonstrates that the entire beta-sheet structure seen in single crystals of the inhibitor is preserved in aqueous solution.     

Polypeptide fold in the two metal clusters of metallothionein-2 by nuclear magnetic resonance in solution

The solution conformation of rabbit liver Cd27+-metallothionein-2 was determined by nuclear magnetic resonance (n.m.r.) and distance geometry. The n.m.r. data are based on complete sequence-specific resonance assignments for the polypeptide chain. This letter describes the global arrangement of the polypeptide chain, which forms two distinct domains containing metal clusters of three and four Cd ions, respectively.     

Sequential resonance assignments as a basis for determination of spatial protein structures by high resolution proton nuclear magnetic resonance

A general scheme is proposed for the determination of spatial protein structures by proton nuclear magnetic resonance. The scheme relies on experimental observation by two-dimensional nuclear magnetic resonance techniques of complete throughbond and through-space proton-proton connectivity maps. These are used to obtain sequential resonance assignments for the individual residues in the amino acid sequence and to characterize the spatial polypeptide structure by a tight network of semi-quantitative, intramolecular distance constraints.     

Two-dimensional NMR spectroscopy of siomycin A. Proton--carbon-13 chemical shift correlation

A trial application of a recent two-dimensional nuclear magnetic resonance experiment to the polypeptide antibiotic siomycin A is described. Proton--carbon-13 chemical shift correlation measures the proton and carbon-13 chemical shift for each directly bonded CH group in a molecule, in a single experiment. The resultant map of correlated chemical shifts enables the carbon-13 spectrum to be assigned directly from the known proton shifts, and allows individual proton signals to be identified without problems of overlap. The signal-to-noise ratio available from such techniques should enable their application to aqueous protein solutions using currently available high-field spectrometers.     

Amide proton exchange rates in cardioactive sea anemone polypeptides

Amide hydrogen exchange rates have been measured using high-resolution 1H nuclear magnetic resonance (NMR) spectroscopy at 300 MHz for three homologous cardioactive polypeptides, anthopleurin-A from Anthopleura xanthogrammica and Anemonia sulcata toxins I and II. There are approximately 15 slowly exchanging hydrogens in each polypeptide, but the slowest exchange rates are found in ATX II, with ATX I and AP-A having rates similar to one another. The exchange rates correlate with the thermal stability of these molecules, but not with the potency and species specificity of their biological activities. The data for AP-A are interpreted in terms of a recent structural model for this polypeptide.     

Three-dimensional structure of human [113Cd7]metallothionein-2 in solution determined by nuclear magnetic resonance spectroscopy

The three-dimensional structure of human [113Cd7]metallothionein-2 was determined by nuclear magnetic resonance spectroscopy in solution. Sequence-specific 1H resonance assignments were obtained using the sequential assignment method. The input for the structure calculations consisted of the metal-cysteine co-ordinative bonds identified with heteronuclear correlation spectroscopy, 1H-1H distance constraints from nuclear Overhauser enhancement spectroscopy, and spin-spin coupling constants 3JHN alpha and 3J alpha beta. The molecule consists of two domains, the beta-domain including amino acid residues 1 to 30 and three metal ions, and the alpha-domain including residues 31 to 61 and four metal ions. The nuclear magnetic resonance data present no evidence for a preferred relative orientation of the two domains. The polypeptide-to-metal co-ordinative bonds in human metallothionein-2 are identical to those in the previously determined solution structures of rat metallothionein-2 and rabbit metallothionein-2a, and the polypeptide conformations in the three proteins are also closely similar.     

Secondary structure determination for the Antennapedia homeodomain by nuclear magnetic resonance and evidence for a helix-turn-helix motif.

The homeodomain encoded by the Antennapedia (Antp) gene of Drosophila was studied in aqueous solution by nuclear magnetic resonance (NMR). Sequence-specific resonance assignments have been obtained for the complete polypeptide chain of 68 amino acid residues. The secondary structure determined from nuclear Overhauser effects (NOE) and information about slowly exchanging amide protons includes three helical segments consisting of the residues 10-21, 28-38 and 42-52, respectively. Combination of the presently available NMR data with computer modeling provided preliminary evidence for the presence of a helix-turn-helix motif in the homeodomain. Near the turn, this supersecondary structure appears to be very similar to the DNA binding site in the 434 and P22 c2 repressors, but both helices in the homeodomain include 2-3 additional residues when compared with these prokaryotic DNA-binding proteins.     

Nonrandom structure in the urea-unfolded Escherichia coli outer membrane protein X (OmpX)

On the basis of sequence-specific resonance assignments for the complete polypeptide backbone and most of the amino acid side chains by heteronuclear nuclear magnetic resonance (NMR) spectroscopy, the urea-unfolded form of the outer membrane protein X (OmpX) from Escherichia coli has been structurally characterized. (1)H-(1)H nuclear Overhauser effects (NOEs), dispersion of the chemical shifts, amide proton chemical shift temperature coefficients, amide proton exchange rates, and (15)N[(1)H]-NOEs show that OmpX in 8 M urea at pH 6.5 is globally unfolded, but adopts local nonrandom conformations in the polypeptide segments of residues 73-82 and 137-145. For these two regions, numerous medium-range and longer-range NOEs were observed, which were used as the input for structure calculations of these polypeptide segments with the program DYANA. The segment 73-82 forms a quite regular helical structure, with only loosely constrained amino acid side chains. In the segment 137-145, the tryptophan residue 140 forms the core of a small hydrophobic cluster. Both nonrandom structures are present with an abundance of about 25% of the protein molecules. The sequence-specific NMR assignment and the physicochemical characterization of urea-denatured OmpX presented in this paper are currently used as a platform for investigations of the folding mechanism of this integral membrane protein.     

Structural characterization of a 39-residue synthetic peptide containing the two zinc binding domains from the HIV-1 p7 nucleocapsid protein by CD and NMR spectroscopy

A 39-residue peptide (p7-DF) containing the two zinc binding domains of the p7 nucleocapsid protein was prepared by solid-phase peptide synthesis. The solution structure of the peptide was characterized using circular dichroic and nuclear magnetic resonance spectroscopy in both the presence and absence of zinc ions. Circular dichroic spectroscopy indicates that the peptide exhibits a random coil conformation in the absence of zinc but appears to form an ordered structure in the presence of zinc. Two-dimensional nuclear magnetic resonance spectroscopy indicates that the two zinc binding domains within the peptide form stable, but independent, units upon the addition of 2 equivalents of ZnCl2 per equivalent of peptide. Structure calculations on the basis of nuclear Overhauser (NOE) data indicate that the two zinc binding domains have the same polypeptide fold within the errors of the coordinates (approximately 0.5 A for the backbone atoms, the zinc atoms and the coordinating cysteine and histidine ligands). The linker region (Arg17-Gly23) is characterized by a very limited number of sequential NOEs and the absence of any non-sequential NOEs suggest that this region of polypeptide chain is highly flexible. The latter coupled with the occurrence of a large number of basic residues (four out of seven) in the linker region suggests that it may serve to allow adaptable positioning of the nucleic acid recognition sequences within the protein.     

Assignment of the 1H nuclear magnetic resonance spectrum of the proteinase inhibitor IIA from bull seminal plasma by two-dimensional nuclear magnetic resonance at 500 MHz

The assignment of the 1H nuclear magnetic resonance (n.m.r.) spectrum of the protease inhibitor IIA from bull seminal plasma is described and documented. The assignments are based entirely on the amino acid sequence and on two-dimensional n.m.r. experiments at 500 MHz. Individual assignments were obtained at 18 degrees C and 45 degrees C for the backbone protons of all 57 amino acid residues, with the single exception of the N-terminal pyroglutamate amide proton. The amino acid side-chain resonance assignments are complete, with the exception of 17 long side-chains, i.e. Pro13, Met43 and all the Glu, Gln, Lys and Arg, where only one or two resonances of C beta H2 and in some cases C gamma H2 could be identified. The sequential assignments showed that the order of the two C-terminal residues in the previously established primary structure had to be changed; this was then confirmed by chemical methods. The chemical shifts for the assigned resonances at 18 degrees C and 45 degrees C are listed for an aqueous solution at pH 4.9. A preliminary characterization of the polypeptide secondary structure was obtained from the observed patterns of sequential connectivities.     

Isolation and sequence-specific DNA binding of the Antennapedia homeodomain.

The homeodomain encoded by the Antennapedia (Antp) gene of Drosophila was overproduced in a T7 expression vector in Escherichia coli. The corresponding polypeptide of 68 amino acids was purified to homogeneity. The homeodomain was analysed by ultracentrifugation and assayed for DNA binding. The secondary structure of the isolated homeodomain was determined by nuclear magnetic resonance spectroscopy. DNA-binding studies indicate that the isolated homeodomain binds to DNA in vitro. It selectively binds to the same sites as a longer Antp polypeptide and a full-length fushi tarazu (ftz) protein. Therefore, the homeodomain represents the DNA-binding domain of the homeotic proteins.     

Conformation of [Cd7]-metallothionein-2 from rat liver in aqueous solution determined by nuclear magnetic resonance spectroscopy

The three-dimensional structure of [Cd7]-metallothionein-2 from rat liver was determined in aqueous solution, using nuclear magnetic resonance spectrometry and distance geometry calculations. The experimental data provided proton-proton distance constraints from measurements of nuclear Overhauser effects, constraints on the geometry of the metal-cysteine clusters determined by heteronuclear correlation spectroscopy, and dihedral angle constraints derived from both coupling constants and nuclear Overhauser effects. The structure calculations were performed with the program DISMAN. As in previous studies with rabbit liver metallothionein-2a, the structure calculations were performed separately for the alpha and beta-domains containing the 4 and 3-metal clusters, respectively, since no interdomain constraints were found. For both domains, the global polypeptide fold, the location of polypeptide secondary structure elements, the architecture of the metal-sulfur cluster and the local chirality of the metal co-ordination are very similar to the solution structure of rabbit metallothionein-2a, but show considerable difference relative to the crystal structure of rat metallothionein-2.     

Analysis of the internal nuclear matrix. Oligomers of a 38 kD nucleolar polypeptide stabilized by disulfide bonds

When rat liver nuclei are treated with the sulfhydryl cross-linking reagent sodium tetrathionate (NaTT) prior to nuclease treatment and extraction with 1.6 M NaCl, residual nucleoli and an extensive non-chromatin intranuclear network remain associated with the nuclear envelope. Subsequent treatment of this structure with 1 M NaCl containing 20 mM dithiothreitol (DTT) solubilizes the intranuclear material, while the nuclear envelope remains structurally intact. We have isolated and partially characterized a major polypeptide of the disulfide-stabilized internal nuclear matrix. The polypeptide, which has an apparent molecular mass 38 kD and isoelectric point 5.3, has been localized to the nucleolus of rat liver nuclei by indirect immunofluorescence using a specific polyclonal chicken antiserum. Based on its molecular mass, isoelectric point, intracellular localization and amino acid composition, the 38 kD polypeptide appears to be analogous to the nucleolar phosphoprotein B23 described by Prestayko et al. (Biochemistry 13 (1974) 1945) [20]. Immunologically related polypeptides have likewise been localized to the nucleoli of both hamster and human tissue culture cell lines as well as the cellular slime mold Physarum polycephalum. By immunoblotting, a single 38 kD polypeptide is recognized by the antiserum in rat, mouse, hamster and human cell lines. The antiserum has been utilized to investigate the oligomeric structure of the 38 kD polypeptide and the nature of its association with the rat liver nuclear matrix. By introducing varying numbers of disulfide bonds, we have found that the 38 kD polypeptide becomes incorporated into the internal nuclear matrix in a two-step process. Soluble disulfide-bonded homodimers of the polypeptide are first formed and then are rendered salt-insoluble by more extensive disulfide cross-linking.     

A 32-kilodalton protein binds to AU-rich domains in the 3'' untranslated regions of rapidly degraded mRNAs.

An AU-rich sequence present within the 3' untranslated region has been shown to mark some short-lived mRNAs for rapid degradation. We demonstrate by label transfer and gel shift experiments that a 32-kDa polypeptide, present in nuclear extracts, specifically interacts with the AU-rich domains present within the 3' untranslated region of human granulocyte-macrophage colony-stimulating factor, c-fos, and c-myc mRNAs and a similar domain downstream of the poly(A) addition site of the adenovirus IVa2 mRNA. Competition experiments and partial protease analysis indicated that the same polypeptide interacts with all four RNAs. A single AUUUA sequence in a U-rich context was sufficient to signal binding of the 32-kDa polypeptide. Insertion of three copies of this minimal recognition site led to markedly reduced accumulation of beta-globin RNA, while the same insert carrying a series of U-to-G changes had little effect on RNA levels. Steady-state levels of beta-globin-specific nuclear RNA, including incompletely processed RNA, and cytoplasmic mRNA were reduced. Cytoplasmic mRNA containing the AU-rich recognition sites for the 32-kDa polypeptide exhibited a half-life shorter than that of mRNA with a mutated insert. We suggest that binding of the 32-kDa polypeptide may be involved in the regulation of mRNA half-life.     

Purification and characterization of a high-molecular-weight protein induced in rat serum during the development of cardiac hypertrophy

A relatively high-molecular-weight polypeptide was found in rat serum within 6 h after aortic constriction in experimental animals. This polypeptide persists for about 7 days of the postoperative period and disappears at later stage of hypertrophy (40%). Further, fractionation and purification of this protein through DEAE-Sepharose and gel filtration chromatography have revealed that this protein is a single polypeptide and its relative molecular weight is 135 kDa. Immunoprecipitation and immunofluorescence microscopic analysis have indicated the presence of the above polypeptide in the nuclear fraction of heart cells. Studies on phosphorylation in vitro have revealed that this protein is a phosphoprotein. DNase I sensitivity and hybridization using a muscle specific gene probe have indicated the involvement of this protein in template associated changes in heart nuclei. Further the possibility of this protein being synthesized by heart cells indicates that this protein could traverse back and forth between heart cells and the extracellular fluid, suggesting an autocrine/paracrine role for this protein during the development of cardiac hypertrophy.     

Sequence-specific 1H NMR assignments and secondary structure in the sea anemone polypeptide Stichodactyla helianthus neurotoxin I

Sequence-specific assignments are reported for the 500-MHz 1H nuclear magnetic resonance (NMR) spectrum of the 48-residue polypeptide neurotoxin I from the sea anemone Stichodactyla helianthus (Sh I). Spin systems were first identified by using two-dimensional relayed or multiple quantum filtered correlation spectroscopy, double quantum spectroscopy, and spin lock experiments. Specific resonance assignments were then obtained from nuclear Overhauser enhancement (NOE) connectivities between protons from residues adjacent in the amino acid sequence. Of a total of 265 potentially observable resonances, 248 (i.e., 94%) were assigned, arising from 39 completely and 9 partially assigned amino acid spin systems. The secondary structure of Sh I was defined on the basis of the pattern of sequential NOE connectivities, NOEs between protons on separate strands of the polypeptide backbone, and backbone amide exchange rates. Sh I contains a four-stranded antiparallel beta-sheet encompassing residues 1-5, 16-24, 30-33, and 40-46, with a beta-bulge at residues 17 and 18 and a reverse turn, probably a type II beta-turn, involving residues 27-30. No evidence of alpha-helical structure was found.     

Solution conformation of proteinase inhibitor IIA from bull seminal plasma by 1H nuclear magnetic resonance and distance geometry

A determination of the solution conformation of the proteinase inhibitor IIA from bull seminal plasma (BUSI IIA) is described. Two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) was used to obtain a list of 202 distance constraints between individually assigned hydrogen atoms of the polypeptide chain, to identify the positions of the three disulfide bridges, and to locate the single cis peptide bond. Supplementary geometric constraints were derived from the vicinal spin-spin couplings and the locations of certain hydrogen bonds, as determined by nuclear magnetic resonance (n.m.r.). Using a new distance geometry program (DISGEO) which is capable of computing all-atom structures for proteins the size of BUSI IIA, five conformers were computed from the NOE distance constraints alone, and another five were computed with the supplementary constraints included. Comparison of the different structures computed from the n.m.r. data among themselves and with the crystal structures of two homologous proteins shows that the global features of the conformation of BUSI IIA (i.e. the overall dimensions of the molecule and the threading of the polypeptide chain) were well-defined by the available n.m.r. data. In the Appendix, we describe a preliminary energy refinement of the structure, which showed that the constraints derived from the n.m.r. data are compatible with a low energy spatial structure.