Sequential NMR resonance assignment and secondary structure of ferrocytochrome c553 from Desulfovibrio vulgaris Hildenborough.

Two-dimensional nuclear magnetic resonance spectroscopy was used to assign the proton resonances of ferrocytochrome c553 from Desulfovibrio vulgaris Hildenbourough at 37 degrees C and pH = 5.9. Only a few side-chain protons were not identified because of degeneracy or overlap. The spin systems of the 79 amino acids were identified by DQF-COSY and HOHAHA spectra in H2O and D2O. Sequential assignments were obtained from NOESY connectivities between adjacent amide, C alpha H, and C beta H protons. From sequential NH(i)----NH(i + 1) and long-range C alpha H(i)----NH(i + 3) connectivities, four stretches of helices were identified (2----8, 34----46, 53----59, 67----77). Long-range NOE between residues in three different helices provide qualitative information on the tertiary structure, in agreement with the general folding pattern of cytochrome c. The heme protons, including the propionate groups, were assigned, and the identification of Met 57 as sixth heme ligand was established. The dynamical behavior of the ring protons of the six tyrosines was analyzed in detail in terms of steric hindrance. The NMR data for ferrocytochrome c553 are consistent with the X-ray structure for the homologous cytochrome from D. vulgaris Miyazaki. On the basis of the secondary structure element and of observed chemical shift due to the heme ring current, a structural alignment of eukaryotic and prokaryotic cytochromes c is proposed.     

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.     

Low resolution structure of interleukin-1 beta in solution derived from 1H-15N heteronuclear three-dimensional nuclear magnetic resonance spectroscopy

A low resolution solution structure of the cytokine interleukin-1 beta, a 153 residue protein of molecular weight 17,400, has been determined on the basis of 446 nuclear Overhauser effect (NOE) derived approximate interproton distance restraints involving solely NH, C alpha H and C beta H protons, supplemented by 90 distance restraints for 45 hydrogen bonds, and 79 phi torsion angle restraints. With the exception of 27 C alpha H-C alpha H NOEs, all the NOEs were assigned from a three-dimensional 1H-1H NOE 15N-1H heteronuclear multiple quantum coherence (HMQC) spectrum. The torsion angle restraints were obtained from accurate 3JHN alpha coupling constants measured from a HMQC-J spectrum, while the hydrogen bonds were derived from a qualitative analysis of the NOE, coupling constant and amide exchange data. A total of 20 simulated annealing (SA) structures was computed using the hybrid distance geometry-dynamical simulated annealing method. The solution structure of IL-1 beta comprises 12 beta-strands arranged in three pseudo-symmetrical topological units (each consisting of 5 anti-parallel beta-strands), joined by turns, short loops and long loops. The core of the structure, which is made up of the 12 beta-strands, together with the turns joining strands I and II, strands VIII and IX and strands X and XI, is well determined with a backbone atomic root-mean-square (r.m.s.) distribution about the mean co-ordinate positions of 1.2(+/- 0.1) A. The loop conformations, on the other hand, are poorly determined by the current data. A comparison of the core of the low resolution solution structure of IL-1 beta with that of the X-ray structure indicates that they are similar, with a backbone atomic r.m.s. difference of only 1.5 A between the co-ordinates of the restrained minimized mean of the SA structures and the X-ray structure.     

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.     

1H nuclear magnetic resonance studies of an integral membrane protein: subunit c of the F1F0 ATP synthase

The membrane-traversing subunit c parallel from the F0 part of the ATP synthase molecule has been studied in chloroform/methanol by high-resolution 1H n.m.r. Various one-dimensional and two-dimensional techniques have been used for assignment purposes, some NOE connectivities were established and some 3JHN alpha coupling constants were measured from spin--echo experiments. The effects of varying pH, solvent composition, lanthanide concentration and temperature have been investigated. Evidence is presented that the molecule has extensive alpha-helical segments, and the hairpin structure suggested by other groups is supported by our n.m.r. data. Only one ionizable group, assigned to the C-terminal carboxyl, is observed to titrate in the pH range 2 to 10; so the conserved residue, Asp61, which binds dicyclohexylcarbodiimide, presumably has (at least in this solvent system) an abnormally high pK value.     

1H NMR studies of the glucocorticoid receptor DNA-binding domain: sequential assignments and identification of secondary structure elements

Two protein fragments containing the DNA-binding domain (DBD) of the glucocorticoid receptor (GR) have been studied by two-dimensional 1H NMR spectroscopy. The two peptides (93 and 115 residues, respectively) contain a common segment corresponding to residues C440-I519 of the rat GR or residues C421-I500 of the human GR and include two Zn-binding "finger" domains. The structures of this segment are almost identical in the two protein fragments, as judged from chemical shifts and sequential NOE connectivities. More than 90% of all observable 1H resonances within a 71-residue segment encompassing C440-R510 (rat GR) could be sequentially assigned by standard techniques, and stereospecific assignments could be made for the methyl groups in four valine residues within this segment. Sequential NOE connectivities indicate several elements of secondary structure including two alpha-helical segments consisting of residues S459-E469 and P493-G504, a type I reverse turn between residues R479 and C482, a type II reverse turn between residues L475 and G478, and several regions of extended peptide conformation. No evidence for alpha-helical conformation was found within the two putative zinc-finger domains, indicating that the structures of these domains differ from that of TFIIIA-type zinc fingers. The observation of some very slowly exchanging amide protons in the N-terminal (CI) domain of the DBD in combination with slow rotation of the Y452 aromatic ring indicates that this domain has a restricted conformational flexibility compared to the C-terminal (CII) domain. We also observe several long-range NOE connectivities within C440-R510, suggesting that the sequential assignments presented here will provide a basis for a complete structure determination of this segment of the GR.     

1H-NMR study of neurotoxin B-IV from the marine worm Cerebratulus lacteus. Solution properties, sequence-specific resonance assignments, secondary structure and global fold.

Sequence-specific resonance assignments are reported for the 500-MHz 1H-NMR spectrum of the 55-residue neurotoxin B-IV, isolated from the heteronemertine worm Cerebratulus lacteus. A range of two-dimensional homonuclear correlated and NOE spectra was used in making these assignments, which include NH, C alpha H and C beta H resonances, as well as most resonances from longer-chain spin systems, with the exception of the ten Lys residues, where spectral overlap prevented complete, unambiguous assignments. The secondary structure of B-IV was identified from the pattern of sequential (i, i + 1) and medium range (i, i + 2/3/4) NOE connectivities and the location of slowly exchanging backbone amide protons. Two helices are present, incorporating residues 13-26 and 33-49, and the C-terminal five residues form a helix-like structure. A type-I reverse turn, involving residues 28-31 is present in a small loop linking the two major helices, and the N-terminus appears to be unordered at 27 degrees C, although it may adopt a more ordered conformation at lower temperatures. These elements of secondary structure, together with the four disulfide bonds in the protein, provide sufficient information to define the global fold of the molecule in solution. The pH and temperature dependence of the toxin have been investigated by 1H-NMR and the pKa values of several ionisable sidechains determined.     

Determination of the secondary structure and folding topology of human interleukin-4 using three-dimensional heteronuclear magnetic resonance spectroscopy.

The secondary structure of human recombinant interleukin-4 (IL-4) has been investigated by three-dimensional (3D) 15N- and 13C-edited nuclear Overhauser (NOE) spectroscopy on the basis of the 1H, 15N, and 13C assignments presented in the preceding paper [Powers, R., Garrett, D. S., March, C. J., Frieden, E. A., Gronenborn, A. M., & Clore, G. M. (1992) Biochemistry (preceding paper in this issue)]. Based on the NOE data involving the NH, C alpha H, and C beta H protons, as well as 3JHN alpha coupling constant, amide exchange, and 13C alpha and 13C beta secondary chemical shift data, it is shown that IL-4 consists of four long helices (residues 9-21, 45-64, 74-96, and 113-129), two small helical turns (residues 27-29 and 67-70), and a mini antiparallel beta-sheet (residues 32-34 and 110-112). In addition, the topological arrangement of the helices and the global fold could be readily deduced from a number of long-range interhelical NOEs identified in the 3D 13C-edited NOE spectrum in combination with the spatial restrictions imposed by three disulfide bridges. These data indicate that the helices of interleukin-4 are arranged in a left-handed four-helix bundle with two overhand connections.     

Two-dimensional NMR investigation of a bent DNA fragment: assignment of the proton resonances and preliminary structure analysis.

The resonances of all the non-exchangeable protons (except 5'H and 5"H) of d(CGAAAAATCGG) + d(CCGATTTTTCG), a putatively bent DNA duplex, have been assigned using 1H two-dimensional nuclear magnetic resonance methods. The nuclear Overhauser effect data indicate an overall B-form structure for this double-helical DNA undecamer. However, several features of the NMR data such as some unusually weak C8/C6 proton to C1' proton NOE cross-peaks, the presence of relatively intense C2H to C1'H NOE cross-peaks, and unusual chemical shifts of some 2", 2', and 1' protons suggest a substantial perturbation of the helix structure at the junctions and along the length of the tract of A residues. These structural deviations are considered in terms of models of DNA bending.     

Dihydrofolate reductase. 1H resonance assignments and coenzyme-induced conformational changes

Lactobacillus casei dihydrofolate reductase has been studied in solution by one and two-dimensional 1H nuclear magnetic resonance (n.m.r.) spectroscopy at 500 MHz. By using a combination of n.m.r. methods in conjunction with the crystal structure of the enzyme-methotrexate-NADPH complex, resonances have been assigned for 32 of the 162 residues of the enzyme. These are widely distributed throughout the structure of the protein, and include all the histidine and tyrosine residues, as well as several valine, leucine, isoleucine and phenylalanine residues. The assignments have been made for the enzyme-methotrexate and enzyme-methotrexate-NADP+ complexes as well as the enzyme-methotrexate-NADPH complex. Comparison of assigned resonances in the spectra of the three complexes has permitted a preliminary assessment of structural differences between them. The beta-sheet "core" of the protein is unaffected by coenzyme binding, but two regions of the structure that undergo coenzyme-induced conformation changes have been identified. These are the loop comprising residues 13 to 23, and alpha-helix C (residues 42 to 49).     

Proton nuclear magnetic resonance study of the B9(Asp) mutant of human insulin. Sequential assignment and secondary structure

The sequence-specific 1H nuclear magnetic resonance (n.m.r.) assignment of 49 of the 51 amino acid residues of human B9(Asp) insulin in water at low pH is reported. Spin systems were identified using a series of two-dimensional n.m.r. techniques. For the majority of the amino acid residues with unique spin systems, particularly Ala, Thr, Val, Leu, Ile and Lys, the complete spin systems were identified. Sequence-specific assignments were obtained from sequential nuclear Overhauser enhancement (NOE) connectivities. The results indicate that the solution structure of the mutant closely resembles the crystal structure of native insulin. Thus, the NOE data reveal three helical domains all consistent with the secondary structure of the native human 2Zn insulin in the crystal phase. Numerous slowly exchanging amide protons support these structural elements, and indicate a relatively stable structure of the protein. A corresponding resemblance of the tertiary structures in the two phases is also suggested by slowly exchanging amide protons, and by the extreme chemical shift values observed for the beta-protons of B15(Leu) that agree with a close contact between this residue and the aromatic rings of B24(Phe) and B26(Tyr), as found in the crystal structure of the 2Zn insulin. Finally, there are clear indications that the B9(Asp) insulin mutant exists primarily as a dimer under the given conditions.     

Proton resonance assignments of horse ferricytochrome c

Two-dimensional nuclear magnetic resonance spectroscopy (2D NMR) was used to obtain extensive resonance assignments in the 1H NMR spectrum of horse ferricytochrome c. Assignments were made for the main-chain and C beta protons of 102 residues (all except Pro-44 and Gly-84) and the majority of side-chain protons. As starting points for the assignment of the oxidized protein, a limited set of protons was initially assigned by use of 2D NMR magnetization transfer methods to correlate resonances in the oxidized form with assigned resonances in the reduced form [Wand, A. J., Di Stefano, D. L., Feng, Y., Roder, H., & Englander, S. W. (1989) Biochemistry (preceding paper in this issue)]. Given the complexity of the spectrum due to the size of this protein (104 residues) and its paramagnetic center, the initial search for side-chain spin systems in J-correlated spectra was successful only for the simplest side chains, but the majority of NH-C alpha H-C beta H subspin systems (NAB sets) could be identified at this stage. The subsequent search for sequential NOE connectivities focused on NAB sets, with use of previously assigned residues to place NOE-connected segments within the amino acid sequence. Selective proton labeling of either the slowly or the rapidly exchanging amide sites was used to simplify the spectra, and systematic work at two temperatures was used to resolve ambiguities in the 2D NMR spectra. These approaches, together with the use of magnetization transfer methods to correlate reduced and oxidized cytochrome c spectra, provide multiple cross-checks to verify assignments.     

Secondary structure changes stabilize the reactive-centre cleaved form of SERPINs. A study by 1H nuclear magnetic resonance and Fourier transform infrared spectroscopy.

Proteinase inhibitor members of the SERPIN superfamily are characterized by the presence of a proteolytically sensitive reactive-site loop. Cleavage within this region results in a conformational transition from an unstable "stressed" native protein to a more stable "relaxed" cleaved molecule. In order to identify the principal molecular aspects of this transition, 1H nuclear magnetic resonance (n.m.r.) and FT-IR spectroscopy were applied to the study of four SERPINs. 1H n.m.r. spectra of approximately 20 high-field ring-current-shifted methyl signals exhibited slightly different chemical shifts in the native and cleaved forms of alpha 1-antitrypsin (alpha 1-AT), alpha 1-antichymotrypsin (alpha 1-ACT) and C1 inhibitor (C1-INH), but not ovalbumin, between 20 degrees C and 90 degrees C. Ring current calculations based on crystal co-ordinates for cleaved alpha 1-AT and alpha 1-ACT and native ovalbumin showed that these signals originate from highly localized interactions between different buried residues corresponding to alpha-helix and beta-sheet segments of the SERPIN fold. The small shift changes correspond to small relative conformational side-chain rearrangements of about 0.01 nm to 0.05 nm in the protein hydrophobic core, i.e. the tertiary structure interactions in the two forms of the SERPIN fold are well-preserved, and changes in this appear unimportant for the stabilization found after reactive centre cleavage. Fourier transform infrared (FT-IR) spectroscopic studies of the amide I band showed that the native and cleaved forms of alpha 1-AT, alpha 1-ACT and C1-INH contain 28% to 36% alpha-helix and 38% to 44% beta-sheet. Second derivative FT-IR spectra using H2O and 2H2O buffers revealed very large differences in the amide I band between the native and cleaved forms of alpha 1-AT, alpha 1-ACT and C1-INH, but not for ovalbumin. The alpha-helix band was most sensitive to 1H-2H exchange, while the beta-sheet bands were not, and greater amounts of antiparallel beta-sheet were detected in the cleaved form. 1H n.m.r. showed that polypeptide amide 1H-2H exchange was greater in the native forms of alpha 1-AT, alpha 1-ACT and C1-INH than in their cleaved forms, whereas for ovalbumin it was unchanged. The FT-IR and 1H-2H exchange data show that alterations in the secondary structure are central to the stabilization of the cleaved SERPIN structure.(ABSTRACT TRUNCATED AT 400 WORDS)     

SMS 201-995, an octapeptide somatostatin analogue. Assignment of the 1H 500 MHZ n.m.r. spectra and conformational analysis of SMS 201-995 in dimethylsulfoxide

The possible conformations of SMS 201-995, an active analogue of somastostatin, have been studied in dimethylsulfoxide solution by 500 MHz proton n.m.r. spectroscopy. The assignments have been made by use of 2D-correlated methods to detect long-range coupling connectivities in aromatic residues and between the alpha protons of consecutive residues. NOESY experiments enabled us to correlate amide and alpha protons of neighbouring amino acid residues, which indicate a less flexible situation than in water. Measurements of temperature coefficients of the amide protons, of NH-C alpha H coupling constants and NOE effects are in favour of one predominant conformation with a beta turn, of type II', involving amino acids Phe3 to Thr6.     

Complete sequence-specific 1H nuclear magnetic resonance assignments for the alpha-amylase polypeptide inhibitor tendamistat from Streptomyces tendae

The 1H nuclear magnetic resonance (n.m.r.) spectrum of the alpha-amylase inhibitor Tendamistat was completely assigned with the use of phase-sensitive homonuclear two-dimensional n.m.r. The assignments include the non-labile protons of the 74 amino acid residues as well as the labile protons which exchange sufficiently slowly to be observed in H2O solution. The proton chemical shifts are listed at 50 degrees C and pH 3.2, which coincides with the conditions used for the determination of the three-dimensional structure of Tendamistat.     

1H NMR studies of porcine calbindin D9k in solution: sequential resonance assignment, secondary structure, and global fold

The 1H nuclear magnetic resonance (NMR) spectrum of Ca2+-saturated porcine calbindin D9k (78 amino acids, Mr 8800) has been assigned. Greater than 98% of the 1H resonances, including spin systems for each amino acid residue, have been identified by using an approach that integrates data from a wide range of two-dimensional scalar correlated NMR experiments [Chazin, Rance, & Wright (1988) J. Mol. Biol. 202, 603-626]. Due to the limited quantity of sample and conformational heterogeneity of the protein, two-dimensional nuclear Overhauser effect (NOE) experiments also played an essential role in the identification of spin systems. On the basis of the pattern of scalar connectivities, 43 of the 78 spin systems could be directly assigned to the appropriate residue type. This provided an ample basis for obtaining the sequence-specific resonance assignments. The elements of secondary structure are identified from sequential and medium-range NOEs, values of 3JNH alpha, and the location of slowly exchanging backbone amide protons. Four well-defined helices and a mini beta-sheet between the two calcium binding loops are present in solution. These elements of secondary structure and a few key long-range NOEs provided sufficient information to define the global fold of the protein in solution. Generally good agreement is found between the crystal structure of the minor A form of bovine calbindin D9k and the solution structure of intact porcine calbindin D9k. The only significant difference is a short one-turn helix in the loop between helices II and III in the bovine crystal structure, which is clearly absent in the porcine solution structure.     

Tertiary structure of conotoxin GIIIA in aqueous solution

The three-dimensional structure of conotoxin GIIIA, an important constituent of the venom from the marine hunting snail Conus geographus L., was determined in aqueous solution by two-dimensional proton nuclear magnetic resonance and simulated annealing based methods. On the basis of 162 assigned nuclear Overhauser effect (NOE) connectivities obtained at the medium field strength frequency of 400 MHz, 74 final distance constraints of sequential and tertiary ones were derived and used together with 18 torsion angle (phi, chi 1) constraints and 9 distance constraints derived from disulfide bridges. A total of 32 converged structures were obtained from 200 runs of calculations. The atomic root-mean-square (RMS) difference about the mean coordinate positions (excluding the terminal residues 1 and 22) is 0.8 A for backbone atoms (N, C alpha, C). Conotoxin GIIIA is characterized by a particular folding of the 22 amino acid peptidic chain, which is stabilized by three disulfide bridges arranged in cage at the center of a discoidal structure of approximately 20-A diameter. The seven cationic side chains of lysine and arginine residues project radially into the solvent and form potential sites of interaction with the skeletal muscle sodium channel for which the toxin is a strong inhibitor. The present results provide a molecular basis to elucidate the remarkable physiological properties of this neurotoxin.     

Heteronuclear 1H-15N nuclear magnetic resonance studies of the c subunit of the Escherichia coli F1F0 ATP synthase: assignment and secondary structure.

Nuclear magnetic resonance (NMR) studies of the c subunit of F1F0 ATP synthase from Escherichia coli are presented. A combination of homonuclear (1H-1H) and heteronuclear (1H-15N) 2D and 3D methods was applied to the 79-residue protein, dissolved in trifluoroethanol. Resonance assignment for all the backbone amide groups and many C alpha H side-chain protons was achieved. Analysis of inter- and intraresidue 1H-1H nuclear Overhauser effect (NOE) data and scalar coupling constant information indicates that this protein contains two extended regions of predominant alpha-helical character (residues 10-40 and 48-77) separated by an eight-residue segment which displays little evidence of ordered secondary structure. This model is consistent with information about the molecular motion of the protein deduced from 15N-1H heteronuclear NOE data and observed pKa values of carboxylic acid groups.     

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.