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.     

A proton nuclear Overhauser effect investigation of the subunit interfaces in human normal adult hemoglobin

High-resolution proton nuclear magnetic resonance spectroscopy and nuclear Overhauser effects for the low-field exchangeable proton resonances of human normal adult hemoglobin in aqueous solvents are being used to confirm and extend the assignments of these resonances to specific protons at the intersubunit interfaces of the molecule. Most of these exchangeable proton resonances of human normal adult hemoglobin have been found to be absent in the spectra of isolated alpha or beta subunits. This finding indicates that they are specific spectral markers for the quaternary structure of the hemoglobin tetramer. Based on the nuclear Overhauser effect results, we have assigned the exchangeable proton resonance at +7.4 ppm downfield from H2O to the hydrogen-bonded proton between alpha 103(G10)His and beta 108(G10)Asn at the alpha 1 beta 1 interface. The nuclear Overhauser effect results have also confirmed the assignments of the exchangeable proton resonances at +9.4 and +8.2 ppm downfield from H2O previously proposed by workers in this laboratory based on a comparison of human normal adult hemoglobin and appropriate mutant hemoglobins. This independent confirmation of previously proposed assignments is necessary in view of the possible long-range conformational effects of single amino-acid substitutions in mutant hemoglobin molecules.     

Proton NMR assignments and secondary structure of the snake venom protein echistatin.

The snake venom protein echistatin is a potent inhibitor of platelet aggregation. The inhibitory properties of echistatin have been attributed to the Arg-Gly-Asp sequence at residues 24-26. In this paper, sequence-specific nuclear magnetic resonance assignments are presented for the proton resonances of echistatin in water. The single-chain protein contains 49 amino acids and 4 cystine bridges. All of the backbone amide, C alpha H, and side-chain resonances, except for the eta-NH of the arginines, have been assigned. The secondary structure of the protein was characterized from the pattern of nuclear Overhauser enhancements, from the identification of slowly exchanging amide protons, from 3JC alpha H-NH coupling constants, and from circular dichroism studies. The data suggest that the secondary structure consists of a type I beta-turn, a short beta-hairpin, and a short, irregular, antiparallel beta-sheet and that the Arg-Gly-Asp sequence is in a flexible loop connecting two strands of the distorted antiparallel beta-sheet.     

1H NMR resonance assignments, secondary structure, and global fold of Apo bovine calbindin D9k

The solution structure and dynamics of apo bovine calbindin D9k have been studied by a wide range of two-dimensional 1H nuclear magnetic resonance experiments. Due to the presence of conformational heterogeneity in the wild-type protein, the sequential resonance assignment was carried out on a Pro43----Gly mutant. By use of a combination of scalar correlation experiments acquired from H2O solution, 61 of the 76 1H spin systems could be assigned to particular amino acid types. The remaining resonances were assigned by a parallel series of experiments acquired from 2H2O solution. These spin system assignments provided a basis for complete sequential resonance assignments from interresidue backbone nuclear Overhauser effects (NOEs). Elements of secondary structure were identified from sequential and medium-range NOEs, backbone spin-spin coupling constants, and slowly exchanging amide protons. Four sections of helix are delineated, together with a short antiparallel beta-sheet interaction between the peptide loops involved in Ca2+ binding. The global fold is provided by combining these elements of secondary structure with a subset of the long-range, interhelix NOEs. Comparison with similar studies on the Ca2(+)-saturated protein indicates that at this crude level the structures are very similar. However, removal of the Ca2+ does dramatically affect the dynamics of the protein, as judged by amide proton exchange rates and aromatic ring rotation. This is particularly evident in the increased flexibility of the residues in the hydrophobic core.     

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.     

Two-dimensional NMR studies of the porcine muscle adenylate kinase

Porcine adenylate kinase was subjected to one- and two-dimensional proton NMR studies in order to identify amino acid spin systems and obtain sequence-specific resonance assignments. With a combination of results from a map of side-chain distances resulting from the refined X-ray crystallographic data and nuclear Overhauser effect spectroscopy (NOESY), assignments are suggested for all the aromatic spin systems.     

Two-dimensional NMR studies of the antimicrobial peptide NP-5

Nearly complete proton resonance assignment of the rabbit antimicrobial peptide NP-5 has been made from two-dimensional NMR data taken at a single temperature. The assignment procedure involved acquisition of phase-sensitive double-quantum-filtered correlation spectra, relayed coherence-transfer spectra, total correlation (homonuclear Hartmann-Hahn) spectra, double- and triple-quantum spectra, and nuclear Overhauser effect spectra. The combination of these complementary experiments simplified and accelerated resonance assignment of the peptide. Individual assignments were made at 20 degrees C for all amide and C alpha protons in the peptide, and for all nonlabile side-chain protons on 26 of the 33 amino acid residues in NP-5. Analysis of the proton-proton nuclear Overhauser effect connectivities, the slowly exchanging amide protons, and the proton chemical shifts in NP-5 indicates that the peptide has a stable, ordered structure in solution. These data also indicate that residues 19-29 in NP-5 are involved in an antiparallel beta-sheet that has a hairpin conformation.     

Epidermal growth factor from the mouse. Structural characterization by proton nuclear magnetic resonance and nuclear overhauser experiments at 500 MHz

Mouse epidermal growth factor (mEGF), a protein hormone effector molecule that regulates cellular development and division, has been investigated by using proton nuclear magnetic resonance techniques at 500 MHz. Well-resolved downfield aromatic and alpha-CH proton resonances (5.0-8.0 ppm) and an upfield ring current shifted isoleucine delta-methyl resonance (approximately 0.5 ppm) have been examined by using principally nuclear Overhauser methods. The data are analyzed in terms of model building based on the predictive Chou-Fasman secondary structure algorithm applied to mEGF [Holladay, L. A., Savage, C. R., Cohen, S., & Puett, D. (1976) Biochemistry 15, 2624-2633], which suggests the existence of some beta-structure and little or no alpha-helicity. Proximity relationships derived from nuclear Overhauser data among Tyr-3, -10, and -13, His-22, and Ile-23 allow refinement of some aspects of the predicted secondary structure and render additional information on how the protein backbone in mEGF is folded (i.e., tertiary structure). Nuclear Overhauser effects (NOEs) from irradiation of several alpha-CH proton resonances give evidence for tiered beta-sheet structure in mEGF. Such proximity relationships derived from NOE data place stringent limitations on possible models for the molecule. pH titration data demonstrate a His-22 pKa of 7.1, indicating either a salt bridge or hydrogen-bond formation between His-22 and another residue. The His-22 pKa is also reflected in the chemical shift changes of several other resonances as a function of pH. Nuclear Overhauser methods, used to differentiate direct (protonation) and indirect (conformation) effects on the chemical shift changes in the spectra of mEGF by varying the pH, yield evidence for a pH-induced conformational transition in the protein hormone associated with the breaking of the His-22 salt bridge or hydrogen bond.     

Assignment of the proton NMR spectrum of reduced and oxidized thioredoxin: sequence-specific assignments, secondary structure, and global fold

Complete proton assignments are reported for the 1H nuclear magnetic resonance (NMR) spectrum of Escherichia coli thioredoxin in the oxidized (with active-site disulfide bridge) and reduced (with two sulfhydryl groups) states. The assignments were obtained by using an integrated assignment strategy in which spin systems were identified from a combination of relayed and multiple quantum NMR techniques prior to sequential assignment. Elements of secondary structure were identified in each protein from characteristic nuclear Overhauser effects (NOE), coupling constants, and slowly exchanging amide protons. In both oxidized and reduced thioredoxin, approximately 33% of the 108 amino acid residues participate in a beta-sheet containing four major strands (three antiparallel and one parallel). A further short beta-strand is connected in a parallel fashion at the N-terminal end of the sheet. Two of the antiparallel beta-strands are connected by a 7-residue beta-bulge loop. Three helical segments, also containing approximately 33% of the amino acid residues, are well-defined in both oxidized and reduced thioredoxin. The remaining third of the molecule apparently consists of reverse turns and loops with little defined secondary structure. The global folds of oxidized and reduced thioredoxin are shown to be essentially identical. Both NOE connectivities and chemical shift values for the two proteins are very similar, except in the immediate vicinity of the active site where significant variations in the chemical shift indicate subtle conformational changes. While the overall fold of oxidized thioredoxin is the same in solution and in the crystalline state, some small differences in local conformation are apparent.     

Epidermal growth factor from the mouse. Physical evidence for a tiered beta-sheet domain: two-dimensional NMR correlated spectroscopy and nuclear Overhauser experiments on backbone amide protons

When H2O-exchanged, lyophilized mouse epidermal growth factor (mEGF) is dissolved in deuterium oxide at low pH (i.e., below approximately 6.0), 13 well-resolved, amide proton resonances are observed in the downfield region of an NMR spectrum (500 MHz). Under the conditions of these experiments, the lifetimes of these amide protons in exchange for deuterons of the deuterium oxide solvent suggest that these amide protons are hydrogen-bonded, backbone amide protons. Several of these amide proton resonances show splittings (i.e., JNH alpha-CH) of approximately 8-10 Hz, indicating that their associated amide protons are in some type of beta-structure. Selective nuclear Overhauser effect (NOE) experiments performed on all amide proton resonances strongly suggest that all 13 of these backbone amide protons are part of a single-tiered beta-sheet structural domain in mEGF. Correlation of 2D NMR correlated spectroscopy data, identifying scaler coupled protons, with NOE data, identifying protons close to the irradiated amide protons, allows tentative assignment of some resonances in the NOE difference spectra to specific amino acid residues. These data allow a partial structural model of the tiered beta-sheet domain in mEGF to be postulated.     

The sequence-specific assignment of the 1H-NMR spectrum of an enzyme, horse-muscle acylphosphatase

A complete range of two-dimensional NMR experiments was used for the assignment of the 1H-NMR spectrum of horse muscle acylphosphatase. Firstly the spin systems of some easily identifiable amino acid side chains were assigned. These side chains involved all the aromatic residues and all the leucine, valine, isoleucine, threonine, alanine, proline as well as some of the glycine residues. Analysis of nuclear Overhauser enhancement spectra in our previous work had identified the sequential and long-range patterns characteristics for secondary structure elements. This result had also provided the identification of the main-chain alpha and amide proton resonances. Several of the completely assigned spin systems were then identified as being part of the secondary structure units which led, after analysis of the primary amino acid sequence, to unambiguous sequence-specific assignments. The identification and assignment of the remaining side-chain resonances was then completed and are reported here. These results provide a complete data base for the three-dimensional structure determination of this enzyme in solution.     

Concerted two-dimensional NMR approaches to hydrogen-1, carbon-13, and nitrogen-15 resonance assignments in proteins

When used in concert, one-bond carbon-carbon correlations, one-bond and multiple-bond proton-carbon correlations, and multiple-bond proton-nitrogen correlations, derived from two-dimensional (2D) NMR spectra of isotopically enriched proteins, provide a reliable method of assigning proton, carbon, and nitrogen resonances. In contrast to procedures that simply extend proton assignments to carbon or nitrogen resonances, this technique assigns proton, carbon, and nitrogen resonances coordinately on the basis of their integrated coupling networks. Redundant spin coupling pathways provide ways of resolving overlaps frequently encountered in homonuclear 1H 2D NMR spectra and facilitate the elucidation of complex proton spin systems. Carbon-carbon and proton-carbon couplings can be used to bridge the aromatic and aliphatic parts of proton spin systems; this avoids possible ambiguities that may result from the use of nuclear Overhauser effects to assign aromatic amino acid signals. The technique is illustrated for Anabaena 7120 flavodoxin and cytochrome c-553, both uniformly enriched with carbon-13 (26%) or nitrogen-15 (98%).     

Carbonyl 13C NMR spectrum of basic pancreatic trypsin inhibitor: resonance assignments by selective amide hydrogen isotope labeling and detection of isotope effects on 13C nuclear shielding

The carbonyl region of the natural abundance 13C nuclear magnetic resonance (NMR) spectrum of basic pancreatic trypsin inhibitor is examined, and 65 of the 66 expected signals are characterized at varying pH and temperature. Assignments are reported for over two-thirds of the signals, including those of all buried backbone amide groups with slow proton exchange and all side-chain carbonyl groups. This is the first extensively assigned carbonyl spectrum for any protein. A method for carbonyl resonance assignments utilizing amide proton exchange and isotope effects on nuclear shielding is described in detail. The assignments are made by establishing kinetic correlation between effects of amide proton exchange observed in the carbonyl 13C region with development of isotope effects and in the amide proton region with disappearance of preassigned resonances. Several aspects of protein structure and dynamics in solution may be investigated by carbonyl 13C NMR spectroscopy. Some effects of side-chain primary amide group hydrolysis are described. The main interest is on information about intramolecular hydrogen-bond energies and changes in the protein due to amino acid replacements by chemical modification or genetic engineering.     

Kringle 4 from human plasminogen: a proton magnetic resonance study via two-dimensional photochemically induced dynamic nuclear polarization spectroscopy

Two-dimensional (2D) proton magnetic resonance techniques used in conjunction with laser photochemically induced dynamic nuclear polarization (photo-CIDNP) spectroscopy have been applied to studying the kringle 4 domain from human plasminogen at 360 MHz. Out of 11 potential CIDNP-sensitive aromatic side chains, only 5 (His3, Tyr41, Tyr50, Trp72, and Tyr74) appear to be accessible to 3-(carboxymethyl)lumiflavin, the dye used to photogenerate spin polarization. Of these, Trp72 and Tyr74 are known to be at, or near, the lysine-binding site. The spin-spin scalar (J) and phase-sensitive dipolar (Overhauser) connectivities in the 2D experiments yield absolute assignments for the aromatic signals stemming from the exposed tyrosyl and tryptophanyl rings. Moreover, a number of side-chain H beta resonances can be identified and assigned to specific types of aromatic amino acid residues.     

Secondary structure in the solution conformation of the proteinase inhibitor IIA from bull seminal plasma by nuclear magnetic resonance

Nuclear magnetic resonance data on the protease inhibitor IIA from bull seminal plasma were used to determine the secondary structure elements in the solution conformation of the protein. The experimental data were obtained from analyses of two-dimensional 1H nuclear magnetic resonance spectra at 500 and 360 MHz and include details of inter-residue nuclear Overhauser enhancements, vicinal spin-spin coupling constants and the sequence location of slowly exchanging amide protons. Accurate measurement of coupling constants and reliable assignments of nuclear Overhauser enhancements were facilitated by the use of absorption mode two-dimensional spectroscopy and large data matrices. It is shown that the peptide backbone is extended from residues 4 to 7, followed by a poorly defined helical region from residues 8 to 13 with a marked change of direction at residue Phe10. Residues 15 to 19 are extended and there is a kink at residue Glu20. Residues 22 to 27 form the central strand of a triple-stranded antiparallel beta-sheet, of which the other two strands are residues 29 to 33 and 49 to 53. Residues 34 to 46 form a helix. The tight turn in the beta-sheet is of type I geometry, and there is a beta-bulge at residue His53.     

Complete assignment of the imino protons of Escherichia coli valine transfer RNA: two-dimensional NMR studies in water

The imino proton spectrum of Escherichia coli valine tRNA has been studied by two-dimensional nuclear Overhauser effect spectroscopy (NOESY) in H2O solution. The small nuclear Overhauser effects from the imino proton of an internal base pair to the imino protons of each nearest neighbor can be observed as off-diagonal cross-peaks. In this way most of the sequential NOE connectivity trains for all the helices in this molecule can be determined in a single experiment. AU resonances can be distinguished from GC resonances by the AU imino NOE to the aromatic adenine C2-H, thus leading to specific base-pair assignments. In general, the NOESY spectrum alone is not capable of assigning every imino proton resonance even in well-resolved tRNA spectra. Multiple proton peaks exhibit more than two cross-peaks, resulting in ambiguous connectivities, and coupling between protons with similar chemical shifts produces cross-peaks that are incompletely resolved from the diagonal. The sequence of the particular tRNA determines the occurrence of the latter problem, which can often be solved by careful one-dimensional experiments. The complete imino proton assignments of E. coli valine tRNA are presented.     

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.     

Two-dimensional 1H NMR study of human ubiquitin: a main chain directed assignment and structure analysis

The 1H resonances of human ubiquitin were studied by two-dimensional nuclear magnetic resonance techniques. A recently introduced assignment algorithm termed the main chain directed (MCD) assignment [Englander, S. W., & Wand, A. J. (1987) Biochemistry 26, 5953-5958] was applied. This approach relies on an ordered series of searches for prescribed patterns of connectivities in two-dimensional J-correlated and nuclear Overhauser effect spectra and centers on the dipolar interactions involving main-chain amide NH, alpha-CH, and beta-CH. Unlike the sequential assignment procedure, the MCD approach does not rest upon definition of side-chain J-coupled networks and is generally not sequential with the primary sequence of the protein. The various MCD patterns and the general algorithm are reiterated and applied to the analysis of human ubiquitin. With this algorithm, the vast majority of amino acid residue amide NH-C alpha H-C beta H J-coupled subspin systems could be associated with and aligned within units of secondary structure without any knowledge of the identity of the side chains. This greatly simplified recognition of side-chain spin systems by restricting their identity. Essentially complete resonance assignments are presented. The MCD method is compared with the sequential assignment method in some detail. The MCD method is highly amenable to automation. Human ubiquitin is found, at pH 5.8 and 30 degrees C, to be composed of an extensive beta-sheet structure involving five strands. Three of these strands form an antiparallel set sharing a common strand and have a parallel orientation to two antiparallel strands. Two helical segments were also observed. The largest, spanning 13 residues, shows dipolar interactions consistent with an alpha-helix while the smaller 4-residue helical segment appears, on the basis of observed nuclear Overhauser effects, to be a 3(10) helix. Five classical tight turns could be demonstrated.     

Assignment of paramagnetically shifted resonances in the 1H NMR spectrum of horse ferricytochrome c.

The proton resonances of the heme, the axial ligands, and other hyperfine-shifted resonances in the 1H nuclear magnetic resonance spectrum of horse ferricytochrome c have been investigated by means of one- and two-dimensional nuclear Overhauser and magnetization transfer methods. Conditions for saturation transfer experiments in mixtures of ferro- and ferricytochrome c were optimized for the cross assignment of corresponding resonances in the two oxidation states. New resonance assignments were obtained for the methine protons of both thioether bridges, the beta and gamma meso protons, the propionate six heme substituent, the N pi H of His-18, and the Tyr-67 OH. In addition, several recently reported assignments were confirmed. All of the resolved hyperfine-shifted resonances in the spectrum of ferricytochrome c are now identified. The Fermi contact shifts experienced by the heme and ligand protons are discussed.