Protein binding chiral discrimination of HPLC stationary phases made with whole, fragmented, and third domain turkey ovomucoid.

Individual protein domains and two domains in combination were prepared by enzymatic and chemical cleavage of turkey ovomucoid followed by isolation and purification by size-exclusion and ion-exchange chromatography. Silica bonded-phase HPLC columns were made from either whole or isolated domains of turkey ovomucoid. The protein columns were tested for chiral recognition by their abilities to resolve enantiomers among a wide range of racemates. The columns made from whole turkey ovomucoid displayed chiral activity toward many racemates, where as a combination of the first and second domain resolved only a selected number of aromatic weak bases. The first and second domains independently gave no appreciable chiral activity. The turkey ovomucoid third domain exhibited enantioselective protein binding for fused-ring aromatic weak acids. Glycosylation of the third domain did not affect chiral recognition. Titration of the third domain with model compounds in conjunction with NMR measurements enabled the identification of the amino acids responsible for binding. Molecular modeling of the ligand-protein complexation provided insights into the ability of a protein surface to discriminate enantiomers on the basis of multiple intermolecular interactions.

Secondary structure and topology of interleukin-1 receptor antagonist protein determined by heteronuclear three-dimensional NMR spectroscopy.

Interleukin-1 (IL-1) proteins, such as IL-1 beta, play a key role in immune and inflammatory responses. Interaction of these cytokines with the IL-1 receptor induces a variety of biological changes in neurologic, metabolic, hematologic, and endocrinologic systems. Interleukin-1 receptor antagonist protein (IRAP) is a naturally occurring inhibitor of the interleukin-1 receptor. The 153-residue protein binds to the receptor with an affinity similar to that of IL-1 beta but does not elicit any physiological responses. As a first step toward understanding IRAP's mode of action, we have used multidimensional, heteronuclear NMR spectroscopy to determine the antagonist's solution secondary structure and global fold. Using a combination of 3D 1H-15N NOESY-HMQC and TOCSY-HMQC and 3D 1H-15N-13C HNCA and HN(CO)CA experiments on uniformly 15N- or doubly 13C/15N-enriched IRAP, we have made resonance assignments for more than 90% of the main-chain atoms. Analysis of short- and long-range NOE's indicates that IRAP is predominantly beta-sheet, with the same overall topology as IL-1 beta but with different regions of the primary sequence comprising the beta-strands. Two short helical segments also were identified. The 14% sequence identity between IL-1 beta and IRAP increases to 25% when differences in the locations of secondary structure elements in the primary sequences are taken into account. Still, numerous differences in side chains, which ultimately play a major role in receptor interaction, exist.(ABSTRACT TRUNCATED AT 250 WORDS)

1H, 13C, and 15N resonance assignments for a ferrocytochrome c553 heme by multinuclear NMR spectroscopy.

A novel strategy has been used to assign the 1H, 13C, and 15N resonances of the heme in Anabaena 7120 ferrocytochrome c553. 13C[13C] double-quantum coherence spectroscopy was used to delineate the heme carbons, 1H[13C] single-bond correlation spectroscopy was used to define the attached protons, and 1H[15N] multiple-bond correlation spectroscopy was used to assign the nitrogens. 1H[13C] multiple-bond correlation spectroscopy confirmed many of the assignments. Proteins were labeled uniformly with 13C or 15N to obtain the required spectral sensitivity.

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%).

Creation of a nuclear magnetic resonance data repository and literature database.

We believe the need exists for an organized and accessible repository of protein nuclear magnetic resonance data. The structure and dynamics of hundreds of proteins are being investigated by NMR. NMR data currently are spread throughout the world and often are not published for lack of journal space. Difficulties in locating, obtaining, and correlating these data with protein structures limits their usefulness to the scientific community. In time, the data may become lost or ignored. To provide a collection point for the results of protein NMR studies and a uniform means of distributing these data, we propose that a data bank be created consisting of two databases: a comprehensive and thoroughly indexed database for the NMR literature and a data repository for the storage of extensive protein NMR data sets. Our current specifications for the types of information to be stored in the NMR databases and their organization for dissemination are defined. The design is intended to be flexible, capable of expanding to include new techniques, new forms of data, and biopolymers other than proteins. This is a proposal to the community of NMR spectroscopists. Only through the active cooperation and support of those in the field of NMR spectroscopy can the proposed data bank succeed.

Hydrogen-1 NMR evidence for three interconverting forms of staphylococcal nuclease: effects of mutations and solution conditions on their distribution.

It has been known for several years that 1H NMR spectra of the enzyme staphylococcal nuclease contain resonances due to conformational heterogeneity [Markley, J. L., Williams, M. N., & Jardetzky, O. (1970) Proc. Natl. Acad. Sci. U.S.A. 65, 645-651]. One source of conformational heterogeneity has been attributed recently to cis/trans isomeriation of the Lys116-Pro117 peptide bond [Evans, P. A., Dobson, C. M., Kautz, R. A., Hatfull, G., & Fox, R. O. (1987) Nature (London) 329, 266-268]. In this paper we present evidence for three interconverting folded forms of nuclease. Forms N and N' are monomeric; form N" appears at higher nuclease concentrations and probably corresponds to dimerized enzyme. Saturation transfer was used to demonstrate that exchange occurs between the denatured state and N". The effects of temperature, pH, and Ca2+ and nucleotide binding on NMR spectra of nuclease were examined. When the temperature is increased or the pH is lowered, form N' is favored relative to N. Binding of a competitive inhibitor (thymidine 3',5'-bisphosphate plus calcium ion) strongly favors one form of nuclease. 1H NMR spectra of wild-type nuclease, the single-mutant nucleases L89F and H124L, and the double-mutant nuclease F76V+H124L were compared. In the unligated proteins, the equilibrium constant for the conformational equilibrium N in equilibrium with N' is approximately 0.1 in wild-type nuclease and nuclease H124L; by contrast, this equilibrium constant is about 0.7 in nuclease L89F and 1.2 in nuclease F76V+H124L under similar conditions.

NMR assignments of the four histidines of staphylococcal nuclease in native and denatured states.

NMR signals from all four histidine ring C epsilon protons and three of the four histidine C delta protons in the protein staphylococcal nuclease have been assigned by comparing spectra of the wild-type (Foggi strain) protein to spectra of three variants that each lack a different histidine residue. All proteins studied were cloned and overproduced in Escherichia coli. The NMR spectra of the three mutant proteins (H8R, H46Y, and H124L) used to make these assignments were similar to one another and to those of the wild type, except for signals from the mutated residues. The pKa values of those histidines conserved between the wild type and the mutants remained essentially unchanged. Multiple histidine C epsilon proton resonances due to non-native forms of nuclease were observed in both thermally induced and acid-induced unfolding. Residue-specific assignments of H epsilon protons in the thermally denatured forms of the mutant H46Y were obtained from connectivities to the native state by saturation transfer.

Location and mobility of ubiquinones of different chain lengths in artificial membrane vesicles.

Ubiquinone (UQn with n = 2, 3, or 10 isoprenoid groups) was incorporated into small, sonicated vesicles made of dipalmitoylphosphatidylcholine (DPPC) or dimyristoylphosphatidylcholine (DMPC). (1) The accessibility of oxidized UQ in DPPC or DMPC vesicles to the reductant sodium borohydride (NaBH4), measured by UV spectroscopy, was UQ2 greater than UQ3 greater than UQ10 (DPPC) and UQ2 greater than UQ3 approximately UQ10 (DMPC). (2) Catalysis of the reduction of entrapped ferricyanide by exogenous NaBH4 was more effective with UQ2 than UQ10 but was slower with all quinones than reduction by added dithionite. (3) The methoxy protons of UQ2 and UQ3 in DPPC and DMPC vesicles exhibited a single NMR resonance centered at approximately 3.95 ppm, whereas the methoxy groups of UQ10 gave rise to two separate proton resonances, at 3.93 ppm and a more narrow resonance at 3.78 ppm. The UQ10 population characterized by the 3.78 ppm resonance was present at a higher concentration in DPPC than in DMPC vesicles and was relatively insensitive to reduction by NaBH4. (4) UQ10 perturbed the melting temperature (Tm) of DPPC vesicles to a smaller extent (delta Tm = -1 degrees C) than did UQ2 and UQ3 (delta Tm = -3 to -4 degrees C). The combined UV and NMR data imply the following: The UQ10 pool characterized by the 3.78 ppm peak corresponds to a more mobile UQ10 fraction that is not reduced by NaBH4 in 2-3 min and is thought to be localized close to the center of the DPPC bilayer since it has little effect on the DPPC Tm.(ABSTRACT TRUNCATED AT 250 WORDS)

Two-dimensional NMR approaches to the study of protein structure and function.

Two-dimensional Fourier transform methods for homonuclear proton NMR spectroscopy have been introduced by Wüthrich and Ernst as a means of extending assignments in spectra of proteins. Multinuclear two-dimensional approaches also appear promising. We are applying current one- and two-dimensional NMR methods to protein family members that differ from one another by one or more amino acid substitutions. The overall goal is to elucidate relationships among the sequences, structures, and functions of these proteins: for example, to delineate primary structural requirements for changes in observable properties such as conformation, amino acid side chain dynamics, hydrogen exchange dynamics, pK'a values, and oxidation-reduction potentials. The ovomucoids from a variety of species of birds, which include a single set of 12 pairs of third-domain proteins (Mr = 6062 for turkey third domain, similar for others) that differ by single amino acid substitutions, provide a favorable system for the study of the structural and dynamic effects of single amino acid replacements. X-ray crystallographic structures of two ovomucoid third domains are available. Other series of proteins being studied by these methods include the photosynthetic electron transport proteins ferredoxin and plastocyanin.

Detailed analysis of protein structure and function by NMR spectroscopy: survey of resonance assignments.

Techniques are now available for making extensive assignments in NMR spectra of proteins of moderate size (molecular weight 20,000 or less). Such assignments provide the first step for experiments designed to extract the full complement of NMR parameters for each group in a protein. The stage is set for exciting research scenarios in protein chemistry involving, for example, the determination of hydrogen exchange kinetics at all exchangeable positions whose half times are on the order of 100 ms (277) or longer than a few minutes (316, 569, 570); the characterization of intermediates in protein folding pathways (318); measurement of the distribution of internal motions within a protein molecule (573); a detailed description of the biophysical consequences of single amino acid replacements in small proteins (387); elucidation of the mechanisms of conformational transitions in proteins; and multiparametric characterization of the parts of an enzyme that participate in catalytic mechanisms. Small proteins for which extensive 1H NMR assignments have been made include lysozyme, several cytochromes, ferredoxins, myelin basic proteins, PTI and related proteinase inhibitors, proteinase inhibitors from seminal plasma and avian eggs, apamin, and several snake venom neurotoxins. (References are given in Table 1).

Imino proton assignments in the proton nuclear magnetic resonance spectrum of the lambda phage OR3 deoxyribonucleic acid fragment.

The 17 base pair duplex d(TATCACCGCAAGGGATAp) . d(TATCCCTTGCGGTGATAp) corresponding to the OR3 operator site of lambda phage has been synthesized and studied by 1H nuclear magnetic resonance spectroscopy at 470 MHz. The 13 imino proton resonances observed at 20 degrees C have been assigned to specific base pairs at positions 3-15 on the basis of nuclear Overhauser effect measurements and studies of the temperature dependence of peak intensities. Resonances from the A-T base pairs at positions 1, 2, 16, and 17 are assumed to be absent from the spectrum because of terminal fraying. Resonance from many of the base pairs suggested by Ohlendorf et al. [Ohlendorf, D. H., Anderson, W. F., Fisher, R. G., Takeda, Y., & Matthews, B. W. (1982) Nature (London) 298, 718-723] to be involved in specific binding of the lambda phage cro repressor are well resolved.

High-resolution proton nuclear magnetic resonance studies of the nickel(II) derivative of azurin.

High-resolution (360 and 470 MHz) 1H NMR studies of Ni(II) azurin, the nickel(II) derivative of the blue copper protein azurin, are reported. The aliphatic resonances of Ni(II) azurin closely parallel those of apoazurin and Cu(I) azurin and indicate that no major structural changes are associated with the binding of nickel(II). The magnetic moment of Ni(II) azurin (mueff = 3.2 muB) is in keeping with a pseudotetrahedral coordination environment like that of Cu(I) azurin. Resonances of protons from the ligand moieties are shifted as far as 125 ppm downfield from 4,4-dimethyl-4-silapentane-1-sulfonate and as far as 20 ppm upfield by internal fields due to the nickel center. One of these strongly shifted resonances is assigned to the methyl protons of the methionine ligand. From spectra of Ni(II) azurin as a function of pH, the pKa' values of histidine-35 and histidine-83 have been measured to be approximately 6.0 and 7.5, respectively. Histidine-35 titrates in a discontinuous fashion, and, significantly, so do several of the isotropically shifted ligand protons, also within experimental error with the same pHmid. This result reinforces the suggestion that the conformational change coupled to the protonation of histidine-35 plays an important role in regulating electron transfer reactions of native azurin [Silvestrini, M. C., Brunori, M., Wilson, M. T., & Darley-Usmar, V. M. (1981) J. Inorg. Biochem. 14, 327-338].

Structure and heme environment of ferrocytochrome c553 from 1H NMR studies.

Cytochrome c553 is a photosynthetic electron transport protein found in algae and cyanobacteria. We have purified cytochromes c553 from five cyanobacteria and studied the structures of the ferrocytochromes by 1H NMR spectroscopy at 360 and 470 MHz. Using standard NMR techniques and by comparing the amino acid sequences of four cytochromes c553 with their 1H NMR spectra, we have assigned in the spectrum of the Aphanizomenon flos-aquae protein 18 resonances to specific amino acid residues and 12 resonances to specific heme protons. Steady state and truncated driven nuclear Overhauser enhancement experiments indicate that a tyrosine and methionine are located near pyrrole ring IV of the heme and that a phenylalanine ring is near the heme alpha-mesoproton. The general folding of the cytochrome c553 protein backbone appears to resemble that of Pseudomonas aeruginosa cytochrome c551, but the chirality of the cytochrome c553 axial methine sulfur is R, the same as that of horse heart cytochrome c.

Isolation of photosynthetic catalysts from cyanobacteria.

Methods are described for the isolation of ferredoxins I and II, cytochrome c-553, cytochrome f, cytochrome c-550 and plastocyanin from large quantities of various cyanobacteria. The amino acid composition of cytochrome c-550 is reported. There is a variation in the relative amounts of these proteins in different batches of cells which may relate to the nutritional status of the organisms.

Nuclear magnetic resonance studies of the copper binding sites of blue copper proteins: oxidized, reduced, and apoplastocyanin.

Proton nuclear resonance spectra at 250 MHz of plastocyanins from spinach (Spinacia oleracea) and a blue green alga (Anabaena variabilis) are reported. Spectra of the reduced plastocyanins contain well-resolved peaks from slowly exchangeable N-H, histidine C2-H tyrosine ring, peptide alpha-CH, and high-field protons. The widths of these peaks indicate that the plastocyanins are monomeric. When the plastocyanins are oxidized, several changes in the spectra are observed including disappearance of peaks assigned to two histidine side chains. The pKa' values of the two histidine residues of reduced spinach plastocyanin are abnormally low (4.9 and less than 4.5). These pKa' values become more normal in apoplastocyanin or plastocyanin inhibited by cyanide. The results suggest that the imidazole groups of the two histidine residues are liganded directly to the copper in plastocyanin. The displacement of copper by cyanide is reversed at low pH. Spectra of apo- and reduced plastocyanins show only minor differences. However, the slowly exchangeable protons of plastocyanin exchange more rapidly in the apoprotein. Copper binding apparently does not cause a major reorganization of the protein structure, but the presence of copper does stabilize this structure.