Nuclear Overhauser Effect Patterns Research Articles

Structure of Tctex-1 and Its Interaction with Cytoplasmic Dynein Intermediate Chain

The minus-ended microtubule motor cytoplasmic dynein contains a number of low molecular weight light chains including the 14-kDa Tctex-1. The assembly of Tctex-1 in the dynein complex and its function are largely unknown. Using partially deuterated, (15)N,(13)C-labeled protein samples and transverse relaxation-optimized NMR spectroscopic techniques, the secondary structure and overall topology of Tctex-1 were determined based on the backbone nuclear Overhauser effect pattern and the chemical shift values of the protein. The data showed that Tctex-1 adopts a structure remarkably similar to that of the 8-kDa light chain of the motor complex (DLC8), although the two light chains share no amino acid sequence homology. We further demonstrated that Tctex-1 binds directly to the intermediate chain (DIC) of dynein. The Tctex-1 binding site on DIC was mapped to a 19-residue fragment immediately following the second alternative splicing site of DIC. Titration of Tctex-1 with a peptide derived from DIC, which contains a consensus sequence R/KR/KXXR/K found in various Tctex-1 target proteins, indicated that Tctex-1 binds to its targets in a manner similar to that of DLC8. The experimental results presented in this study suggest that Tctex-1 is likely to be a specific cargo adaptor for the dynein motor complex.

Anti-HIV and anti-tumor protein MAP30, a 30 kDa single-strand type-I RIP, shares similar secondary structure and beta-sheet topology with the A chain of ricin, a type-II RIP.

MAP30 is a 30 kDa single-stranded, type-I ribosome inactivating protein (RIP) possessing anti-tumor and anti-HIV activities. It binds both ribosomal RNA and the HIV-1 long-terminal repeat DNA. To understand the structural basis for MAP30 activities, we undertook the study of MAP30 by solution NMR spectroscopy. We report nearly complete 1H, 13C, and 15N chemical shift assignments of its 263 amino acids. Based upon an analysis of secondary 13C chemical shifts, 3J(HNHA) coupling constants, hydrogen exchange data, and nuclear Overhauser effect patterns, we find that the secondary structure and beta-sheet topology of MAP30 are very similar to those of the ricin A chain, a subunit of the well-known type-II RIP, even though two proteins display distinct activities. We therefore suggest that MAP30 and ricin A chain share a similar three-dimensional fold, and that the reported functional differences between two proteins arise primarily from differences in local three-dimensional structure and other structural properties such as surface electrostatic potentials.

Backbone dynamics of sequence specific recognition and binding by the yeast Pho4 bHLH domain probed by NMR.

Backbone dynamics of the basic/helix-loop-helix domain of Pho4 from Saccharomyces cerevisae have been probed by NMR techniques, in the absence of DNA, nonspecifically bound to DNA and bound to cognate DNA. Alpha proton chemical shift indices and nuclear Overhauser effect patterns were used to elucidate the secondary structure in these states. These secondary structures are compared to the co-crystal complex of Pho4 bound to a cognate DNA sequence (Shimizu T. Toumoto A, Ihara K, Shimizu M, Kyogou Y, Ogawa N, Oshima Y, Hakoshima T, 1997, EMBO J 15: 4689-4697). The dynamic information provides insight into the nature of this DNA binding domain as it progresses from free in solution to a specifically bound DNA complex. Relative to the unbound form, we show that formation of either the nonspecific and cognate DNA bound complexes involves a large change in conformation and backbone dynamics of the basic region. The nonspecific and cognate complexes, however, have nearly identical secondary structure and backbone dynamics. We also present evidence for conformational flexibility at a highly conserved glutamate basic region residue. These results are discussed in relation to the mechanism of sequence specific recognition and binding.

NMR studies of adrenocorticotropin hormone peptides in sodium dodecylsulfate and dodecylphosphocholine micelles: proline isomerism and interactions of the peptides with micelles.

Three adrenocorticotropin hormone (ACTH) fragments (1-10, 1-24, and 11-24) have been studied in water and in sodium dodecylsulfate (SDS) and dodecylphosphocholine (DPC) micelles by nuclear magnetic resonance spectroscopy. The trans-cis isomerism at all three proline sites (at positions 12, 19, and 24) was found in the 11-24 segment of the peptide. The population of the cis isomers changes with the environment of the peptide. Specifically, the presence of the DPC micelle does not affect the trans-cis equilibrium in the 11-24 segment from that in water. In contrast, the presence of the SDS micelles decreases the population of the cis isomer at Pro(24), but increases its population at Pro(12) and Pro(19). The effect of SDS micelles on the trans-cis equilibrium at these proline sites was discussed. Intermolecular nuclear Overhauser effect (NOE) correlations between the ACTH peptides and the micelles were observed. These correlations occurred only in the 1-10 segment of the peptides, and the hydrophobic side chains contributed most to the intermolecular NOE. The intermolecular NOE pattern corroborates the suggestion that the 1-10 segment of the ACTH peptides bind to these micelles via a surface-binding mode, with most of the interactions coming from the insertion of the hydrophobic side chains.

NMR solution structure of the theta subunit of DNA polymerase III from Escherichia coli.

The catalytic core of Escherichia coli DNA polymerase III contains three tightly associated subunits (alpha, epsilon, and theta). The theta subunit is the smallest, but the least understood of the three. As a first step in a program aimed at understanding its function, the structure of the theta subunit has been determined by triple-resonance multidimensional NMR spectroscopy. Although only a small protein, theta was difficult to assign fully because approximately one-third of the protein is unstructured, and some sections of the remaining structured parts undergo intermediate intramolecular exchange. The secondary structure was deduced from the characteristic nuclear Overhauser effect patterns, the 3J(HN alpha) coupling constants and the consensus chemical shift index. The C-terminal third of the protein, which has many charged and hydrophilic amino acid residues, has no well-defined secondary structure and exists in a highly dynamic state. The N-terminal two-thirds has three helical segments (Gln10-Asp19, Glu38-Glu43, and His47-Glu54), one short extended segment (Pro34-Ala37), and a long loop (Ala20-Glu29), of which part may undergo intermediate conformational exchange. Solution of the three-dimensional structure by NMR techniques revealed that the helices fold in such a way that the surface of theta is bipolar, with one face of the protein containing most of the acidic residues and the other face containing most of the long chain basic residues. Preliminary chemical shift mapping experiments with a domain of the epsilon subunit have identified a loop region (Ala20-Glu29) in theta as the site of association with epsilon.

Exploration of the conformational space of oxytocin and arginine-vasopressin using the electrostatically driven Monte Carlo and molecular dynamics methods.

Conformational analysis of the neurohypophyseal hormones oxytocin (OT) and arginine-vasopressin (AVP) has been carried out using two different computational approaches and three force fields, namely by the Electrostatically Driven Monte Carlo (EDMC) method, with the Empirical Conformational Energy Program for Peptides (ECEPP/3) force field or with the ECEPP/3 force field plus a hydration-shell model, and by simulated-annealing molecular dynamics with the Consistent Valence Force Field (CVFF). The low-energy conformations obtained for both hormones were classified using the minimal-tree clustering algorithm and characterized according to the locations of beta-turns in the cyclic moieties. Calculations with the CVFF force field located conformations with a beta-turn at residues 3 and 4 as the lowest energy ones both for OT and for AVP. In the ECEPP/3 force field the lowest energy conformation of OT contained a beta-turn at residues 2 and 3, conformations with this location of the turn being higher in energy for AVP. The latter difference can be attributed to the difference in the size of the side chain in position 3 of the sequences: the bulkier phenylalanine residue of AVP in combination with the bulky Tyr2 residue hinders the formation of a turn at residues 2 and 3. Conformations of OT and AVP with a turn at residues 3,4 were in the best agreement with the x-ray structures of deaminooxytocin and pressinoic acid (the cyclic moiety of vasopressin), respectively, and with the nmr-derived distance constraints. Generally, the low-energy conformations obtained with the hydration-shell model were in a better agreement with the experimental data than the conformations calculated in vacuo. It was found, however, that the obtained low-energy conformations do not satisfy all of the nmr-derived distance constraints and the nuclear Overhauser effect pattern observed in nmr studies can be fully explained only by assuming a dynamic equilibrium between conformations with beta-turns at residues 2,3, 3,4, and 4,5. The low-energy structures of OT with a beta-turn at residues 2,3 have the disulfide ring conformations close to the model proposed recently for a potent bicyclic antagonist of OT [M. D. Shenderovich et al. (1994) Polish Journal of Chemistry, Vol. 25, pp. 921-927], although the native hormone differs from the bicyclic analogue by the conformation of the C-terminal tripeptide. This finding confirms the hypothesis of different receptor-bound conformations of agonists and antagonists of OT.

Structure of the IGF-binding domain of the insulin-like growth factor-binding protein-5 (IGFBP-5): implications for IGF and IGF-I receptor interactions.

Binding proteins for insulin-like growth factors (IGFs) IGF-I and IGF-II, known as IGFBPs, control the distribution, function and activity of IGFs in various cell tissues and body fluids. Insulin-like growth factor-binding protein-5 (IGFBP-5) is known to modulate the stimulatory effects of IGFs and is the major IGF-binding protein in bone tissue. We have expressed two N-terminal fragments of IGFBP-5 in Escherichia coli; the first encodes the N-terminal domain of the protein (residues 1-104) and the second, mini-IGFBP-5, comprises residues Ala40 to Ile92. We show that the entire IGFBP-5 protein contains only one high-affinity binding site for IGFs, located in mini-IGFBP-5. The solution structure of mini-IGFBP-5, determined by nuclear magnetic resonance spectroscopy, discloses a rigid, globular structure that consists of a centrally located three-stranded anti-parallel beta-sheet. Its scaffold is stabilized further by two inside packed disulfide bridges. The binding to IGFs, which is in the nanomolar range, involves conserved Leu and Val residues localized in a hydrophobic patch on the surface of the IGFBP-5 protein. Remarkably, the IGF-I receptor binding assays of IGFBP-5 showed that IGFBP-5 inhibits the binding of IGFs to the IGF-I receptor, resulting in reduction of receptor stimulation and autophosphorylation. Compared with the full-length IGFBP-5, the smaller N-terminal fragments were less efficient inhibitors of the IGF-I receptor binding of IGFs.

1H NMR studies of azide binding to cytochrome c

The binding of azide ion to the heme iron of ferricytochrome c in D 2O is studied using 1H NMR methods at pH 7.0 and 300 K or 315 K. Some hyperfine shifted resonances arising from heme peripheral protons and resonances of side-chain protons of some amino acid residues in N 3-cyt c have been assigned using 2D EXSY and DQF-COSY methods. The majority of the heme pocket side-chain proton signals have been identified. The 1D nuclear overhauser effect (NOE) difference spectra and 2D NOESY spectrum are presented and changes in NOE patterns between the heme and certain residues, and several residues around the axial ligand are interpreted in terms of changes in the pocket structure. Interpretation of NOE data indicates that conformation changes are obvious on the Met80 side of the heme cavity in the environment of the axial ligand in N 3-cyt c. In addition, kinetics analysis of azide binding to cyt c is studied using 2D EXSY method.

Studies of the Binding and Structure of Adrenocorticotropin Peptides in Membrane Mimics by NMR Spectroscopy and Pulsed-Field Gradient Diffusion

The partition and structure of three adrenocorticotropic hormone peptides ACTH(1–10), ACTH(1–24), and ACTH(11–24) in water and in sodium dodecylsulfate (SDS) and dodecylphosphocholine (DPC) micelles were studied by 2D NMR and NMR gradient diffusion measurements. The diffusion rates, the NH chemical shifts, and the nuclear Overhauser effect patterns provided a coherent picture of binding of these peptides. All three peptides are significantly partitioned in the negatively charged SDS micelles and possess definite secondary structure, as opposed to random structures in water. For ACTH (1–24), the hydrophobic 1–10 segment is partitioned in DPC micelles, but the charged 11–24 segment prefers to remain in the aqueous region. ACTH(11–24) does not bind significantly to the DPC micelles. The binding of the ACTH peptides in these two widely used “membrane mimics” are substantially different from that in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayers obtained by attenuated total reflection infrared spectroscopy and from our preliminary diffusion studies of the same peptides in POPC vesicles. This study showed that, in a given micellar medium, all corresponding segments of these peptides are located in the same membrane environment in the system, regardless of whether these segments exist by themselves or are attached to other segments. This result may contradict the membrane-compartments concept of Schwyzer, which suggests that ACTH(1–10) and ACTH(1–24) are located in different membrane compartments because they have different address segments, and consequently, bind to different receptors. The present results also suggest that the assumption that micelles are good membrane mimics should be carefully examined.

Aggregation and conformational transition in aqueous solution of a bombolitin III analogue containing a photoreactive side-chain group.

The peptide toxin bombolitin III [B(III)], originally isolated from bumblebee venom, has been shown to undergo a concentration-dependent conformational change from a random structure to an alpha-helix induced by aggregation. The aggregation process and the consequent folding results from a delicate balance of electrostatic and hydrophobic interactions. The conformational change is strongly dependent on pH and salt concentration. In order to gain insight on the structure of the aggregates, and in particular, on the aggregation number and relative orientation of helices in the molecular complexes, the following analogue of bombolitin III was designed and synthesized: Ile-Lys-Bpa-Met-Asp-Ile-Leu-Ala-Lys-Leu-Gly-Lys-Val-Leu-Ala-His-Val-NH2 Bpa3-B(III) where Bpa is benzoylphenylalanine. Bpa3-B(III) aggregates were investigated by CD and nmr techniques. The observed nuclear Overhauser effect pattern accounts for an antiparallel orientation of two distinct helices. The Bpa side chain allows for the photoinduced cross reaction with any aliphatic proton in spatial proximity. After irradiation, the reaction mixture was analyzed by high performance liquid chromatography and electrospray mass spectrometry. The results confirmed the presence of dimeric and trimeric complexes of bombolitin III formed upon interhelix cross-linking.

Interaction of the receptor binding domains of Pseudomonas aeruginosa pili strains PAK, PAO, KB7 and P1 to a cross-reactive antibody and receptor analog: implications for synthetic vaccine design

The four synthetic peptide antigens, PAK 128-144, PAO 128-144, KB7 128-144 and P1 126-148, correspond in amino acid sequence to the C-terminal receptor binding regions of four strains (PAK, PAO, KB7, P1) of Pseudomonas aeruginosa pilin. The NMR solution structures of the trans forms of the peptides show conserved β-turns which have been implicated in antibody and receptor recognition. The interactions between these peptides and a cross-reactive monoclonal antibody, PAK-13, have been studied using two-dimensional 1H NMR spectroscopy in order to map the antigenic determinants recognized by the antibody. Residues for which spectral changes were observed upon antibody binding differed from peptide to peptide but were mostly confined to one or both of the turn regions and to the hydrophobic pockets. Conformational changes in the β-turns and hydrophobic pockets of these peptides upon antibody binding were also monitored by examination of the pattern of nuclear Overhauser effects (NOEs) versus transferred nuclear Overhauser effects (TRNOEs) for the free versus the bound peptides. Although TRNOEs developed strongly between side chain resonances in the hydrophobic pockets of the peptides, no additional backbone TRNOEs were observed in the presence of antibody, suggesting no major conformational changes in the secondary structures of the peptides upon binding. This implies a flexible antibody combining site, a feature which is discussed with respect to cross-reactivity, strain specificity, and the design of a synthetic peptide vaccine effective against a broad spectrum of P. aeruginosa strains. The binding of the PAK peptide to a disaccharide receptor analog, (βGalNAc(1-4)βGal), was also studied using 1H NMR in order to map the “adhesintope” recognized by the receptor. Spectral changes observed in the peptide spectrum with the binding of receptor were similar to those seen for the binding of antibody, suggesting that the epitope recognized by the antibody is structurally coincident with the adhesintope recognized by the receptor. The relevancy of this result is discussed with respect to immunogenicity versus pathogenicity, and the proper design of a vaccine which could prevent the mutational escape of the pathogen away from the host’s defence systems.

Structural characterization of the active site of Brucella abortus Cu-Zn superoxide dismutase: a 15N and 1H NMR investigation.

Prokaryotic Cu-Zn superoxide dismutases (SODs) are rare and poorly characterized compared to their eukaryotic counterparts. To better characterize the structure of the prokaryotic enzyme, an NMR investigation of Brucella abortus Cu-Zn SOD in the reduced form was undertaken. The enzyme studied was a recombinant form, expressed in Escherichia coli. The enzyme initially lacked a full complement of Cu and Zn ion. After demetallation and remetallation with a stoichiometric amount of Cu and Zn ion, the specific activity of the recombinant B. abortus Cu-Zn SOD was comparable to the specific activity of the bovine enzyme. The 15N and 1H resonances of seven active site histidine imidazole rings were assigned using two-dimensional NMR methods. A self-consistent set of nuclear Overhauser effects between imidazole ring protons was observed, which was in agreement with the predictions of a model based on the X-ray crystallographic structure of the oxidized bovine enzyme (Tainer, J.A., Getzoff, E. D., Beem, K. M., Richardson, J.S., & Richardson, D.C. (1982) J. Mol. Biol. 160, 181-217). These observations strongly suggest that the structure of the active site of the prokaryotic enzyme is similar to that of the eukaryotic enzyme. Differences in the observed and predicted nuclear Overhauser effects could be ascribed to differences in the oxidation state of the Cu ion (Cu(I) in the reduced B. abortus enzyme and Cu(II) in the oxidized bovine enzyme), as much as they could to the different origins of the enzymes. The NMR data were also compared to a similar 1H NMR study of the human enzyme (Bertini, I., Capozzi, F., Luchinat, C., Piccioli, M., & Viezzoli, M. S. (1991) Eur. J. Biochem. 197, 691-697). The pattern of nuclear Overhauser effects and the chemical shifts of corresponding resonances were very similar in 1H NMR spectra of the human and B. abortus enzymes. Significant differences in the chemical shifts or exchange behavior of a few resonances indicated differences in the environments of several histidines in the active sites of reduced B. abortus and human Cu-Zn SODs. This is consistent with the presence of a number of insertions and deletions in the loop regions that make up the active site as indicated by amino acid sequence alignment studies. The tautomeric and protonation states of the active site histidines were also determined in this study, and the results were in agreement with previous studies. The resonances of nitrogen atoms coordinated to metal ions were found to fall between those of protonated and unprotonated nitrogens on histidine imidazoles.

Fungal metabolites. Part 7. Solution structure of an antibiotic peptide, trichosporin B-V, from Trichoderma polysporum

The secondary structure of the peptaibol trichosporin B-V in methanol was investigated in detail by 600 MHz nuclear magnetic resonance spectroscopy. Its fundamental secondary structure was characterized as a helix by the circular dichroism spectrum. Inter-residual nuclear Overhauser effect patterns, 3JNH-CaH amide coupling constants, hydrogen–deuterium (H–D) exchange rates of the amide protons, and inspection of molecular models showed that the secondary structure of this peptide consists of two major α-helical structures because of a bent structure around a Pro residue, and that the first three amino acid residues of the N-terminal α-helix are arranged predominantly in a 310-helical fashion.

Solution conformation of endothelin and point mutants by nuclear magnetic resonance spectroscopy.

Two-dimensional NMR techniques were utilized to determine the secondary structural elements of endothelin-1 (ET-1), a potent vasoconstrictor peptide, and two of its point mutants, Met-7 to Ala-7 (ETM7A), and Asp-8 to Ala-8 (ETD8A) in acetic acid-d3/water solution. Sequence specific NMR assignments were determined for all three peptides, as well as chemical shifts and NOE connectivity patterns. The chemical shifts of ET-1 and ETM7A are identical (+/- 0.05 ppm) except for the site of substitution, whereas marked shift changes were detected between ET-1 and ETD8A. These chemical shift differences imply that the Asp-8 to Ala-8 mutation has induced a conformational change relative to the parent conformation. All three molecules show the same basic nuclear Overhauser effect (NOE) pattern, which suggests that the gross conformation of all three molecules is the same. Small changes in sequential NOE intensities and changes in medium-range NOE patterns indicate that there are subtle conformational differences between ET-1 and ETD8A.

Proton nuclear Overhauser effect study of the heme active site structure of chloroperoxidase

Chloroperoxidase, a glycoprotein from the mold Caldariomyces fumago, has been investigated in its ferric low-spin cyanide-ligated form through use of nuclear Overhauser effect (NOE) spectroscopy to provide information on the heme pocket electronic/molecular structure. Spin-lattice relaxation times for the hyperfine-shifted heme resonances were found to be three times less than those in horseradish peroxidase. This must reflect a slower electronic relaxation rate for chloroperoxidase than for horseradish peroxidase as a consequence of axial ligation of cysteine in the former versus histidine in the latter enzyme. Isoenzymes A1 and A2 of chloroperoxidase show the largest chemical shift differences near the heme propionate on the basis of NOE measurements. This suggests that the primary structure differences for the two isoenzymes are communicated to the heme group through the ring propionate substituents. A downfield peak has been detected in chloroperoxidase with chemical shift, T1, and line width characteristics similar to those of the C epsilon-H proton of the distal histidine residue. The NOE pattern and T1's of the peaks in the 0.0 to -5.0 ppm upfield region are consistent with the presence of an arginine amino acid residue in the heme pocket near either the 1-CH3 or 3-CH3 group. Existence of catalytically important distal histidine and arginine amino acid residues in chloroperoxidase shows it to be structurally similar to peroxidases rather than to the often compared monooxygenase, cytochrome P-450. This result supports the earlier conclusions of Sono et al. [Sono, M., Dawson, J.H., Hall, K., & Hager, L.P. (1986) Biochemistry 25, 347-356].

Characterization of the secondary structure of calmodulin in complex with a calmodulin-binding domain peptide

The interaction between calcium-saturated chicken calmodulin and a peptide corresponding to the calmodulin-binding domain of the chicken smooth muscle myosin light chain kinase has been studied by multinuclear and multidimensional nuclear magnetic resonance methods. Extensive 1H and 15N resonance assignments of calmodulin in the complex have been obtained from the analysis of two- and three-dimensional nuclear magnetic resonance spectra. The assignment of calmodulin in the complex was facilitated by the use of selective labeling of the protein with alpha-15N-labeled valine, alanine, lysine, leucine, and glycine. These provided reference points during the main-chain-directed analysis of three-dimensional spectra of complexes prepared with uniformly 15N-labeled calmodulin. The pattern of nuclear Overhauser effects (NOE) seen among main-chain amide NH, C alpha H, and C beta H hydrogens indicates that the secondary structure of the globular domains of calmodulin in the complex closely corresponds to that observed in the calcium-saturated state of the protein in the absence of bound peptide. However, the backbone conformation of residues 76-84 adopts an extended chain conformation upon binding of the peptide in contrast to its helical conformation in the absence of peptide. A sufficient number of NOEs between the globular domains of calmodulin and the bound peptide have been found to indicate that the N- and C-terminal regions of the peptide interact with the C- and N-terminal domains of calmodulin, respectively. The significance of these results are discussed in terms of recently proposed models for the structure of calmodulin-peptide complexes.

Proton NMR sequential assignments and identification of secondary structural elements in oxidized putidaredoxin, an electron-transfer protein from Pseudomonas

Sequential 1H resonance assignments and secondary structural features of putidaredoxin (Pdx), a 106-residue globular protein consisting of a single polypeptide chain and a [2Fe-2S] cluster, are reported. No crystal structure has been obtained for Pdx or for any closely homologous protein. The sequentially assigned resonances represent ca. 83% of all the protons in Pdx and a large majority of those protons which are unaffected by the paramagnetism of the iron-sulfur cluster. A total of three alpha-helices, two beta-sheets, and two type I beta-turns have been identified from NOE (nuclear Overhauser effect) patterns. Besides the extensive beta-sheet described previously, a second sheet is identified, consisting of two short antiparallel strands (Ile 89-Thr 91 and Val 21-Leu 23), one of which ends in a tight type I turn (Thr 91-Pro 92-Glu 93-Leu 94). One short helix (Ala 26-Gly 31) and a second longer helical region (Glu 54-Cys 73) are present. This second helical region is discontinuous, breaking at Pro 61, resuming at Glu 65, and ending at Cys 73. The functionally important C-terminal tryptophan residue has been identified, and some structural constraints on this residue are described. Previously reported functional data concerning Pdx are discussed in light of present structural information. Finally, approaches to the determination of a high-resolution solution structure of the protein are discussed.

Proton NMR identification of a .beta.-sheet structure and description of folding topology in putidaredoxin

Putidaredoxin (Pdx), a 106-residue globular protein consisting of a single polypeptide chain and a [2Fe-2S] cluster, is the physiological reductant of P-450cam, which in turn catalyzes the monohydroxylation of camphor by molecular oxygen. No crystal structure has been obtained for Pdx or for any closely homologous protein. The application of two-dimensional 1H NMR methods to the problem of structure determination in Pdx is reported. A beta-sheet consisting of five short strands and one beta-turn has been identified from distinctive nuclear Overhauser effect patterns. All of the backbone resonances and a majority of the side-chain resonances corresponding to protons in the beta-sheet have been assigned sequence specifically. The sheet contains one parallel and three antiparallel strand orientations. Hydrophobic side chains in the beta-sheet face primarily toward the protein interior, except for a group of three valine side chains that are apparently solvent exposed. The potential significance of this "hydrophobic patch" in terms of biological activity is discussed. The folding topology, as determined by the constraints of the beta-sheet, is compared with that of other [2Fe-2S] proteins for which folding topologies are known.

Secondary structure of the phosphocarrier protein IIIGlc, a signal-transducing protein from Escherichia coli, determined by heteronuclear three-dimensional NMR spectroscopy.

IIIGlc is a signal-transducing phosphocarrier protein of the phosphoenolpyruvate:glycose phosphotransferase system of Escherichia coli. The secondary structure of IIIGlc is determined by heteronuclear (15N, 13C) three-dimensional NMR spectroscopy. Sequential, medium-range, and long-range nuclear Overhauser effects seen in NMR spectra are used to elucidate 11 antiparallel beta-strands and four helical segments. The medium-range nuclear Overhauser effect patterns suggest that the helices are either distorted alpha-helices or are of the 3(10) class. The amino acids separating the active-site histidine residues (His75 and His90) form two strands (Ala76-Ser81 and Val85-Phe91) of a six-stranded antiparallel beta-sheet that brings His90 and His75 in close proximity. Sequence similarities in IIIGlc and several other sugar-transport proteins suggest that the histidine residues within these proteins may be arranged in a similar manner. The 18-residue N-terminal peptide that precedes beta-strand Thr19-Ile22 in native IIIGlc is disordered and does not interact with the rest of the protein. Furthermore, removal of the N-terminal heptapeptide by a specific endopeptidase does not affect the structure of the remaining protein, thus explaining the phospho-acceptor activity of modified IIIGlc with the phospho-histidine-containing phosphocarrier protein of this system.

Two-dimensional 1H nuclear magnetic resonance studies on the gene V-encoded single-stranded DNA-binding protein of the filamentous bacteriophage IKe : I. Structure elucidation of the DNA-binding wing

Two-dimensional nuclear magnetic resonance techniques were used to obtain residue- and sequence-specific assignments in the 1H spectrum of the single-stranded DNA-binding protein encoded by gene V of the filamentous phage IKe (IKe GVP). The residue-specific assignments are based on the analysis of J-correlated spectra, i.e. correlated spectroscopy and homonuclear-Hartmann-Hahn total correlated spectroscopy. Complete assignments of side-chain spin systems, e.g. long side-chains, were, to a major part, derived from two-dimensional spectra obtained by means of the latter technique. Sequence-specific residue assignments were obtained for the two neighbouring residues V41 and Y42, and the amino acid sequence segment encompassing residues S17 through I29. The structure of this segment, a β-loop, was deduced from the interresidue nuclear Overhauser effect pattern. Residues S17 through V19 and P26 through I29 form an anti-parallel β-ladder segment, whereas residues Q21 to K25 constitute the loop region. The β-loop is expected to project into the solution and is intimately involved in binding to single-stranded DNA; it is therefore designated the “DNA-binding wing”. By analogy with the structure of the DNA-binding wing deduced from IKe GVP, a similar structure is proposed for the corresponding domain of the gene V protein encoded by the filamentous phage Ff for which, from X-ray diffraction studies, a three-dimensional structure has been deduced. Essential differences appear to exist between the DNA-binding domain in the X-ray structure and that proposed in this paper. Possible reasons for these differences are discussed.