Sequence-dependent conformation of DNA duplexes. The AATT segment of the d(G-G-A-A-T-T-C-C) duplex in aqueous solution

The nonexchangeable base and sugar protons of the octanucleotide d(G-G-A-A-T-T-C-C) have been assigned by two-dimensional correlated (COSY) and nuclear Overhauser effect (NOESY) methods in aqueous solution. The assignments are based on distance connectivities of less than 4.5 A established from NOE effects between base and sugar protons on the same strand and occasionally between strands, as well as, coupling connectivities within the protons on each sugar ring. We observe the NOEs to exhibit directionality and are consistent with the d(G-G-A-A-T-T-C-C) duplex adopting a right-handed helix in solution. The relative magnitude of the NOEs between base and sugar H2' protons of the same and 5'-adjacent sugars characterizes the AATT segment to the B-helix type in solution.     

Sequence-dependent conformations of DNA duplexes: the TATA segment of the d(G-G-T-A-T-A-C-C) duplex in aqueous solution

The base and sugar protons of the d(G-G-T-A-T-A-C-C) duplex have been assigned from two-dimensional correlated (COSY) and nuclear Overhauser effect (NOESY) measurements in D₂O solution at 25°C. The nucleic acid protons have been assigned from NOEs between protons on adjacent bases on the same and partner strands, as well as from NOEs between the base protons and their own and 5′-flanking H1′, H2′, H2″, H3′, and H4′ sugar protons. These assignments are confirmed from coupling constant and NOE connectivities within the sugar protons of a given residue. Several of these NOEs exhibit directionality and demonstrate that the d(G-G-T-A-T-A-C-C) duplex is a right-handed helix. The relative magnitude of the NOEs between the base protons and the sugar H2′ protons of its own and 5′-flanking sugar demonstrate that the TATA segment of the d(G-G-T-A-T-A-C-C) duplex adopts a B-DNA type helix geometry in solution, in contrast to the previous observation of a A-type helix for the same octanucleotide duplex in the crystalline state.     

Intracomplex alkylation of octanucleotide pd(TGTTTGGC) by a 5'-(4-N-methyl-N-(2-chloroethyl)-amino)benzylphosphamide derivative of heptanucleotide pd(CCAAACA). Preparation of a covalent adduct and study of its spatial structure in an aqueous solution by two-dimensional (1)H-NMR spectroscopy]

Covalent adduct--the product of intracomplex alkylation at N-3-position of dC-8-nucleoside residue of target octanucleotide pd[TGTTTGGC] was completely synthesized by means of 4-[N-methyl-N-(2-chloroethyl)amino]benzyl-5'-phosphamido derivative of heptanucleotide pd[CCAAACA]. Its melting temperature was shown to be 70 degrees C. Tm did not depend on covalent adduct concentration and was by 40 degrees C higher than that for unmodified duplex pd[TGTTTGGC].pd[CCAAACA] at concentration of 0.5 x 10(-4) M. The spatial structure of the covalent adduct in aqueous solution was investigated by two-dimensional 3H-NMR spectroscopy. The assignment of oligonucleotide protons as well as protons of a modifying group was carried out using COSY, COSY-DQF and NOESY experiments. Conformational analysis of proton-proton coupling constants for H1', H2'a, H2'b and H3' protons showed the sugar residues to be in 2'-endo conformation. Analysis of NOE connectivities observed between the protons of the alkylating group and oligonucleotide protons yielded conclusion, regarding the 4-[N-methyl-N-(2-chloroethyl)amino]benzylamido 5'-residue being localized in the region of the lacked nucleoside residue of the heptanucleotide chain about 5 A apart from the dC-1 residue and from cytosine base of the alkylated dC-8 residue.     

Two-dimensional 1H-NMR study of the 1-34 fragment of human parathyroid hormone

The complete assignment of resonances in the proton nmr spectrum of the 1-34 amino acid fragment of human parathyroid hormone [hPTH(1-34)], determined using a combination of one- and two-dimensional nmr techniques at 500 MHz, is described. In particular, homonuclear Hartmann-Hahn experiments, recorded in H2O and D2O, are used to resolve ambiguities in the connectivities between the highly overlapped resonances in the aliphatic region of the spectrum. One-dimensional multiple quantum filtering experiments are used to identify serine and phenylalanine spin systems. Analyses of the through-bond and through-space connectivities in the alpha H-NH fingerprint regions of the correlated spectroscopy (COSY) and nuclear Overhauser effect spectroscopy (NOESY) spectra lead to the assignment of resonances to specific amino acid residues in the polypeptide. Examination of the observed NOE cross peaks indicates that hPTH(1-34) has no detectable secondary structural elements in aqueous solution.     

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 NMR studies of plastocyanin from Scenedesmus obliquus: complete sequence-specific assignment, secondary structure analysis, and global fold

Two-dimensional 1H NMR methods have been used to make sequence-specific resonance assignments for the 97 amino acid residues of the plastocyanin from the green alga Scenedesmus obliquus. Assignments were obtained for all backbone protons and the majority of the side-chain protons. Spin system identification relied heavily on the observation of relayed connectivities to the backbone amide proton. Sequence-specific assignments were made by using the sequential assignment procedure. During this process, an extra valine residue was identified that had not been detected in the original amino acid sequence. Elements of regular secondary structure were identified from characteristic NOE connectivities between backbone protons, 3JHN alpha coupling constant values, and the observation of slowly exchanging amide protons. The protein in solution contains eight beta-strands, one short segment of helix, five reverse turns, and five loops. The beta-strands may be arranged into two beta-sheets on the basis of extensive cross-strand NOE connectivities. The chain-folding topology determined from the NMR experiments is that of a Greek key beta-barrel and is similar to that observed for French bean plastocyanin in solution and poplar plastocyanin in the crystalline state. While the overall structures are similar, several differences in local structure between the S. obliquus and higher plant plastocyanins have been identified.     

NMR studies of complex formation between the modified oligonucleotide d(T*TCTGT) covalently linked to an acridine derivative and its complementary sequence d(GCACAGAA)

The oligodeoxynucleotide d(TTCTGT) was covalently attached to the 9-amino group of 2-methoxy-6-chloro-9-aminoacridine (Acr) through its 3'-phOsphate via a pentamethylene linker (m5). In order to avoid its hydrolysis by nucleases inside the cel., one of its phosphates (TpT) was substituTed with a neopentyl group. Complex formation between each of the two purified isomers and the complementary strand d(GCACAGAA) was investigated by nuclear magnetic resonance. The COSY and NOESY connectivities allowed us to assign all the proton resonances of the bases, the sugars (except the overlapping 5'-5' resonances), the acridine, and the pentamethylene chain. Structural information derived from the relative intensity of COSY and NOESY maps revealed that the duplex d(T*TCTGT).d(GCACAGAA) adopts a B-type conformation and that the deoxyriboses preferentially adopt a 2'-endo conformation. The NOE connectivities observed between the protons of the bases or the sugars and the protons of the dye show the intercalation of the acridine between the base pairs. NOE connectivities as well as imino proton resonances show that, at room temperature, the C7 base and the G8 base belonging to two different duplexes are paired. The pseudoaxial and pseudoequatorial isomers were assigned, and the differences in stability of their complex with the complementary strand are discussed.     

Complete assignment of the 500 MHz 1H-NMR spectra of bleomycin A2 in H2O and D2O solution by means of two-dimensional NMR spectroscopy

1H-NMR spectra of bleomycin A2 recorded at 500 MHz in D2O and H2O at 24 degrees C and 3 degrees C were investigated. Resonances of the individual spin systems were identified by using two-dimensional correlation spectroscopy (COSY), two-dimensional spin echo correlated spectroscopy (SECSY) and by the application of two-dimensional Nuclear Overhauser Effect spectroscopy (NOESY). Employment of these techniques allowed the assignment of 113 exchangeable and 59 non-exchangeable protons in the 1H NMR spectrum of bleomycin A2. By means of 2D NOE spectroscopy also interresidual connectivities could be observed. Comparison of the NOESY spectra at 3 degrees C and 24 degrees C suggest that at low temperatures the central party of the bleomycin A2 molecule tends to adopt an extended conformation.     

Conformation of a bulge-containing oligomer from a hot-spot sequence by NMR and energy minimization

Two-dimensional nmr data on a bulge-containing oligodeoxyribonucleotide, 5'dGATGGGCAG.dCTGACCCATC, and a regular oligomer of similar sequence, 5'dGATGGCAG.dCTGCCATC, are presented. The nonexchangeable protons are assigned from sequential nuclear Overhauser effect spectroscopy (NOESY) connectivities. The two-dimensional NOE (NOESY) and correlated (COSY) spectra of the bulge-containing oligomer are compared to those of the perfect 8-mer. Experimental proton-proton distances are determined from NOESY spectra acquired with mixing times of 100, 150, and 200 ms, using comparable distances in the B-DNA region of the molecule as a calibration. With this approach, measured distances do not depend systematically on mixing time. Energy minimization techniques are used to calculate a three-dimensional structure for the bulge-containing oligomer in agreement with the nmr data. The helix is of the B family, with the extra adenine stacked into the helix, and the helix axis is bent by 20 degrees.     

1H and 15N NMR characterization of free and bound states of an amphiphilic peptide interacting with calmodulin.

A peptide of 17 amino acid residues Ac-L-K-W-K-K-L-L-K-L-L-K-K-L-L-K-L-G-NH2, designed to form an amphiphilic basic alpha-helix [DeGrado, W.F., Prendergast, F. G., Wolfe, H. R., Jr., & Cox, J. A. (1985) J. Cell. Biochem. 29, 83-93], was labeled with 15N at positions 1, 7, 9, and 10. Homo- and heteronuclear NMR techniques were used to characterize the conformational changes of the peptide when it binds to calmodulin in the presence of Ca2+ ions. The spectrum of the free peptide in aqueous solution at pH 6.3 and 298 K was completely assigned by a combined application of several two-dimensional proton NMR methods. Analysis of the short- and medium-range NOE connectivities and of the secondary chemical shifts indicated that the peptide populates, to a significant extent, an alpha-helix conformational state, in agreement with circular dichroism measurements under similar physicochemical conditions. 15N-edited 1D spectra and 15N(omega 2)-half-filtered two-dimensional NMR experiments on the peptide in a 1:1 complex with calmodulin allowed assignment of half of the amide proton resonances and three C alpha H resonances of the bound peptide. The observed NOE connectivities between the peptide backbone protons are indicative of a stable helical secondary structure spanning at least the fragment L1-K11. The equilibrium and dynamic NMR parameters of the bound peptide are discussed in terms of a molecular interaction model.     

Assignment of the backbone 1H and 15N NMR resonances of bacteriophage T4 lysozyme

The proton and nitrogen (15NH-H alpha-H beta) resonances of bacteriophage T4 lysozyme were assigned by 15N-aided 1H NMR. The assignments were directed from the backbone amide 1H-15N nuclei, with the heteronuclear single-multiple-quantum coherence (HSMQC) spectrum of uniformly 15N enriched protein serving as the master template for this work. The main-chain amide 1H-15N resonances and H alpha resonances were resolved and classified into 18 amino acid types by using HMQC and 15N-edited COSY measurements, respectively, of T4 lysozymes selectively enriched with one or more of alpha-15N-labeled Ala, Arg, Asn, Asp, Gly, Gln, Glu, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, or Val. The heteronuclear spectra were complemented by proton DQF-COSY and TOCSY spectra of unlabeled protein in H2O and D2O buffers, from which the H beta resonances of many residues were identified. The NOE cross peaks to almost every amide proton were resolved in 15N-edited NOESY spectra of the selectively 15N enriched protein samples. Residue specific assignments were determined by using NOE connectivities between protons in the 15NH-H alpha-H beta spin systems of known amino acid type. Additional assignments of the aromatic proton resonances were obtained from 1H NMR spectra of unlabeled and selectively deuterated protein samples. The secondary structure of T4 lysozyme indicated from a qualitative analysis of the NOESY data is consistent with the crystallographic model of the protein.     

Solution structural characteristics of cyanometmyoglobin: resonance assignment of heme cavity residues by two-dimensional NMR

Steady-state nuclear Overhauser effects (NOE), two-dimensional (2D) nuclear Overhauser effect spectroscopy (NOESY), and 2D spin correlation spectroscopy (COSY) have been applied to the fully paramagnetic low-spin, cyanide-ligated complex of sperm whale ferric myoglobin to assign the majority of the heme pocket side-chain proton signals and the remainder of the heme signals. It is shown that the 2D NOESY map reveals essentially all dipolar connectivities observed in ordinary 1D NOE experiments and expected on the basis of crystal coordinates, albeit often more weakly than in a diamagnetic analogue. For extremely broad (approximately 600-Hz) and rapidly relaxing (Tf1 approximately 3 ms) signals which show no NEOSY peaks, we demonstrate that conventional steady-state NOEs obtained under very rapid pulsing conditions still allow detection of the critical dipoar connectivities that allow unambiguous assignments. The COSY map was found to be generally less useful for the hyperfine-shifted residues, with cross peaks detected only for protons greater than 6 A from the iron. Nevertheless, numerous critical COSY cross peaks between strongly hyperfine-shifted peaks were resolved and assigned. In all, 95% (53 of 56 signals) of the total proton sets within approximately 7.5 A of the iron, the region experiencing the strongest hyperfine shifts and paramagnetic relaxation, are now unambiguously assigned. Hence it is clear that the 2D methods can be profitably applied to paramagnetic proteins. The scope and limitations of such application are discussed. The resulting hyperfine shift pattern for the heme confirmed expectations based on model compounds. In contrast, while exhibiting fortuitous 1H NMR spectral similarities, a major discrepancy was uncovered between the hyperfine shift pattern of the axially bound (F8 histidyl) imidazole in the protein and that of the imidazole in a relevant model compound [Chacko, V.P., & La Mar, G. N. (1982) J. Am. Chem. Soc. 104, 7002-7007], providing direct evidence for a protein-based deformation of axial bonding in the protein.     

NMR studies of abasic sites in DNA duplexes: deoxyadenosine stacks into the helix opposite the cyclic analogue of 2-deoxyribose

Proton and phosphorus NMR studies are reported for the complementary d(C-A-T-G-A-G-T-A-C).d(G-T-A-C-F-C-A-T-G) nonanucleotide duplex (designated APF 9-mer duplex) which contains a stable abasic site analogue, F, in the center of the helix. This oligodeoxynucleotide contains a modified tetrahydrofuran moiety, isosteric with 2-deoxyribofuranose, which serves as a structural analogue of a natural apurinic/apyrimidinic site [Takeshita, M., Chang, C.N., Johnson, F., Will, S., & Grollman, A.P. (1987) J. Biol. Chem. 262, 10171-10179]. Exchangeable and nonexchangeable base and sugar protons, including those located at the abasic site, have been assigned in the complementary APF 9-mer duplex by recording and analyzing two-dimensional phase-sensitive NOESY data sets in H2O and D2O solution at low temperature (0 degrees C). These studies indicate that A5 inserts into the helix opposite the abasic site F14 and stacks with flanking G4.C15 and G6.C13 Watson-Crick base pairs. Base-sugar proton NOE connectivities were measured through G4-A5-G6 on the unmodified strand and between the base protons of C15 and the sugar protons of the 5'-flanking residue F14 on the modified strand. These studies establish that all glycosidic torsion angles are anti and that the helix is right-handed at and adjacent to the abasic site in the APF 9-mer duplex. Two of the 16 phosphodiester groups exhibit phosphorus resonances outside the normal spectral dispersion indicative of altered torsion angles at two of the phosphate groups in the backbone of the APF 9-mer duplex.     

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.     

One- and two-dimensional 1H-NMR investigations of two 19-base-pair analogues of the tet operator

Two 19-base-pair oligodeoxynucleotides, analogues of one of the operators which specifically bind the repressor protein in the regulatory part of the transposon Tn10 tetracycline-resistance (tet) determinant, have been studied by 1H-NMR spectroscopy. The analogues contain a mismatch in the central base pair of the double helix (T.T or A.A). The imino protons have been assigned to the base pairs by one-dimensional NOE measurements, and the thermally induced transition from the duplex to the single strand has been followed. The cytidine amino resonances have been assigned by means of two-dimensional NOE spectroscopy in H2O. Two-dimensional phase-sensitive NOE and magnitude-correlated spectra have been recorded in 2H2O; all nonexchangeable protons, with the exception of some of H5', H5" protons, have been assigned. The NMR data made it possible to carry out a qualitative analysis of the structures of both oligodeoxynucleotides. The general structures close to B-DNA, show irregularities in the mismatch areas.     

Dihydrofolate reductase: sequential resonance assignments using 2D and 3D NMR and secondary structure determination in solution

Three-dimensional (3D) heteronuclear NMR techniques have been used to make sequential 1H and 15N resonance assignments for most of the residues of Lactobacillus casei dihydrofolate reductase (DHFR), a monomeric protein of molecular mass 18,300 Da. A uniformly 15N-labeled sample of the protein was prepared and its complex with methotrexate (MTX) studied by 3D 15N/1H nuclear Overhauser-heteronuclear multiple quantum coherence (NOESY-HMQC), Hartmann-Hahn-heteronuclear multiple quantum coherence (HOHAHA-HMQC), and HMQC-NOESY-HMQC experiments. These experiments overcame most of the spectral overlap problems caused by chemical shift degeneracies in 2D spectra and allowed the 1H-1H through-space and through-bond connectivities to be identified unambiguously, leading to the resonance assignments. The novel HMQC-NOESY-HMQC experiment allows NOE cross peaks to be detected between NH protons even when their 1H chemical shifts are degenerate as long as the amide 15N chemical shifts are nondegenerate. The 3D experiments, in combination with conventional 2D NOESY, COSY, and HOHAHA experiments on unlabelled and selectively deuterated DHFR, provide backbone assignments for 146 of the 162 residues and side-chain assignments for 104 residues of the protein. Data from the NOE-based experiments and identification of the slowly exchanging amide protons provide detailed information about the secondary structure of the binary complex of the protein with methotrexate. Sequential NHi-NHi+1 NOEs define four regions with helical structure. Two of these regions, residues 44-49 and 79-89, correspond to within one amino acid to helices C and E in the crystal structure of the DHFR.methotrexate.NADPH complex [Bolin et al. (1982) J. Biol. Chem. 257, 13650-13662], while the NMR-determined helix formed by residues 26-35 is about one turn shorter at the N-terminus than helix B in the crystal structure, which spans residues 23-34. Similarly, the NMR-determined helical region comprising residues 102-110 is somewhat offset from the crystal structure's helix F, which encompasses residues 97-107. Regions of beta-sheet structure were characterized in the binary complex by strong alpha CHi-NHi+1 NOEs and by slowly exchanging amide protons. In addition, several long-range NOEs were identified linking together these stretches to form a beta-sheet. These elements align perfectly with corresponding elements in the crystal structure of the DHFR.methotrexate.NADPH complex, which contains an eight-stranded beta-sheet, indicating that the main body of the beta-sheet is preserved in the binary complex in solution.     

1H NMR studies of lambda cro repressor. 2. Sequential resonance assignments of the 1H NMR spectrum

The cro repressor protein from bacteriophage lambda has been studied in solution by two-dimensional nuclear magnetic resonance spectroscopy (2D NMR). Following the approach of Wüthrich and co-workers [Wüthrich, K., Wider, G., Wagner, G., & Braun, W. (1982) J. Mol. Biol. 155, 311-319], individual spin systems were identified by J-correlated spectroscopy (COSY) supplemented, where necessary, by relayed coherence transfer spectroscopy (RELAY). Nuclear Overhauser effect spectroscopy (NOESY) was used to obtain sequence-specific assignments. From the two-dimensional spectra, the peptide backbone resonances (NH and C alpha H) for 65 of the 66 amino acids were assigned, as well as most of the side chain resonances. The chemical shifts for the assigned protons are reported at 35 degrees C in 10 mM potassium phosphate, pH 6.8, and in 10 mM potassium phosphate, pH 4.6, 0.2 M KCl, and 0.1 mM EDTA. Small shifts were observed for some resonances upon addition of salt, but no major changes in the spectrum were seen, indicating that no global structural change occurs between these ionic strengths. NOE patterns characteristic of alpha-helices, beta-strands, and turns are seen in various regions of the primary sequence. From the location of these regions the secondary structure of cro in solution appears to be virtually identical with the crystal structure [Anderson, W. F., Ohlendorf, D. H., Takeda, Y., & Matthews, B. W. (1981) Nature (London) 290, 754-758]. Missing assignments include the Pro-59 resonances and the peripheral protons of the eight lysine, the three arginine, and three of the five isoleucine residues.     

Two-dimensional NMR studies of staphylococcal nuclease. 1. Sequence-specific assignments of hydrogen-1 signals and solution structure of the nuclease H124L-thymidine 3',5'-bisphosphate-Ca2+ ternary complex

Staphylococcal nuclease H124L is a recombinant protein produced in Escherichia coli whose sequence is identical with that of the nuclease produced by the V8 variant of Staphylococcus aureus. The enzyme-metal ion activator-nucleotide inhibitor ternary complex, nuclease H124L-thymidine 3',5'-bisphosphate-Ca2+, was investigated by two-dimensional (2D) NMR techniques. Efficient overproduction of the enzyme facilitated the production of random fractionally deuterated protein, which proved essential for detailed NMR analysis. 1H NMR spin systems were analyzed by conventional 2D 1H[1H] methods: COSY, relayed COSY, HOHAHA, and NOESY. Assignments obtained by 1H NMR experiments were confirmed and extended by 1H-13C and 1H-15N heteronuclear NMR experiments [Wang, J., Hinck, A. P., Loh, S. N., & Markley, J. L. (1990) Biochemistry (following paper in this issue)]. Spectra of the ternary complexes prepared with protein at natural abundance and at 50% random fractional deuteration provided the information needed for sequence-specific assignments of 121 of the 149 amino acid residues. Short- and intermediate-range NOE connectivities allowed the determination of secondary structural features of the ternary complex: three alpha-helical domains and three antiparallel beta-pleated sheets with several reverse turns. A number of nonsequential long-range HN-HN and H alpha-HN connectivities revealed additional information about the spatial arrangement of these secondary structural elements. The solution structure of this ternary complex shows a close correspondence to the crystal structure of the nuclease wt-thymidine 3',5'-bisphosphate-Ca2+ ternary complex [Cotton, F. A., Hazen, E. E., & Legg, M. J. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 2551-2555].     

Solution conformational preferences of immunogenic peptides derived from the principal neutralizing determinant of the HIV-1 envelope glycoprotein gp120

With standard one- and two-dimensional proton NMR techniques, a common structural motif has been identified in water solutions of short peptide sequences derived from the envelope glycoprotein gp120 of HIV-1. Three peptides of lengths 12, 24, and 40 residues (termed RP342, RP142, and RP70, respectively) were synthesized, each containing a central amino acid sequence common to many HIV-1 isolates. In addition, RP70 contained a disulfide bond between cysteine residues close to the ends of the molecule, forming a loop that is thought to constitute an important structural and immunological component of the intact glycoprotein. Peptides RP70 and RP142 showed evidence for the presence of a significant population of conformations containing a beta-turn in the conserved sequence Gly-Pro-Gly-Arg. Strong nuclear Overhauser effect (NOE) connectivities were observed between the amide protons of the arginine and the adjacent glycine. A weak NOE connectivity was observed between the C alpha H of the proline residue and the NH of the Arg [a d alpha N(i,i + 2) NOE connectivity], confirming the presence of a conformational preference for a turn conformation in this sequence. The remainder of the peptide showed evidence of conformational averaging: no NMR evidence for a uniquely folded structure was obtained for any of the peptides in water solution. Circular dichroism (CD) spectra indicated that no ordered helix was present in water solutions of RP70, although a CD spectrum that indicated the presence of approximately 30% helix could be induced by the addition of trifluoroethanol.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.