The use of two-dimensional correlated spectroscopy to obtain new assignments in the high-resolution 1H nuclear magnetic resonance spectrum of the biantennary complex oligosaccharide isolated from human serum transferrin by hydrazinolysis

The asialo biantennary complex type oligosaccharide from human serum transffrrin was isolated by hydrazinolysis, a method which results in the quantitative release of the intact oligosaccharide free of all amino acids. The ¹H-NMR chemical shifts of the previously assigned anomeric and H-2 protons from the peripheral residues of the glycopeptide are identical to the corresponding values for the reduced oligosaccharide. The chemical shift of GlcNAc-1 H-1 proton in the reduced oligosaccharide was assigned by selective deuteration. Proton J connectivities were determined using two-dimensional ¹H-¹H correlated high resolution NMR spectroscopy. Twelve new assignments were made within the central envelope of the NMR spectrum and a further six were tentatively proposed. The ability to assign proton resonances in this way should allow further conformational studies of the oligosaccharide using nuclear Overhauser effects between the relevant assigned protons on different saccharide residues (Homans, S.W., Dwek, R.A., Fernandes, D.L. and Rademacher, T.W. (1982) FEBS Lett. 150, 503–506).     

Two-dimensional NMR studies of Kazal proteinase inhibitors. 2. Sequence-specific assignments and secondary structure of reactive site modified turkey ovomucoid third domain

The solution structure of modified turkey ovomucoid third domain (OMTKY3*) was investigated by high-resolution proton NMR techniques. OMTKY3* was obtained by enzymatic hydrolysis of the scissile reactive site peptide bond (Leu18-Glu19) in turkey ovomucoid third domain (OMTKY3). All of the backbone proton resonances were assigned to sequence-specific residues except the NH's of Leu1 and Glu19, which were not observed. Over 80% of the side-chain protons also were assigned. The secondary structure of OMTKY3*, as determined from assigned NOESY cross-peaks and identification of slowly exchanging amide protons, contains antiparallel beta-sheet consisting of three strands (residues 21-25, 28-32, and 49-54), one alpha-helix (residues 33-44), and one reverse turn (residues 26-28). This secondary structure closely resembles that of OMTKY3 in solution [Robertson, A. D., Westler, W. M., & Markley, J. L. (1988) Biochemistry (preceding paper in this issue)]. On the other hand, changes in the tertiary structure of the protein near to and remote from the cleavage site are indicated by differences in the chemical shifts of numerous backbone protons of OMTKY3 and OMTKY3*.     

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.     

Two-dimensional NMR studies of Kazal proteinase inhibitors. 1. Sequence-specific assignments and secondary structure of turkey ovomucoid third domain

Two-dimensional proton NMR experiments have been used to sequentially assign resonances to all of the peptide backbone protons of turkey ovomucoid third domain (OMTKY3) except those of the N-terminal alpha-amino group whose signal was not resolved owing to exchange with the solvent. Assignments also have been made for more than 80% of the side-chain protons. Two-dimensional chemical shift correlated spectroscopy (COSY), relayed coherence transfer spectroscopy (RELAY), and two-dimensional homonuclear Hartmann-Hahn spectroscopy (HOHAHA) were used to identify the spin systems of almost half of the residues prior to sequential assignment. Assignments were based on two-dimensional nuclear Overhauser enhancements observed between adjacent residues. The secondary structure of OMTKY3 in solution was determined from additional assigned NOESY cross-peaks; it closely resembles the secondary structure determined by single-crystal X-ray diffraction of OMTKY3 in complex with Streptomyces griseus proteinase B [Fujinaga, M., Read, R.J., Sielecki, A., Ardelt, W., Laskowski, M., Jr., & James, M.N.G. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 4868-4872]. The NMR data provide evidence for three slowly exchanging amide protons that were not identified as hydrogen-bond donors in the crystal structure.     

1H NMR (500 MHz) identification of aromatic residues of gene 32 protein involved in DNA binding by use of protein containing perdeuterated aromatic residues and by site-directed mutagenesis

Preparation of gene 32 protein containing perdeuterated tyrosyl and phenylalanyl residues has allowed the resolution of separate 1H NMR signals for the Tyr and Phe residues of the protein by NMR difference spectra. Upfield shifts in the chemical shifts of a number of aromatic protons previously observed to accompany deoxyoligonucleotide complex formation with gene 32 protein [Prigodich, R. V., Casas-Finet, J., Williams, K. R., Konigsberg, W., & Coleman, J. E. (1984) Biochemistry 23, 522-529] can be assigned to five Tyr and two Phe residues that must form part of the DNA binding domain. Site-directed mutation of Tyr-115 to Ser-115 results in the disappearance of a set of 2,6 and 3,5 tyrosyl protons that are among those moved upfield by oligonucleotide complex formation. These findings suggest that the amino acid sequence from Tyr-73 to Tyr-115 which contains six of the eight Tyr residues of the protein forms part of the DNA binding surface.     

Study of self-association of molecules of deoxyhexanucleotides 5'-d(CpGpTpApCpG) and 5'-d(CpGpCpGpCpG) in water solutions by NMR

The self-association of deoxyribohexanucleoside pentaphosphates 5'-d(CpGpTpApCpG) and 5'-d(CpGpCpGpCpG) in aqueous salt solutions was studied by 1 D- and 2 D homonuclear PMR and heteronuclear 1H-31P-spectroscopies. Signals from nonexchangeable protons of hexamers in NMR spectra were assigned using the available 2M-TOCSY, 2M-NOESY, and 1H-31P-(HMBS) spectra. The dependences of proton chemical shifts of deoxyhexanucleotides on concentration and temperature were measured. In terms of the two-states model (monomer-duplex), constants and thermodynamic parameters of self-association of hexamer molecules in solution were obtained based on these dependences. The values obtained correlate well with theoretical values calculated using the model of the "nearest neighbor" for the formation of duplexes of sequences d(CGTACG) and d(CGCGCG).     

Two-dimensional magnetization exchange spectroscopy of Anabaena 7120 ferredoxin. Nuclear Overhauser effect and electron self-exchange cross peaks from amino acid residues surrounding the 2Fe-2S* cluster

Hyperfine 1H NMR signals of the 2Fe-2S* vegetative ferredoxin from Anabaena 7120 have been studied by two-dimensional (2D) magnetization exchange spectroscopy. The rapid longitudinal relaxation rates of these signals required the use of very short nuclear Overhauser effect (NOE) mixing times (0.5-20 ms). The resulting pattern of NOE cross-relaxation peaks when combined with previous 1D NOE results [Dugad, L. B., La Mar, G. N., Banci, L., & Bertini, I. (1990) Biochemistry 29, 2263-2271] led to elucidation of the carbon-bound proton spin systems from each of the four cysteines ligated to the 2Fe-2S* cluster in the reduced ferredoxin. Additional NOE cross peaks were observed that provide information about other amino acid residues that interact with the iron-sulfur cluster. NOE cross peaks were assigned tentatively to Leu27, Arg42, and Ala43 on the basis of the X-ray coordinates of oxidized Anabaena 7120 ferredoxin [Rypniewski, W.R., Breiter, D.R., Benning, M.M., Wesenberg, G., Oh, B.-H., Markley, J.L., Rayment, I., & Holden, H. M. (1991) Biochemistry 30, 4126-4131]. Three chemical exchange cross peaks were detected in magnetization exchange spectra of half-reduced ferredoxin and assigned to the 1H alpha protons of Cys49 and Cys79 [both of whose sulfur atoms are ligated to Fe(III)] and Arg42 (whose amide nitrogen is hydrogen-bonded to one of the inorganic sulfurs of the 2Fe-2S* cluster). The chemical exchange cross peaks provide a means of extending assignments in the spectrum of reduced ferredoxin to assignments in the spectrum of the oxidized protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Differences between the electronic environments of reduced and oxidized Escherichia coli DsbA inferred from heteronuclear magnetic resonance spectroscopy.

DsbA is the strongest protein disulfide oxidant yet known and is involved in catalyzing protein folding in the bacterial periplasm. Its strong oxidizing power has been attributed to the lowered pKa of its reactive active site cysteine and to the difference in thermodynamic stability between the oxidized and the reduced form. However, no structural data are available for the reduced state. Therefore, an NMR study of DsbA in its two redox states was undertaken. We report here the backbone 1HN, 15N, 13C(alpha) 13CO, 1H(alpha), and 13Cbeta NMR assignments for both oxidized and reduced Escherichia coli DsbA (189 residues). Ninety-nine percent of the frequencies were assigned using a combination of triple (1H-13C-15N) and double resonance (1H-15N or 1H-13C) experiments. Secondary structures were established using the CSI (Chemical Shift Index) method, NOE connectivity patterns, 3(J)H(N)H(alpha) and amide proton exchange data. Comparison of chemical shifts for both forms reveals four regions of the protein, which undergo some changes in the electronic environment. These regions are around the active site (residues 26 to 43), around His60 and Pro 151, and also around Gln97. Both the number and the amplitude of observed chemical shift variations are more substantial in DsbA than in E. coli thioredoxin. Large 13C(alpha) chemical shift variations for residues of the active site and residues Phe28, Tyr34, Phe36, Ile42, Ser43, and Lys98 suggest that the backbone conformation of these residues is affected upon reduction.     

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.     

Determination of the secondary structure of the DNA binding protein Ner from phage Mu using 1H homonuclear and 15N-1H heteronuclear NMR spectroscopy

The sequential resonance assignment of the 1H and 15N NMR spectra of the DNA binding protein Ner from phage Mu is presented. This is carried out by using a combination of 1H-1H and 1H-15N two-dimensional experiments. The availability of completely labeled 15N protein enabled us to record a variety of relayed heteronuclear multiple quantum coherence experiments, thereby enabling the correlation of proton-proton through-space and through-bond connectivities with the chemical shift of the directly bonded 15N atom. These heteronuclear experiments were crucial for the sequential assignment as the proton chemical shift dispersion of the Ner protein is limited and substantial overlap precluded unambiguous assignment of the homonuclear spectra in several cases. From a qualitative interpretation of the NOE data involving the NH, C alpha H, and C beta H protons, it is shown that Ner is composed of five helices extending from residues 11 to 22, 27 to 34, 38 to 45, 50 to 60, and 63 to 73.     

Structure analyses of DNA oligomers by use of heteronuclear 2D NMR.

DNA oligomer d(CGGAAGACTCTCCTCCG):d(CGGAGGAGAGTCTTCCG) named UASG (17mer M.W. = 11 kDa) was studied by 1H NMR and heteronuclear two dimensional (2D) NMR. All the labile protons and half of the non-exchangeable protons were assigned by use of conventional 1H 2D experiments including NOESY using 1-1 echo excitation for water suppression. Signal degeneracy in the sugar proton region made it difficult to make assignments of the remaining half of the non-exchangeable protons of the oligomer in 1H 2D spectra. Here we report a new strategy using 1H/13C and 1H/31P heteronuclear single-quantum correlation spectroscopy combined with homonuclear three dimensional NOESY-TOCSY. By this strategy, most of the proton resonances of the oligomer have been assigned, and it turned out that the whole conformation of the oligomer is B-form like.     

Proton nuclear magnetic resonance studies of intact native bovine parathyroid hormone

Native intact bovine PTH was studied by proton nuclear magnetic resonance (NMR) techniques, at pH 3.5 and pH 6.3. The 1H-NMR spectra had good resolution and many multiplet structures were observed. Assignment of the NMR resonances corresponding to specific amino acids was approached using 1H chemical shifts, coupling constants, and pH dependence in the one-dimensional spectra and the 1H-1H connectivities revealed in two-dimensional homonuclear correlated spectroscopy (COSY) experiments. All the aromatic proton resonances were assigned. Two histidine residues had lower pK than the other two. The methyl groups of two residues were moved significantly downfield: using COSY and two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) correlations, these were assigned to an alanine residue close to both Trp-23 and Tyr-43, and a valine residue in close spatial proximity to Trp-23. The NOESY spectrum also showed cross-peaks between the residues of the upfield valine-leucine-isoleucine methyl envelope. Many of the H alpha protons moved upfield as the pH was increased. These results indicate that intact native PTH exists in a preferred conformation in solution at pH 6.5. Our studies have provided new information on the three-dimensional spatial proximity of several amino acids along the polypeptide chain. The observed interactions are consistent with the currently accepted model suggesting that the hormone has two separate structural domains associated with the amino- and carboxy-terminal regions of the molecule respectively. The potential implications of this model for the expression of biological activity are discussed.     

Structure of the toxic domain of the Escherichia coli heat-stable enterotoxin ST I

Active fragments of the heat-stable enterotoxin ST I of Escherichia coli were chemically synthesized with the sequence Cys-Cys-Glu-Leu-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-(Tyr) and studied by proton (1H NMR) and carbon-13 (13C NMR) nuclear magnetic resonance spectroscopy as a function of pH and temperature. All of the nonexchangeable protons in the 1H NMR spectrum were assigned. Although all amide protons were present at temperatures below 25 degrees C and and pH values below 6, some of the resonances are broad and could not be assigned. The temperature dependence of these broad resonances indicates a change in conformation that is localized in the N-terminus. Other amide protons disappear at higher temperatures owing to chemical exchange with the solvent. Sufficient resonance assignments can be made at high and low temperatures to permit structural conclusions to be made. The chemical shifts of the alpha-carbon protons indicate the presence of substantial structure, which was further defined with the observed pattern of nuclear Overhauser enhancements (NOEs), coupling constants, and exchange rates. The NMR data identify a turn from Ala-14 to Cys-18. A second likely turn is centered around the proline residue. An interresidue NOE between the alpha-carbon protons of Asn-12 and Gly-17 indicates that the molecule folds back on itself. The NMR information is sufficient to define the structure of the C-terminal region of ST I. Manual model building then indicated that one arrangement of the three disulfides is particularly compatible with the NMR data and van der Waals constraints. A model incorporating the disulfide arrangement proposed by Houghten and his co-workers [Houghten, R.A., Ostresh, J.M., & Klipstein, F.A. (1984) Eur. J. Biochem. 145, 157-162] and the NMR constraints was derived with the programs PROTO [Frayman, F. (1985) Ph.D. Thesis, Northwestern University] and NOEMOT [Lane, A.N., Lefévre, J.-F., & Jardetsky, O. (1986) Biochim. Biophys. Acta 867, 45-56].     

NMR study of the exchange rates of allosterically responsive labile protons in the heme pockets of hemoglobin A.

1H NMR spectroscopy has been used to measure the proximal histidyl labile ring proton (NH) rates of exchange with bulk solvent in the individual subunits of hemoglobin (Hb) A. These protons displayed a substantial decrease in their exchange rates in comparison with related monomeric proteins and exhibited sensitivity to the quarternary state. With the beta subunit NH, the exchange behaviour was similar to an allosterically responsive subset of protons, which have been identified using 1H-3H methods (Englander, J.J., R. Rogero, and S. W. Englander, 1983, J. Mol. Biol. 169:325-344). Assuming similar exchange mechanisms for the two subunits, the NMR data suggested a more flexible alpha than beta subunit in Hb A.     

Two-dimensional proton nuclear magnetic resonance investigation of the synthetic deoxyribonucleic acid decamer d(ATATCGATAT)2

In this study two-dimensional NMR techniques (COSY and NOESY) have been used in conjunction with one-dimensional NMR results to complete the assignment of the proton NMR spectrum of the double-stranded DNA decamer, d(ATATCGATAT)2, and to obtain qualitative information about numerous interproton distances in this molecule and some limited information about conformational dynamics. COSY and NOESY measurements have been combined to systematically assign many of the resonances from the H1' and H2',2" sugar protons to specific nucleotides in the double helix. This method relies on the fact that sugar protons within a specific nucleotide are scalar coupled and that base protons (AH8, GH8, TH6, and CH6) in right-handed helices can interact simultaneously with their own H2',2" sugar protons and those of the adjacent (5'-3') nucleotide attached to its 5' side (i.e., XpA not ApX). A COSY experiment is used to identify sugar resonances within a residue whereas the NOESY experiment allows the neighboring sugar to be connected (linked). The CH5 and CH6 resonances in the spectrum can immediately be identified by the COSY experiment. The methyl protons of thymine residues exhibit strong through-space interbase interactions both with their own TH6 proton and with AH8 proton on the adjacent (5'-3') adenine residue. These interactions are used both to make assignments of the spectra and to establish that the thymine methyl groups are in close proximity to the AH8 protons of adjacent adenine residues [Feigon, J., Wright, J. M., Leupin, W., Denny, W. A., & Kearns, D. R. (1982) J. Am. Chem. Soc. 104, 5540].(ABSTRACT TRUNCATED AT 250 WORDS)     

A comparison by 220-MHz NMR of histidine hydronium ion titrations in porcine pancreatic ribonuclease and an extensively deglycosylated derivative.

220-MHz NMR was used to observe the titration behavior of the 5 histidine residues in porcine pancreatic ribonuclease (ribonucleate pyrimidine-nucleotido-2'-transferase (cyclizing), EC 3.1.4.22) and a derivative prepared by removal of 80% of the attached carbohydrate from this glycoprotein. Resonances due to histidine C-2 protons were observed over the full pH range for 3 of the residues; such resonances for the remaining 2 histidine residues broadened out as the pH was increased. Resonances due to histidine C-4 protons were also observed for 2 of the residues. The titration curves for both proteins were identical within experimental error. Resonances were assigned by comparison with histidine NMR titrations in ribonucleases from other species. Histidine 105, immediately adjacent to the site of attachment of a heterosaccharide side chain, has a C-2 proton chemical shift and pK that are insensitive to the large alteration in the bulk of the carbohydrate side chain. The chemical shifts of the C-2 proton of histidine 48 and of the C-4 proton of histidine 80, histidine residues that are close to one another and to another heterosaccharide side chain, show a similar insensitivity. The observations are direct evidence in support of the thesis that the heterosaccharides in porcine ribonuclease project away from the surface of the protein into the solution environment.     

Deoxyguanosine-deoxyadenosine pairing in the d(C-G-A-G-A-A-T-T-C-G-C-G) duplex: conformation and dynamics at and adjacent to the dG X dA mismatch site

Nuclear magnetic resonance (NMR) has been used to monitor the conformation and dynamics of the d-(C1-G2-A3-G4-A5-A6-T6-T5-C4-G3-C2-G1) self-complementary dodecanucleotide (henceforth called 12-mer GA) that contains a dG X dA purine-purine mismatch at position 3 in the sequence. These results are compared with the corresponding d(C-G-C-G-A-A-T-T-C-G-C-G) dodecamer duplex (henceforth called 12-mer) containing standard Watson-Crick base pairs at position 3 [Patel, D.J., Kozlowski, S.A., Marky, L.A., Broka, C., Rice, J.A., Itakura, K., & Breslauer, K.J. (1982) Biochemistry 21, 428-436]. The dG X dA interaction at position 3 was monitored at the guanosine exchangeable H-1 and nonexchangeable H-8 protons and the nonexchangeable adenosine H-2 proton. We demonstrate base-pair formation between anti orientations of the guanosine and adenosine rings on the basis of nuclear Overhauser effects (NOE) observed between the H-2 proton of adenosine 3 and the imino protons of guanosine 3 (intra base pair) and guanosines 2 and 4 (inter base pair). The dG(anti) X dA(anti) pairing should result in hydrogen-bond formation between the guanosine imino H-1 and carbonyl O-6 groups and the adenosine N-1 and NH2-6 groups, respectively. The base pairing on either side of the dG X dA pair remains intact at low temperature, but these dG X dC pairs at positions 2 and 4 are kinetically destabilized in the 12-mer GA compared to the 12-mer duplex. We have estimated the hydrogen exchange kinetics at positions 4-6 from saturation-recovery measurements on the imino protons of the 12-mer GA duplex between 5 and 40 degrees C. The measured activation energies for imino proton exchange in the 12-mer GA are larger by a factor of approximately 2 compared to the corresponding values in the 12-mer duplex. This implies that hydrogen exchange in the 12-mer GA duplex results from a cooperative transition involving exchange of several base pairs as was previously reported for the 12-mer containing a G X T wobble pair at position 3 [Pardi, A., Morden, K.M., Patel, D.J., & Tinoco, I., Jr. (1982) Biochemistry 21, 6567-6574]. We have assigned the nonexchangeable base protons by intra and inter base pair NOE experiments and monitored these assigned markers through the 12-mer GA duplex to strand transition.(ABSTRACT TRUNCATED AT 400 WORDS)     

Sequence-specific 1H NMR assignments and secondary structure in solution of Escherichia coli trp repressor

Sequence-specific 1H NMR assignments are reported for the active L-tryptophan-bound form of Escherichia coli trp repressor. The repressor is a symmetric dimer of 107 residues per monomer; thus at 25 kDa, this is the largest protein for which such detailed sequence-specific assignments have been made. At this molecular mass the broad line widths of the NMR resonances preclude the use of assignment methods based on 1H-1H scalar coupling. Our assignment strategy centers on two-dimensional nuclear Overhauser spectroscopy (NOESY) of a series of selectively deuterated repressor analogues. A new methodology was developed for analysis of the spectra on the basis of the effects of selective deuteration on cross-peak intensities in the NOESY spectra. A total of 90% of the backbone amide protons have been assigned, and 70% of the alpha and side-chain proton resonances are assigned. The local secondary structure was calculated from sequential and medium-range backbone NOEs with the double-iterated Kalman filter method [Altman, R. B., & Jardetzky, O. (1989) Methods Enzymol. 177, 218-246]. The secondary structure agrees with that of the crystal structure [Schevitz, R., Otwinowski, Z., Joachimiak, A., Lawson, C. L., & Sigler, P. B. (1985) Nature 317, 782], except that the solution state is somewhat more disordered in the DNA binding region and in the N-terminal region of the first alpha-helix. Since the repressor is a symmetric dimer, long-range intersubunit NOEs were distinguished from intrasubunit interactions by formation of heterodimers between two appropriate selectively deuterated proteins and comparison of the resulting NOESY spectrum with that of each selectively deuterated homodimer. Thus, from spectra of three heterodimers, long-range NOEs between eight pairs of residues were identified as intersubunit NOEs, and two additional long-range intrasubunits NOEs were assigned.     

360-MHz 1H nuclear-magnetic-resonance spectroscopy of sialyl-oligosaccharides from patients with sialidosis (mucolipidosis I and II)

360-MHz proton nuclear magnetic resonance spectra were recorded of 10 sialyl-oligosaccharides isolated from urine of sialidosis patients. Their structures are related to the complex asparagine-linked glycan chains of glycoproteins. By correlation of these spectra and comparison with spectra of reference glycopeptides and sialyl-lactose isomers it was possible to assign all signals belonging to anomeric, mannose H-2, sialic acid H-3 and N-acetyl protons. The number of the consituting monosaccharide residues of the oligomers can be obtained by integration of the above-mentioned signals. The chemical shifts of the anomeric and mannose H-2 protons give information about the type of glycan structure (mono-, bi-, triantennary) and the presence of terminal sialic acid at each of the antennas. The chemical shifts of sialic acid H-3 protons are typical for sialic acid residues in 2 leads to 3 or 2 leads to 6 linkage to galactose.