15N-labeled tRNA. Identification of 4-thiouridine in Escherichia coli tRNASer1 and tRNATyr2 by 1H-15N two-dimensional NMR spectroscopy

Uridine is uniquely conserved at position 8 in elongator tRNAs and binds to A14 to form a reversed Hoogsteen base pair which folds the dihydrouridine loop back into the core of the L-shaped molecule. On the basis of 1H NMR studies, Hurd and co-workers (Hurd, R. E., Robillard, G. T., and Reid, B. R. (1977) Biochemistry 16, 2095-2100) concluded that the interaction between positions 8 and 14 is absent in Escherichia coli tRNAs with only 3 base pairs in the dihydrouridine stem. We have taken advantage of the unique 15N chemical shift of N3 in thiouridine to identify 1H and 15N resonances for the imino units of S4U8 and s4U9 in E. coli tRNASer1 and tRNATyr2. Model studies with chloroform-soluble derivatives of uridine and 4-thiouridine show that the chemical shifts of the protons in the imino moieties move downfield from 7.9 to 14.4 ppm and from 9.1 to 15.7 ppm, respectively; whereas, the corresponding 15N chemical shifts move downfield from 157.5 to 162.5 ppm and from 175.5 to 180.1 ppm upon hydrogen bonding to 5'-O-acetyl-2',3'-isopropylidene adenosine. The large difference in 15N chemical shifts for U and s4U allows one to unambiguously identify s4U imino resonances by 15N NMR spectroscopy. E. coli tRNASer1 and tRNATyr2 were selectively enriched with 15N at N3 of all uridines and modified uridines. Two-dimensional 1H-15N chemical shift correlation NMR spectroscopy revealed that both tRNAs have resonances with 1H and 15N chemical shifts characteristic of s4UA pairs. The 1H shift is approximately 1 ppm upfield from the typical s4U8 resonance at 14.8 ppm, presumably as a result of local diamagnetic anisotropies. An additional s4U resonance with 1H and 15N shifts typical of interaction of a bound water or a sugar hydroxyl group with s4U9 was discovered in the spectrum of tRNATyr2. Our NMR results for tRNAs with 3-base pair dihydrouridine stems suggest that these molecules have an U8A14 tertiary interaction similar to that found in tRNAs with 4-base pair dihydrouridine stems.     

Preparation of platinum(IV) complexes with dipeptide and diimine. X-ray crystal structure and 195Pt NMR spectra

We prepared platinum(IV) complexes containing dipeptide and diimine or diamine, the [PtCl(dipeptide-N,N,O)(diimine or diamine)]Cl complex, where -N,N,O means dipeptide coordinated as a tridentate chelate, dipeptide=glycylglycine (NH(2)CH(2)CON(-)CH(2)COO(-), digly, where two protons of dipeptide are detached when the dipeptide coordinates to metal ion as a tridentate chelate), glycyl-L-alanine (NH(2)CH(2)CON(-)CHCH(3)COO(-), gly-L-ala), L-alanylglycine (NH(2)CH CH(3)CON(-)CH(2)COO(-), L-alagly), or L-alanyl-L-alanine (NH(2)CHCH(3)CON(-)CHCH(3)COO(-), dil-ala), and diimine or diamine=bipyridine (bpy), ethylenediamine (en), N-methylethylenediamine (N-Me-en), or N,N'-dimethylethylenediamine (N,N'-diMe-en). In the complexes containing gly-L-ala or dil-ala, two separate peaks of the (195)Pt NMR spectra of the [PtCl(dipeptide-N,N,O)(diimine or diamine)]Cl complexes appeared in, but in the complexes containing digly or L-alagly, one peak which contained two overlapped signals appeared. One of the two complexes containing gly-L-ala and bpy, [PtCl(gly-L-ala-N,N,O)(bpy)]NO(3), crystallized and was analyzed. This complex has the monoclinic space group P2(1)2(1)2(1) with unit cell dimensions of a=9.7906(3)A, b=11.1847(2)A, c=16.6796(2)A, Z=4. The crystal data revealed that this [PtCl(gly-L-ala-N,N,O)(bpy)]NO(3) complex has the near- (Cl, CH(3)) configuration of two possible isomers. Based on elemental analysis, the other complex must have the near- (Cl, CH(3))-[PtCl(gly-L-ala-N,N,O)(bpy)]NO(3) configuration. The (195)Pt NMR chemical shifts of the near- (Cl, CH(3))-[PtCl(gly-L-ala-N,N,O)(bpy)]NO(3) complex and the far- (Cl, CH(3))-[PtCl(gly-L-ala-N,N,O)(bpy)]NO(3) complex are 0 ppm and -19 ppm, respectively (0 ppm for the Na(2)[PtCl(6)] signal). The additive property of the (195)Pt NMR chemical shift is discussed. The (195)Pt NMR chemical shifts of [PtCl(dipeptide-N,N,O)(bpy)]Cl appeared at a higher field when the H attached to the dipeptide carbon atom was replaced with a methyl group. On the other hand, the (195)Pt NMR chemicals shifts of [PtCl(dipeptide-N,N,O)(diamine)] appeared at a lower field when the H attached to the diamine nitrogen atom was replaced with a methyl group, in the order of [PtCl(digly-N,N,O)(en)]Cl, [PtCl(digly-N,N,O)(N-Me-en)]Cl, and [PtCl(digly-N,N,O)(N,N'-diMe-en)]Cl.     

NMR identification of left-handed polyproline type II helices

NMR characteristics of a model left-handed 3(1)-helical peptide are reported in this study. With temperature and sequence corrections on the predicted random coil (15)N chemical shifts, a significant (15)N chemical shift deviation is observed for the model 3(1) peptide. The (15)N chemical shift differences also correlate well with the molar ellipticities (at 220 nm) of the CD spectra at different temperatures, indicating that the (15)N chemical shift is a sensitive probe for 3(1)-helices. The average (3)J(HNalpha) and (1)J(CalphaHalpha) values of the model peptide are determined to be 6.5 and 142.6 Hz, respectively, which are consistent with the values calculated from the geometry of 3(1)-helices. With careful measurements of amide (15)N chemical shifts and incorporating temperature and sequence effect corrections, the (15)N chemical shifts can be used together with (3)J(HNalpha) and (1)J(CalphaHalpha) to differentiate 3(1)-helices from random coils with high confidence. Based on the observed NMR characteristics, a strategy is developed for probing left-handed 3(1)-helical structures from other secondary structures.     

Cross-peptide bond 13C--15N coupling constants by 13C and J cross-polarization 15N NMR

Comparative 13C--15N coupling constants are reported for the linear dipeptide tBoc-L-[U-13C]Ala-[15N]GlyOMe and the corresponding cyclic diketopiperazine, both in dimethylsulfoxide (DMSO) and, upon removal of the tBoc group, in water solutions. Spectra were obtained by 13C NMR and by the first application of J cross-polarization (JCP) 15N NMR, which greatly reduces the time required to accumulate 15N NMR spectra. In DMSO there was evidence for the formation of complexed species which were not present in water. The values obtained for the cross-peptide bond coupling constant 2J13C alpha--15N were consistently less (by 2.2 Hz in DMSO, 4.3 Hz in water) for the cyclic than for the linear peptide, which may be related to the cross-peptide bond conformation. The 15N resonance for the cyclic peptide was shifted only 2 ppm downfield from the linear peptide chemical shift value in both solvents.     

Nuclear magnetic resonance studies of the old yellow enzyme. 2. 13C NMR of the enzyme recombined with 13C-labeled flavin mononucleotides

The apoenzyme of NADPH oxidoreductase, 'old yellow enzyme', was reconstituted with selectively 13C-enriched flavin mononucleotides and investigated by 13C NMR spectroscopy. The 13C NMR results confirm the results obtained by 15N NMR spectroscopy and yield additional information about the coenzyme-apoenzyme interaction. A strong deshielding of the C(2) and C(4) atoms of enzyme-bound FMN both in the oxidized and reduced state is observed, which is supposed to be induced by hydrogen-bond formation between the protein and the two carbonyl groups at C(2) and C(4) of the isoalloxazine ring system. The chemical shifts of all 13C resonances of the flavin in the two-electron-reduced state indicate that the N(5) atom is sp3-hybridized. From 31P NMR measurements it is concluded that the FMN phosphate group is not accessible to bulk solvent. The unusual 31P chemical shift of FMN in old yellow enzyme seems to indicate a different binding mode of the FMN phosphate group in this enzyme as compared to the flavodoxins. The 13C and 15N NMR data on the old-yellow-enzyme--phenolate complexes show that the atoms of the phenolate are more deshielded whereas the atoms of the enzyme-bound isoalloxazine ring are more shielded upon complexation. A non-linear correlation exists between the chemical shifts of the N(5) and the N(10) atoms and the pKa value of the phenolate derivative bound to the protein. Since the chemical shifts of N(5), N(10) and C(4a) are influenced most on complexation it is suggested that the phenolate is bound near the pyrazine ring of the isoalloxazine system. 15N NMR studies on the complex between FMN and 2-aminobenzoic acid indicate that the structure of this complex differs from that of the old-yellow-enzyme--phenolate complexes.     

15N NMR and electronic properties of S-nitrosothiols.

Investigation of the 15N NMR of S-nitrosothiols showed that primary and tertiary RSNOs have distinct 15N chemical shifts around 730 and 790 ppm, respectively. Using 15N NMR technique, the equilibrium constant of NO transfer between SNAP and GSH was found to be 0.74. For primary RSNOs, linear relationships exist among 15N NMR chemical shifts, reduction potentials, and the pK(a)s of their parent thiols.     

NMR study of the metabolic 15N isotopic enrichment of cyanophycin synthesized by the cyanobacterium Synechocystis sp. strain PCC 6308

1H, 13C and 15N nuclear magnetic resonance (NMR) spectroscopy has been used to characterize cyanophycin, a multi-l-arginyl-poly-[l-aspartic acid] polypeptide from the cyanobacterium Synechocystis sp. strain PCC 6308. 1H, 13C and 15N chemical shifts and 1JHN and 1JCN coupling constants were measured in isolated 15N-labeled cyanophycin, and showed chemical shift values and J-couplings consistent with the reported polypeptide structure. 15N enrichment levels were determined from the extent of 1H-15N J-coupling in 1H NMR spectra of cyanophycin. Similar experiments using 13C-15N coupling in 13C NMR spectra were not useful in determining enrichment levels.     

Inhibition of thermolysin by phosphonamidate transition-state analogues: measurement of 31P-15N bond lengths and chemical shifts in two enzyme-inhibitor complexes by solid-state nuclear magnetic resonance

31P and 15N chemical shifts and 31P-15N bond lengths have been measured with solid-state NMR techniques in two inhibitors of thermolysin, carbobenzoxy-Glyp-L-Leu-L-Ala (ZGpLA) and carbobenzoxy-L-Phep-L-Leu-L-Ala (ZFpLA), both as free lithium salts and when bound to the enzyme. Binding of both inhibitors to thermolysin results in large changes in the 31P chemical shifts. These changes are more dramatic for the tighter binding inhibitor ZFpLA, where a approximately 20 ppm downfield movement of the 31P isotropic chemical shift (sigma iso) is observed. This shift is due to changes in the shift tensor elements sigma 11 and sigma 22, while sigma 33 remains essentially constant. We observed a similar pattern for ZGpLA, but only a approximately 5 ppm change occurs in sigma iso. The changes in the 15N chemical shifts for both inhibitors are small upon binding, amounting to downfield shifts of 2 and 4 ppm for ZGpLA and ZFpLA, respectively. This indicates that there are no changes in the protonation state of the 15N in either the ZFpLA- or the ZGpLA-thermolysin complex. NMR distance measurements yield a P-N bond length rP-N = 1.68 +/- 0.03 A for the tight binding inhibitor ZFpLA both in its free lithium salt form and in its thermolysin-ZFpLA complex, a distance that is much shorter than the 1.90-A distance reported by X-ray crystallography studies [Holden et al. (1987) Biochemistry 26, 8542-8553].(ABSTRACT TRUNCATED AT 250 WORDS)     

Active site contacts in the purine nucleoside phosphorylase--hypoxanthine complex by NMR and ab initio calculations

Hypoxanthine (Hx) with specific (15)N labels has been used to probe hydrogen-bonding interactions with purine nucleoside phosphorylase (PNP) by NMR spectroscopy. Hx binds to human PNP as the N-7H tautomer, and the N-7H (1)H and (15)N chemical shifts are located at 13.9 and 156.5 ppm, respectively, similar to the solution values. In contrast, the (1)H and (15)N chemical shifts of N-1H in the PNP.Hx complex are shifted downfield by 3.5 and 7.5 ppm to 15.9 and 178.8 ppm, respectively, upon binding. Thus, hydrogen bonding at N-1H is stronger than at N-7H in the complex. Ab initio chemical shift calculations on model systems that simulate Hx in solution and bound to PNP are used to interpret the NMR data. The experimental N-7H chemical shift changes are caused by competing effects of two active site contacts. Hydrogen bonding of Glu201 to N-1H causes upfield shifts of the N-7H group, while the local hydrogen bond (C=O to N-7H from Asn243) causes downfield shifts. The observed N-7H chemical shift can be reproduced by a hydrogen bond distance approximately 0.13 A shorter (but within experimental error) of the experimental value found in the X-ray crystal structure of the bovine PNP.Hx complex. The combined use of NMR and ab initio chemical shift computational analysis provides a novel approach to understand enzyme-ligand interactions in PNP, a target for anticancer agents. This approach has the potential to become a high-resolution tool for structural determination.     

Ligand-induced changes in dynamics in the RT loop of the C-terminal SH3 domain of Sem-5 indicate cooperative conformational coupling

We report the effects of peptide binding on the (15)N relaxation rates and chemical shifts of the C-SH3 of Sem-5. (15)N spin-lattice relaxation time (T(1)), spin-spin relaxation time (T(2)), and ((1)H)-(15)N NOE were obtained from heteronuclear 2D NMR experiments. These parameters were then analyzed using the Lipari-Szabo model free formalism to obtain parameters that describe the internal motions of the protein. High-order parameters (S(2) > 0.8) are found in elements of regular secondary structure, whereas some residues in the loop regions show relatively low-order parameters, notably the RT loop. Peptide binding is characterized by a significant decrease in the (15)N relaxation in the RT loop. Concomitant with the change in dynamics is a cooperative change in chemical shifts. The agreement between the binding constants calculated from chemical shift differences and that obtained from ITC indicates that the binding of Sem-5 C-SH3 to its putative peptide ligand is coupled to a cooperative conformational change in which a portion of the binding site undergoes a significant reduction in conformational heterogeneity.     

Structure of an elastin-mimetic polypeptide by solid-state NMR chemical shift analysis

The conformation of an elastin-mimetic recombinant protein, [(VPGVG)4(VPGKG)]39, is investigated using solid-state NMR spectroscopy. The protein is extensively labeled with 13C and 15N, and two-dimensional 13C-13C and 15N-13C correlation experiments were carried out to resolve and assign the isotropic chemical shifts of the various sites. The Pro 15N, 13Calpha, and 13Cbeta isotropic shifts, and the Gly-3 Calpha isotropic and anisotropic chemical shifts support the predominance of type-II beta-turn structure at the Pro-Gly pair but reject a type-I beta-turn. The Val-1 preceding Pro adopts mostly beta-sheet torsion angles, while the Val-4 chemical shifts are intermediate between those of helix and sheet. The protein exhibits a significant conformational distribution, shown by the broad line widths of the 15N and 13C spectra. The average chemical shifts of the solid protein are similar to the values in solution, suggesting that the low-hydration polypeptide maintains the same conformation as in solution. The ability to measure these conformational restraints by solid-state NMR opens the possibility of determining the detailed structure of this class of fibrous proteins through torsion angles and distances.     

Expression of doubly labeled Saccharomyces cerevisiae iso-1 ferricytochrome c and (1)H, (13)C and (15)N chemical shift assignments by multidimensional NMR

We have expressed [U-(13)C,(15)N]-labeled Saccharomyces cerevisiae iso-1 cytochrome c C102T;K72A in Escherichia coli with a yield of 11 mg/l of growth medium. Nuclear magnetic resonance (NMR) studies were conducted on the Fe(3+) form of the protein. We report herein chemical shift assignments for amide (1)H and (15)N, (13)C(omicron), (13)C(alpha), (13)C(beta), (1)H(alpha) and (1)H(beta) resonances based upon a series of three-dimensional NMR experiments: HNCA, HN(CO)CA, HNCO, HN(CA)CO, HNCACB, HCA(CO)N, HCCH-TOCSY and HBHA(CBCA)NH. An investigation of the chemical shifts of the threonine residues was also made by using density functional theory in order to help solve discrepancies between (15)N chemical shift assignments reported in this study and those reported previously.     

Assignment of the side-chain 1H and 13C resonances of interleukin-1 beta using double- and triple-resonance heteronuclear three-dimensional NMR spectroscopy

The assignment of the aliphatic 1H and 13C resonances of IL-1 beta, a protein of 153 residues and molecular mass 17.4 kDa, is presented by use of a number of novel three-dimensional (3D) heteronuclear NMR experiments which rely on large heteronuclear one-bond J couplings to transfer magnetization and establish through-bond connectivities. These 3D NMR experiments circumvent problems traditionally associated with the application of conventional 2D 1H-1H correlation experiments to proteins of this size, in particular the extensive chemical shift overlap which precludes the interpretation of the spectra and the reduced sensitivity arising from 1H line widths that are often significantly larger than the 1H-1H J couplings. The assignment proceeds in two stages. In the first step the 13C alpha chemical shifts are correlated with the NH and 15N chemical shifts by a 3D triple-resonance NH-15N-13C alpha (HNCA) correlation experiment which reveals both intraresidue NH(i)-15N(i)-13C alpha (i) and some weaker interresidue NH(i)-15N(i)-C alpha (i-1) correlations, the former via intraresidue one-bond 1JNC alpha and the latter via interresidue two-bond 2JNC alpha couplings. As the NH, 15N, and C alpha H chemical shifts had previously been sequentially assigned by 3D 1H Hartmann-Hahn 15N-1H multiple quantum coherence (3D HOHAHA-HMQC) and 3D heteronuclear 1H nuclear Overhauser 15N-1H multiple quantum coherence (3D NOESY-HMQC) spectroscopy [Driscoll, P.C., Clore, G.M., Marion, D., Wingfield, P.T., & Gronenborn, A.M. (1990) Biochemistry 29, 3542-3556], the 3D triple-resonance HNCA correlation experiment permits the sequence-specific assignments of 13C alpha chemical shifts in a straightforward manner. The second step involves the identification of side-chain spin systems by 3D 1H-13C-13C-1H correlated (HCCH-COSY) and 3D 1H-13C-13C-1H total correlated (HCCH-TOCSY) spectroscopy, the latter making use of isotropic mixing of 13C magnetization to obtain relayed connectivities along the side chains. Extensive cross-checks are provided in the assignment procedure by examination of the connectivities between 1H resonances at all the corresponding 13C shifts of the directly bonded 13C nuclei. In this manner, we were able to obtain complete 1H and 13C side-chain assignments for all residues, with the exception of 4 (out of a total of 15) lysine residues for which partial assignments were obtained. The 3D heteronuclear correlation experiments described are highly sensitive, and the required set of three 3D spectra was recorded in only 1 week of measurement time on a single uniformly 15N/13C-labeled 1.7 mM sample of interleukin-1 beta.(ABSTRACT TRUNCATED AT 400 WORDS)     

13C and 15N NMR studies on the interaction between 6,7-dimethyl-8-ribityllumazine and lumazine protein

The interaction between the prosthetic group 6,7-dimethyl-8-(1'-D-ribityl)lumazine and the lumazine apoproteins from two marine bioluminescent bacteria, one from a relatively thermophilic species, Photobacterium leiognathi, and the other from a psychrophilic species, Photobacterium phosphoreum, was studied by 13C and 15N NMR using various selectively enriched derivatives. It is shown that the electron distribution in the protein-bound 6,7-dimethyl-8-ribityllumazine differs from that of free 6,7-dimethyl-8-ribityllumazine in buffer. The 13C and 15N chemical shifts indicate that the protein-bound 6,7-dimethyl-8-ribityllumazine is embedded in a polar environment and that the ring system is strongly polarized. It is concluded that the two carbonyl groups play an important role in the polarization of the molecule. The N(3)-H group is not accessible to bulk solvent. The N(8) atom is sp2 hybridized and has delta+ character. Nuclear Overhauser effect studies indicate that the 6,7-dimethyl-8-ribityllumazine ring is rigidly bound with no internal mobility. The NMR results indicate that the interaction between the ring system and the two apoproteins is almost the same.     

An 15N NMR study of adenine-uracil base pair in a non-aqueous solvent

[1,3,7,9,10-15N]-2',3',5'-Tri-O-acetyl adenosine (A) and its 8-D and 8-Br derivatives (AD and ABr) were prepared from 95% 15N enriched adenosine obtained from microbial fermentation. The chemical shifts and nuclear Overhauser effects of 15N resonances were measured as a function of the concentration of the mixed 1-cyclohexyluracil. The limiting shift of each 15N resonance was calculated and the structure of the A-U dimer was estimated. From the shifts of 15N-1 and 15N-7 signals it is determined that ABr-U dimers prefer the Watson-Crick type hydrogen bond while the Hoogsteen type pairs are predominant in the A-U dimers.     

Partial site-specific assignment of a uniformly (13)C, (15)N enriched membrane protein, light-harvesting complex 1 (LH1), by solid state NMR

Partial site-specific assignments are reported for the solid state NMR spectra of light-harvesting complex 1, a 160 kDa integral membrane protein. The assignments were derived from 600 MHz (15)N-(13)CO-(13)Calpha and (15)N-(13)Calpha-(13)CX correlation spectra, using uniformly (13)C, (15)N enriched hydrated material, in an intact and precipitated form. Sequential assignments were verified using characteristic (15)N-(13)Calpha-(13)Cbeta side chain chemical shifts observed in 3D experiments. Tertiary contacts found in 2D DARR spectra of the selectively (13)C enriched sample provided further confirmatory evidence for the assignments. The assignments include the region of the Histidine ligands binding the Bacteriochlorophyll chromophore. The chemical shifts of Calpha and Cbeta resonances indicated the presence of typical alpha-helical secondary structure, consistent with previous studies.     

NMR mapping of the recombinant mouse major urinary protein I binding site occupied by the pheromone 2-sec-butyl-4,5-dihydrothiazole.

The interactions between the mouse major urinary protein isoform MUP-I and the pheromone 2-sec-butyl-4,5-dihydrothiazole have been characterized in solution. (15)N-labeled and (15)N, (13)C-doubly-labeled recombinant MUP-I were produced in a bacterial expression system and purified to homogeneity. Racemic 2-sec-butyl-4, 5-dihydrothiazole was produced synthetically. An equilibrium diffusion assay and NMR titration revealed that both enantiomers of the pheromone bind to the recombinant protein with a stoichiometry of 1 equiv of protein to 1 equiv of racemic pheromone. A micromolar dissociation constant and slow-exchange regime dissociation kinetics were determined for the pheromone-protein complex. (1)H, (15)N, and (13)C chemical shifts of MUP-I were assigned using triple resonance and (15)N-correlated 3D NMR experiments. Changes in protein (1)H(N) and (15)N(H) chemical shifts upon addition of pheromone were used to identify the ligand binding site. Several amide signals, corresponding to residues on one side of the binding site, were split into two peaks in the saturated protein-ligand complex. Similarly, two overlapping ligand spin systems were present in isotope-filtered NMR spectra of labeled protein bound to unlabeled pheromone. The two sets of peaks were attributed to the two possible chiralities of the pheromone. Intermolecular NOEs indicated that the orientation of the pheromone in the MUP-I binding cavity is opposite to that modeled in a previous X-ray structure.     

1H, 13C and 15N backbone assignments of cyclophilin when bound to cyclosporin A (CsA) and preliminary structural characterization of the CsA binding site.

The backbone 1H, 13C and 15N chemical shifts of cyclophilin (CyP) when bound to cyclosporin A (CsA) have been assigned from heteronuclear two- and three-dimensional NMR experiments involving selectively 15N- and uniformly 15N- and 15N,13C-labeled cyclophilin. From an analysis of the 1H and 15N chemical shifts of CyP that change upon binding to CsA and from CyP/CsA NOEs, we have determined the regions of cyclophilin involved in binding to CsA.     

The binding of ATP and AMP to Escherichia coli adenylate kinase investigated by 1H and 15N NMR spectroscopy

[N6 15N]ATP and [N6 15N]AMP, complexed with E.coli adenylate kinase (AKe), were observed with 15N isotope-filtered NMR pulse sequences and 1H[15N] heterocorrelated experiments to determine differences between binding sites based on chemical shifts and competition by substrate analogs. The chemical shifts of the N6 amino proton and nitrogen signals changed significantly after mixing with adenylate kinase. Differences in chemical shifts between the bound ATP and AMP signals are slight. The response of these shifts to further addition of other substrates or Mg2+ supports the view that the unchelated nucleotides can bind to both the sites, whereas the metal complexed species are restricted to the MgATP/MgADP binding site.     

13C and 15N chemical shift assignments and secondary structure of the B3 immunoglobulin-binding domain of streptococcal protein G by magic-angle spinning solid-state NMR spectroscopy

Complete ¹³C and ¹⁵N assignments of the B3 IgG-binding domain of protein G (GB3) in the microcrystalline solid phase, obtained using 2D and 3D MAS NMR, are presented. The chemical shifts are used to predict the protein backbone conformation and compared with solution-state shifts. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.