F.J.M. van de Ven

Chemically relayed nuclear overhauser effects. Connectivities between resonances of nonexchangeable protons and water

Abstract In NOESY spectra recorded in H 2 O, cross peaks between nonexchangeable protons and water can be observed. From theoretical and experimental studies of this phenomenon we conclude that it is brought about by a “relay” mechanism which involves dipolar cross relaxation between a nonexchangeable proton and an exchangeable one followed by chemical exchange of the latter with water. This process dominates strongly over the “direct” mechanism which is caused by translational diffusion of water molecules to and from the solute. It was found that the “chemically relayed nuclear Overhauser effect” does not stand in the way of proper determination of exchange rates of exchangeable protons or of interproton distance, if one uses measured relaxation rates for the diagonal terms of the relaxation matrix.

1H, 13C and 15N NMR backbone assignments and secondary structure of the 269-residue protease subtilisin 309 from Bacillus lentus

Summary1H, 13C and 15N NMR assignments of the backbone atoms of subtilisin 309, secreted by Bacillus lentus, have been made using heteronuclear 3D NMR techniques. With 269 amino acids, this protein is one of the largest proteins to be sequentially assigned by NMR methods to date. Because of the size of the protein, some useful 3D correlation experiments were too insensitive to be used in the procedure. The HNCO, HN(CO)CA, HNCA and HCACO experiments are robust enough to provide most of the expected correlations for a protein of this size. It was necessary to use several experiments to unambiguously determine a majority of the α-protons. Combined use of HCACO, HN(COCA)HA, HN(CA)HA, 15N TOCSY-HMQC and 15N NOESY-HMQC experiments provided the Hα chemical shifts. Correlations for glycine protons were absent from most of the spectra. A combination of automated and interactive steps was used in the process, similar to that outlined by Ikura et al. [(1990) J. Am. Chem. Soc., 112, 9020–9022] in the seminal paper on heteronuclear backbone assignment. A major impediment to the linking process was the amount of overlap in the Cα and Hα frequencies. Ambiguities resulting from this redundancy were solved primarily by assignment of amino acid type, using Cα chemical shifts and ‘TOCSY ladders’. Ninety-four percent of the backbone resonances are reported for this subtilisin. The secondary structure was analyzed using 3D 15N NOESY-HMQC data and Cα secondary chemical shifts. Comparison with the X-ray structure [Betzel et al. (1992) J. Mol. Biol., 223, 427–445] shows no major differences.

Residue-specific assignments of resonances in the 1H nuclear magnetic resonance spectrum of ribosomal protein E-L30 by systematic application of two-dimensional fourier transform nuclear magnetic resonance methods☆

A two-dimensional Fourier transform nuclear magnetic resonance study of the ribosomal protein E-L30 is reported. Five two-dimensional techniques, namely: nuclear magnetic resonance J-resolved spectroscopy, correlated spectroscopy, double quantum spectroscopy, relayed coherence transfer and nuclear Overhauser enhancement spectroscopy were used. Qualitative inspection of the spectra obtained by these techniques provided evidence that the E-L30 molecule has a well-defined structure in solution. This analysis indicated that, despite the fact that the protein is stable only at moderate temperatures and neutral pH, a structural analysis of the molecule would be feasible. A detailed analysis of the spectra permitted unambiguous discrimination between the spin systems of different amino acids, resulting in residue-specific resonance assignments. We were able to assign all resonances of all six threonine, four valine, five alanine, two histidine, two serine, one phenylalanine, one asparagine and one aspartic acid residue of E-L30. Complete resonance assignment was obtained for two glycine residues. Partial assignments became available for all six isoleucine, three glycine and one glutamine residue. These results form a sound basis for the structure determination of the protein described in the accompanying paper.

Sequential resonance assignments as a basis for the determination of a three-dimensional structure of protein E-L30 of Escherichia coli.

Nuclear Overhauser enhancement spectra of ribosomal protein E-L30 were searched for interresidual connectivities involving peptide bond amide protons in order to establish sequential neighbourships between amino acid residues. By comparing these data with the actual amino acid sequence of the protein, sequential resonance assignments became available for almost 90% for the amino acids in E-L30. With the aid of these assignments, some 30 nuclear Overhauser connectivities could be interpreted in terms of short interproton distances involving remote sites in the polypeptide chain. It turned out that these contacts between residues generated enough constraints to permit construction of a three-dimensional structure for the protein.

Double-quantum NOESY. Coupled coherent and incoherent transfer of magnetization

The application of 2D Ff NMR to the structural analysis of biological macromolecules has revolutionized the field. On the one hand networks of scalar coupled spins in complicated molecules such as proteins and nucleic acids can be analyzed by making use of coherent magnetization transfer in two-dimensional correlated spectroscopy (COSY) (l-4), on the other hand the proximity of specific nuclei in such molecules can be investigated by detecting incoherent magnetization transfer by means of nuclear Overhauser enhancement spectroscopy (NOESY) (5-7). During the last five years the number of techniques using coherent magnetization transfer has grown substantially: examples are relayed coherence spectroscopy and doublequantum spectroscopy (8-13). The latter techniques were shown to be a prerequisite in the unraveling of networks of scalar coupled spins which, due to ambiguities arising from extensive overlap, could not be resolved within COSY or SECSY (14-16). The process of incoherent transfer of magnetization, taking place via dipolar cross relaxation, is fundamentally different from coherent magnetization transfer which occurs via the J coupling between spins. In methods developed so far, the two phenomena have been employed strictly independently from one another; this is true even in the so-called COCONOSY experiment (I 7). However, there is no reason why a 2D FT NMR experiment should be based solely on either coherent or incoherent magnetization transfer (18). In this paper an experiment is described which couples coherent and incoherent transfer. It will be shown that such an experiment can be used as an aid in resolving ambiguities that may arise in NOESY spectra. To illustrate the basic principles of this new experiment, which we call doublequantum NOESY (DQ NOESY), we shall compare it with the conventional NOESY experiment (see Table 1). Both experiments can be divided in six periods and both experiments start by creating coherences which are frequency labeled during the evolution period I,. In a conventional NOESY experiment a 7r/2 pulse is used to create in-phase single-quantum coherence (i.e., transverse magnetization (5)). In DQ NOESY the preparation sequence of double-quantum spectroscopy (II, 19, 20), namely r/2-r-r-r--1r/2 is used, which gives rise to double-quantum coherences that are frequency labeled during the evolution period t, . After the evolution period,

Two‐dimensional Fourier transform 1H NMR studies of ribosomal protein E‐L30

Two‐dimensional Fourier transform 1H NMR spectra of ribosomal protein L30 of Escherichia coli MRE 600 were recorded at 500 MHz both in H2O and in 2H2O. From the available data we infer (i) the existence of one or more hydrophobic domains in the molecule, (ii) at least two helical regions and (iii) the presence of an antiparallel β‐sheet involving two threonines.

Studies on (K+ + H+)-ATPase: III. Binding of adenylyl imidodiphosphate

1. Adenylyl imidodiphosphate (AMPPNP) binds to (K+ + H+)-ATPase from pig gastric mucosa with a dissociation constant (Kd) of 50 microM for the AMPPNP-enzyme complex. 2. Monovalent cations reduce the amount of AMPPNP bound in the following order of effectiveness Tl+ greater than K+ greater than Rb+ greater than Cs+ greater than Na+, Li+, choline+. 3. AMPPNP binding to the enzyme has a pH optimum at pH 7.0--7.5 in the absence of added ions, which is shifted to pH 8 upon addition of MgCl2. 4. Cyclodiaminotetraacetic acid (CDTA, Tris salt) inhibits binding of AMPPNP. This inhibition is not due to chelation of Mg2+. It may be due to direct binding of CDTA to the enzyme or to removal of stabilizing cations other than Mg2+. 5. Binding curves determined in the presence of various concentrations of Mg2+ show that at low Mg2+ concentrations (less than 0.5 mM), the apparent number of binding sites is reduced, while at higher Mg2+ concentrations (greater than or equal to 0.5 mM), the binding of AMPPNP is inhibited in a competitive way. 6. From these observations it is concluded that the enzyme has two binding sites for AMPPNP and only one for Mg-AMPPNP (or two with strong anti-cooperativity), and that Mg2+ inhibits binding of Mg-AMPPNP. This finding is interpreted in terms of a model involving a dimeric form of the enzyme.