C.W. Hilbers

Recommendations for the presentation of NMR structures of proteins and nucleic acids – IUPAC-IUBMB-IUPAB Inter-Union Task Group on the Standardization of Data Bases of Protein and Nucleic Acid Structures Determined by NMR Spectroscopy

The recommendations presented here are designed to support easier communication of NMR data and NMR structures of proteins and nucleic acids through unified nomenclature and reporting standards. Much of this document pertains to the reporting of data in journal articles; however, in the interest of the future development of structural biology, it is desirable that the bulk of the reported information be stored in computer-accessible form and be freely accessible to the scientific community in standardized formats for data exchange. These recommendations stem from an IUPAC-IUBMB-IUPAB inter-union venture with the direct involvement of ICSU and CODATA. The Task Group has reviewed previous formal recommendations and has extended them in the light of more recent developments in the field of biomolecular NMR spectroscopy. Drafts of the recommendations presented here have been examined critically by more than 50 specialists in the field and have gone through two rounds of extensive modification to incorporate suggestions and criticisms.

Structure, kinetics and thermodynamics of DNA hairpin fragments in solution.

The hairpin-to-coil equilibrium of the hexadecadeoxynucleotide d(ATCCTATTTTTAGGAT) was extensively studied by means of NMR, T-jump and UV. The thermodynamic and kinetic parameters for this equilibrium were determined, yielding a consistent picture of the dynamical behavior of this hairpin structure, which is shown to be a clear example of a situation in which the linebroadening of the imino proton resonances is not determined by the lifetime of the double helix. A comparative study of the homologous hairpins in which the size of the loop was elongated from 4 to 7 thymidine residues shows a monotonous decrease in Tm for the hairpin-to-coil transitions. This finding is in contrast with the view that the stability of hairpins reaches a maximum with a loop size of 6-7 residues. The NMR results indicate that the accessibility of the thymine bases in the loop towards solvent molecules or complementary nucleotides greatly depends on the size of the loop.

Fluorescence studies of the complex formation between the gene 5 protein of bacteriophage M13 and polynucleotides

The binding characteristics of the interaction of gene 5 protein with polynucleotides, i.e. poly(dA), poly(dT) and M13 DNA, have been determined by following the quenching of the protein fluorescence. In general, the binding is highly co-operative and for the binding of the protein to poly(dA) and M13 DNA the co-operativity parameter ω is estimated to have values between 50 and 300. Under comparable experimental conditions, the intrinsic binding constant Kint is at least two orders of magnitude higher for poly(dT) than for poly(dA), while the value for M13 DNA is intermediate. For poly(dA), the binding has been studied as a function of ionic strength and temperature. From these experiments it can be concluded that ionic interactions as well as van der Waals interactions (e.g. stacking interactions) are important for the complex formation of the protein with polynucleotides. From a comparison of the binding of the protein to poly(dA) and poly(dT), it is concluded that stacking interactions in the polynucleotide have a negative influence on protein binding. This conclusion, in conjunction with the weak temperature dependence of Kint. indicates that ionic interactions play a major role in the stabilization of the protein-poly(dA) complex. The co-operativity factor ω is little or not dependent on the ionic strength or the type of polynucleotide involved in binding. It is determined by interactions between complexed protein molecules. These interactions are primarily non-electrostatic. The binding characteristics obtained for the gene 5 protein-polynucleotide complexes are compared with those we have found for the binding to small oligonucleotides. It appears that oligonucleotide and polynucleotide binding differ in many aspects; i.e. there is a difference in Kint, ω and the number of nucleotides covered. The validity of linear lattice binding theories is discussed in this context. By comparing the binding parameters found for the gene 5 protein with those of the Escherichia coli DNA binding protein I. it is possible to explain the displacement of the E. coli protein by the gene 5 protein that occurs in vivo.

NMR studies of lantibiotics: assignment of the 1H-NMR spectrum of nisin and identification of interresidual contacts

Nisin is a 34 residue long antimicrobial polypeptide, which contains unusual α,β‐unsaturated amino acids as well as lanthionines. By making use of 2D‐NMR methods, the complete 1H‐NMR spectrum of the polypeptide could be assigned. The NMR data indicate that the molecule adopts a well‐defined three‐dimensional structure.

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.

Three-dimensional homonuclear TOCSY-NOESY of nucleic acids. Possibilities for improved assignments

Abstract The application of 3D TOCSY-NOESY spectroscopy to the analysis of nucleic acids is presented, using a TOCSY-NOESY spectrum of the DNA hairpin 5′GTTCCA-AAC-TGGAAC3′. An easy and concise method for describing the magnetization transfer in 3D experiments as applied to the TOCSY-NOESY experiment of nucleic acids is presented. It is shown that a 3D TOCSY-NOESY experiment can be considered as a collection of spin-lock patterns. In particular the repeating spin-lock pattern present in the f 1 f 3 planes is an extremely useful feature of a TOCSY-NOESY experiment since it can be used to distinguish between protons whose resonances overlap. The application of TOCSY-NOESY makes it possible also to assign the very crowded H4′, H5′, H5″ resonances.

Structure of the DNA binding wing of the gene-V encoded single-stranded DNA binding protein of the filamentous bacteriophage M13

The structure in solution of a β‐loop in mutant Y41H of the single‐stranded DNA binding protein encoded by gene‐V of the filamentous phage M13 has been elucidated using 2‐dimensional 1H‐nuclear magnetic resonance techniques. Furthermore, these studies enabled us to demonstrate that an identical structural element is present in wild‐type gene‐V‐protein and that this element intimately is involved in the binding of gene‐V‐protein to single‐stranded DNA. It is shown that the structure of the DNA binding wing deviates from that proposed for the same amino acid sequence on the basis of X‐ray diffraction data. The structure is, however, identical to that of the DNA binding wing present in the single‐stranded DNA binding protein encoded by the genome of the evolutionary distantly related filamentous phage IKe. The latter observations support our current view that in the binding of these proteins to single‐stranded DNA a common structural motif is involved.

The solution structure of yeast tRNAPhe as studied by nuclear Overhauser effects in NMR.

Recently, the imino proton spectrum of yeast tRNAPhe has been assigned by means of the application of the nuclear Overhauser effect (NOE). In the present paper it will be shown that even for tRNA (MW 28000) connectivities between the imino proton spins can be observed using two-dimensional NOE spectroscopy. In this way the imino proton resonances of the D-stem region are assigned. The results are discussed in relation to those obtained by the classical one-dimensional nuclear Overhauser effect. It turns out that in 2D-NOE experiments connectivities from overlapping resonances can be observed which cannot be determined by one-dimensional Overhauser experiments. Moreover, the total assignment of the imino proton spectrum of yeast tRNAPhe is used to relate the three-dimensional crystal structure of the tRNA to its solution structure. It is shown that the principle elements of the X-ray structure, i.e. the hydrogen bonding network and the stacking of the stems upon one another, are also found in solution. This is true for the presence as well as for the absence of magnesium ions. However, in absence of magnesium ions the tRNA structure appears to differ in details from that in the presence of magnesium ions. Finally, the influence of the elongation factor Tu from B.stearothermophilus on the tRNA structure is discussed.

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.