S.S. Wijmenga

The solution structure of the circular trinucleotide cr(GpGpGp) determined by NMR and molecular mechanics calculation

The 3-5 circular trinucleotide cr(GpGpGp) was studied by means of 1D and 2D high resolution NMR techniques and molecular mechanics calculations. Analysis of the J-couplings, obtained from the 1H and 13C-NMR spectra, allowed the determination of the conformation of the sugar rings and of the circular phosphate backbone. In the course of the investigations it was found that the Karplus-equation most recently parametrized for the CCOP J-coupling constants could not account for the measured J(C4P) of 11.1 Hz and a new parametrization for both HCOP and CCOP coupling constants is therefore presented. Subsequent analysis of the coupling constants yielded fixed values for the torsion angles beta and delta (with beta = 178 degrees and delta = 139 degrees). The value of the latter angle corresponds to an S-type sugar conformation. The torsion angles gamma and epsilon are involved in a rapid equilibrium in which they are converted between the gauche(+) and trans and between the trans and gauche(-) domain respectively. We show that the occurrence of epsilon in the gauche(-) domain necessitates S-type sugar conformations. Given the aforementioned values for beta, gamma, delta and epsilon the ring closure constraints for the ring, formed by the phosphate backbone can only be fulfilled if alpha and zeta adopt some special values. After energy minimization with the CHARMm force field only two combinations of alpha and zeta result in energetically favourable structures, i.e. the combination alpha (t)/zeta(g-) in case gamma is in a gauche(+) and epsilon is in a trans conformation, and the combination alpha (t)/zeta (g+) for the combination gamma (t)/epsilon (g-). The results are discussed in relation to earlier findings obtained for cd(ApAp) and cr(GpGp), the latter molecule being a regulator of the synthesis of cellulose in Acetobacter xylinum.

Through Bond Sugar-Phosphate Backbone Assignment in 13C labeled RNA by Triple Resonance 1H,13C, and 31P NMR Spectroscopy

Successful studies of the structure of biomolecules by means of NMR depend crucially on the ability to interpret their complicated spectra and to derive as many structural constraints from those spectra as possible. In protein NMR studies big strides forward have been made by the introduction of molecules uniformly enriched in 15N and/or in 13C isotopes (Clore and Gronenbom 1991). This allows, by means of multidimensional NMR spectroscopy, the spreading of spectral information along 13C and/or 15N frequency axes in addition to or in place of the traditional 1H axes. This increases the spectral resolution appreciably and since in these experiments use is made of coherence transfer methods spectral assignment does not depend on NOE effects and thus not on assumptions about structure (Ikura et al. 1990).

Multidimensional nuclear magnetic resonance methods to probe metal environments in proteins

Publisher Summary This chapter discusses methods to obtain information about metal sites in proteins by means of nuclear magnetic resonance (NMR) techniques. NMR spectroscopy has developed into a powerful method to study structures both of metal environments in proteins and of proteins in general. The application of NMR in chemistry and biochemistry is discussed in the chapter. An introduction to general NMR methodology used for protein structure determination is also presented. The chapter also discusses the structure of metal environments in proteins. The use of pH variations to identify metal ligands, techniques to probe the environment of a metal inside a protein for paramagnetic and diamagnetic metal ions, and extrinsic ligands are described. It discusses time-dependent phenomena—namely, conformational equilibria, NH exchange rates, and NMR relaxation times. It also focuses on probing the oxidation state of a metal site and changes thereof. Finally, modeling metal sites in proteins is also presented in the chapter.

A Conformational Study of the Calixspherand and Its Complexes with Alkali-Metal Cations

The calixpherand 2 forms kinetically very stable complexes with alkalimetal cations. This molecule is not completely preorganized for binding of a cation, as is evidenced from the results of NOESY spectroscopy and X-ray diffraction measurements. Both in CDCl3 solution and in the solid state the free ligand adopts a cone conformation, whereas the Na+ complex adopts a flattened partial cone conformation. Molecular-mechanics calculations with different programs give rather biased results. Calculations with QUANTA(the all atom CHARMM-force field) correctly predict the conformation of the free ligand but not of the complexes, whereas with MACROMODEL(the united atom AMBER-force field) the experimentally observed conformation had the lowest energy only for the Na+ complex. The calculated geometries of the experimentally found conformations of the free ligand and the Na+ complex agree well with the X-ray structures, especially for the structures that were obtained with QUANTA. A comparison of the calculated structures of the Na+, K+ and Rb+ complexes showed that larger cations force the terphenyl bridge to bend away, thereby opening up the cage of the ligand and making the cation more accessible to solvent molecules. This might explain the considerably lower kinetic and thermodynamic stability of the Rb+ complex compared with those of the Na+ and K+ complexes.