ABSTRACT The H3'-C3'-C4'-H4' t^orsional angles of two microcrystalline 2'-deoxynucleosides, thymidine and 2'-- deoxycytidine-HCl, doubly ^sup 13C-labeled at the C3' and C4' positions of the sugar ring, have been measured by solid-state magic-angle-spinning nuclear magnetic resonance (NMR). A double-quantum heteronuclear local field experiment with frequency-switched Lee-Goldberg homonuclear decoupling was used. The H3'-C3'-C4'-H4' torsional angles were obtained by comparing the experimental curves with numerical simulations, including the two ^sup 13^C nuclei, the directly bonded ^sup 1^H nuclei, and five remote protons. The H3'-C3'-C4'-H4' angles were converted into sugar pucker angles and compared with crystallographic data. The delta torsional angles determined by solid-state NMR and x-ray crystallography agree within experimental error. Evidence is also obtained that the proton positions may be unreliable in the x-ray structures. This work confirms that double-quantum solid-state NMR is a feasible tool for studying sugar pucker conformations in macromolecular complexes that are unsuitable for solution NMR or crystallography.
INTRODUCTION
The conformations of individual monomers in the polynucleic acids DNA and RNA are decisively important for their biological function. In particular, protein-DNA recognition is thought to involve the detailed local conformation of the DNA molecule through the so-called indirect recognition mechanism (Travers, 1993). Spectroscopic methods that are capable of obtaining information on the individual nucleotide conformations are therefore most important, particularly if they are applicable to large macromolecular assemblies.
A particularly important conformational parameter in nucleotides, nucleosides, and nucleic acids is the angle delta, defined as the torsional angle C5'-C4'-C3'-03' of the ribofuranose unit (Fig. 1). Together with the pucker amplitude, which is highly conserved in nucleosides (see Saenger, 1984, page 55), the torsional angle delta defines the pucker of the ribofuranose ring, which affects the entire nucleotide fragment and potentially the conformation of adjacent units. For instance, the sugar pucker changes from C2'-endo to C3'-endo in the transition from the B-form to the A-form of DNA, representing a change in delta from around gauche (60 deg) to around trans (180 deg).
The principal methods for examining nucleotide conformations are x-ray crystallography, solution NMR, and solidstate NMR. High-resolution x-ray crystallography gives direct information on the molecular structure. Solution-state NMR, in contrast, gives indirect information on the torsional angles through chemical shifts (Santos et al., 1989; Gorenstein, 1992; Xu et al., 1998; Rossi and Harbison, 2001), scalar J-couplings (Davies, 1978; Ippel et al., 1996), and cross-correlated relaxation effects (Boisbouvier et al., 2000; Felli et al., 1999). However, as the DNA and RNA molecules grow larger, and for the interesting cases of polynucleotide-protein complexes, both x-ray crystallography and solution-state NMR frequently encounter difficulties, the former due to imperfect crystallization, and the latter because of spectral line broadening due to slow molecular rotation.
Solid-state NMR does not require long-range crystallinity or rapid molecular motion. Several solid-state NMR methods have found application in the structural investigations of nucleic acids (Alam and Drobny, 1991; Lee et al., 2000; van Dam and Levitt, 2000). In particular, the torsional angle delta may be estimated from isotropic ^sup 13^C chemical shift values (Santos et al., 1989; Rossi and Harbison, 2001). However, chemical shift information can be difficult to interpret in structured macromolecules due to nonlocal effects, so a complementary method for estimating delta would be useful.
Torsion angles may also be estimated by solid-state NMR, if experiments are used that are sensitive to the relative orientations of nuclear spin interaction tensors (Feng et al., 1996; Ishii et al., 1996; Schmidt-Rohr, 1996a,b; Tycko et al., 1996; Weliky and Tycko, 1996; Costa et al., 1997; Feng et al., 1997; Fujiwara et al., 1997; Gregory et al., 1997; Hong et al., 1997; Feng et al., 1998; Bower et al., 1999; Feng et al., 2000; Middleton et al., 2000; Ravindranathan et al., 2000; Takegoshi et al., 2000). A particular useful class of experiments is called double-quantum heteronuclear local field (2Q-HLF) spectroscopy. These experiments exploit the evolution of a correlated two-spin state, double-quantum coherence (2QC), under the heteronuclear local fields of neighboring spins. The evolution of the 2QC is sensitive to the correlation of the heteronuclear local fields, and therefore to the relative orientation of the heteronuclear dipolar coupling tensors. In particular, the HCCH-2Q-HLF experiment was designed to measure the torsional angle in an ^sup 1^H-^sup 13^C-^sup 13^C-^sup 1^H molecular fragment by allowing ^sup 13^C 2QC to evolve under the ^sup 13^C-^sup 1^H heteronuclear dipolar couplings (Feng et al., 1996). This experiment has found several applications in biologically relevant molecules. For example, the H-C10-C11-H molecular torsional angle in the isomerization region of the retinal chromophore was determined in the ground state of rhodopsin (Feng et al., 1997) and in the metarhodopsin-I photointer-- mediate (Feng et al., 2000). The HCCH-2Q-HLF experiment has also been applied to mono and disaccharides (Ravindranathan et al., 2000, 2001), the drug compound cimetidine (Middleton et al., 2000) and bacteriorhodopsin (Lansing et al., 2002).
The HCCH-2Q-HLF experiment is an appropriate spectroscopic tool for studying nucleic acid conformations because each of the C4' and C3' sites has one attached proton, and selective ^sup 13^C labeling of the C4' and C3' sites is technically feasible using known synthetic routes (Ouwerkerk et al., 2000, 2002). An attractive feature of the 2Q-HLF experiment is that NMR signals from the natural abundance ^sup 13^C background are effectively suppressed, making the experiment feasible even in large molecular assemblies. The information provided by this experiment is complementary to that provided by chemical shift data. In preparation for experiments on macromolecular nucleic acid complexes, we have first applied the method to two different microcrystalline ^sup 13^C^sub 2^-labeled nucleosides, where a direct comparison with x-ray structures may be made. This comparison is the subject of this paper.
CONCLUSIONS
These investigations have shown that the 2Q-HLF NMR method is a potentially useful method for the investigation of nucleic acid sugar pucker. However, it is necessary to include remote protons in the analysis, at least up to a distance range of 250 pm. Although our results for the sugar pucker angle delta are in good agreement with x-ray studies for both thymidine and 2'-deoxycytidine*HCl, there are significant discrepancies for the H3'-C3'-C4'-H4' torsional angle phi, which could be associated with the difficulties of locating the protons by x-ray diffraction.
The experiment should be feasible on macromolecular systems that are beyond the reach of x-ray diffraction or solution NMR. In its simplest form, the method is restricted to systems with well-defined local conformations, but we anticipate that the method may be extended, in suitable cases, to systems that possess a distribution of torsional angles. This might involve combining the 2Q evolution with the conformational information contained in broadened chemically-shifted lineshapes (Zhang et al., 1998).
The authors thank Dr. Peter J. M. Verdegem for preliminary NMR work and Jasper R. Plaisier for participation in the x-ray diffraction measurements. Dr. Marjan Steenweg is thanked for help with the synthesis of 2'-deoxycytidine*HCl.
This work was sponsored by the Goran Gustafsson foundation for Research in the Natural Sciences and Medicine. We are grateful to Prof. Dr. J. Lugtenburg and Prof. Dr. J. H. van Boom for their participation in this investigation.
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[Author Affiliation]
Lorens van Dam,* Niels Ouwerkerk,^ Andreas Brinkmann,* Jan Raap,^ and Malcolm H. Levitt*^^
*Physical Chemistry, Arrhenius Laboratory, Stockholm University, 106 91 Stockholm, Sweden, ^Leiden Institute of Chemistry, Leiden University, 2300 RA Leiden, The Netherlands, and ^^Chemistry Department, Southampton University, Southampton SO17 1BJ, United Kingdom
[Author Affiliation]
Submitted March 22, 2002, and accepted for publication July 10, 2002.
[Author Affiliation]
Dr. Brinkmann's present address is Physical Chemistry, Univ. of Nijmegen, 6525ED, The Netherlands.
[Author Affiliation]
Address reprint requests to Malcolm H. Levitt, Chemistry Department, Southampton University, University Rd., Southampton SO17 1BJ, U.K. Tel.: +44-23-80596753; Fax: +44-23-80593781; E-mail: mhl@soton.ac.uk.
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