NEW NMR METHODS FOR CONFORMATIONAL STUDIES OF BIOMOLECULES

R. Fiala and L. Žídek

National Centre for Biomolecular Research, Masaryk University, Kotlářska 2, CZ-611 37 Brno, Czech Republic

In the last three decades, high resolution NMR has taken its place beside X-ray crystallography as a means to obtain high-resolution structures of biomolecules. Traditionally, the structure determination by NMR relies on the measurement of interatomic distances that are accessible by the nuclear Overhauser effect (NOE) and the determination of torsional angles that can be derived from scalar coupling constants. The analysis of NMR spectra consist of two main steps – identification of the signals in the spectra corresponding to individual atoms in the molecule (assignment), and extracting the constraints that can be used for the calculation of the structure.

Originally, the NMR studies of biomolecules were based almost exclusively on the resonances of ¹H [1], since the nucleus provides the best sensitivity due to the high gyromagnetic ratio and a high (99.98%) natural abundance. However, the relatively low dispersion of the proton NMR spectra led to severe signal overlap even with molecules of moderate complexity. This problem was overcome by the introduction of multidimensional spectroscopic techniques and by the use of samples isotopically enriched by ¹³C and ¹⁵N. These advances made the assignment procedure more reliable by following the through-bond interactions rather than conformationally dependent interatomic distances. The structural constraints, however, are still based mainly on the ¹H -¹H NOE and J-couplings. While their values can characterize the local structure with very good accuracy, they provide virtually no information on the relative orientations of distant regions of a molecule (NOEs are typically observable only for the distances shorter than 6 Å). The current research in NMR methods therefore concentrates on obtaining additional structural constraints, preferably those providing an absolute configuration with respect to a fixed common frame of reference.

Recently, high resolution NMR started using interactions that were previously the domain of solid-state NMR and in liquids manifested themselves only in the relaxation properties, namely residual dipolar couplings (RDC) [2] and chemical shift anisotropy (CSA) [3]. In order to obtain structurally dependent RDCs and CSA in the high-resolution liquid NMR spectra, it is usually necessary to introduce certain degree of orientation into the solution of the biomolecule. While the high magnetic field itself induces certain amount of orientation of the molecules due to their inherent anisotropies in magnetic susceptibility, an alignment medium has to be added to the sample in most cases to make the measurement of RDCs a chemical shift changes due to CSA practically feasible. A great variety of alignment media has been proposed so far with lipid bicelles, filamentous bacteriophage particles and polyacrylamide gels being the most commonly used. In the partially oriented samples, the effects of dipolar couplings and CSA are typically scaled down by about three orders of magnitude, allowing thus their measurement with sufficient accuracy while retaining the simplicity and relaxation properties of liquid-phase spectra.

In practice, the RDCs are measured as changes of scalar couplings. Their values are a function of orientation of the internuclear vector with respect to the magnetic field, which represents here the reference direction common to all parts of the molecule. The constraints obtained from RDCs can be complemented by the changes of chemical shift that arise from CSA. The changes in the chemical shifts between and isotropic and oriented phases provide information on the orientation of chemical shielding tensors relative to the molecule's alignment frame. Angular information can also be obtained from the cross-correlated relaxation interference [4]. In this case, no partial alignment of the sample is necessary. Angles between two interatomic vectors can be determined from the measured effects of the cross-correlated relaxation directly, without the need of an empirical calibration curve. The interference between the dipolar and CSA relaxation mechanisms is also used in TROSY (transverse relaxation optimized spectroscopy) techniques [5] that, while not providing any additional structural constraints by themselves, greatly expanded the range of molecules amenable to studies by the methods of NMR spectroscopy towards higher molecular weights.

[1] K. Wüthrich: NMR of Proteins and Nucleic Acids, New York 1986, Wiley.

[2] J. H. Prestegard, C. M. Bougault, and A. I. Kishore, Chem. Rev. 104 (2004) 3519-3540.

[3] G. Cornilescu and A. Bax, J. Am. Chem. Soc. 122 (2000) 10143-10154.

[4] B. Reif, M. Hennig, and C. Griesinger, Science 276 (1997) 1230-1233.

[5] K. Pervushin, R. Riek, G. Wider, and K. Wüthrich, Proc. Natl. Acad. Sci. USA 94 (1997) 12366-12371.