PI: Wijmenga (UmU)
Project: Development of methods for improved structure determination of larger systems by means of NMR
The project aims at improving the structure determination of larger systems by means of NMR by using 1) chemical shifts as additional constraints in structure refinement and 2) reverse labeling (deuteration) approaches.
1. Chemical Shifts.
The use of chemical shifts as a tool in NMR structure refinement of proteins and nucleic acids is a recent development1-4. We have recently demonstrated that for DNAs H chemical shifts can be calculated with good accuracy and can be valuable tools for NMR structural studies4. Only in the past three to five years have a number (around 25) of well-determined RNA solution structures come available. We have analysed them and compared their calculated and experimental 1H chemical shifts5,6. We conclude: 1) 1H shifts can be calculated with good accuracy (0.2 ppm rmsd). 2) The experimental shifts contain a wealth of valuable structural information. 3) The patterns in the shift distribution can be used to simplify resonance assignment. 4) Calculated shifts are very good indicators of errors in derived structures. We have now incorporated chemical restraints for nucleic acids into X-PLOR and tested their usefulness in structure calculations. A refinement protocol has been established, which uses chemical shifts and torsion angle dynamics7. The results show that chemical shifts are most useful in refinement and can in fact partially replace traditional restraints derived from NOEs and J-couplings5,7.
A labeling scheme suitable for NMR structure studies of larger RNAs ( see below) has been devised and will be tested on PBS (31 nucleotides), which is the primer binding site in viral RNA for reverse transcriptase of Hepatitus B Virus. NMR quantities of PBS have been made via large-scale enzymatic synthesis by means of T7 Polymerase, which uses NTPs as building blocks. The NMR spectra show via the assigned imino resonances, that the correct secondary fold is obtained. For the 13C/15N/2H labeling of PBS the glucose pathway8 will be used. Here, glucose is enzymatically converted into ribose and subsequently enzymatically connected to its base to obtain NTPs. In this way a variety of different labeling patterns can be set up (e.g. NTPs with deuterated ribose only protonated at the 2' position, with either 13C/15N labeled or unlabelled base). The deuteration and partial labeling lead to narrower line widths and a simplified proton network. This new strategy for RNA structure determination by NMR is suitable for larger systems (size limit > 100 nucleotides2, present limit 50 nucleotides9). The enzymes required for NTP synthesis have been isolated from plasmids obtained from Williamson c.s. and their activity has been tested. The next step will be the synthesis of the RNAs from the differently labeled NTPs.
1Szilagyi, Prog.Magn.Res.Spec, 27, 325-443, 1996; 2Wijmenga & van Buuren, Progr.Magn.Res.Spec., 32, 287-387, 1998; 3Kuszewski et al., J.Magn.Reson. B, 107, 293-297, 1995; 4Wijmenga et al., J.Biomol.NMR, 10, 337-350, 1997; 5J.Cromsigt et al., in Magnetic Resonance and Related Phenomena, Berlin, 132, 1998; 6J.Cromsigt, & S.Wijmenga, in preparation; 7J.Zdunek & S.Wijmenga, in preparation; 8Tolbert & Williamson, J.Am.Chem.Soc., 119, 12100-12108, 1997; 9Kolk et al., Science, 280, 434-438 (1998), J.Biomol.NMR, 12, 375-387 (1998).
J.A.M.T.C. Cromsigt, B.N.M. van Buuren, J. Zdunek, J. Schleucher, C.W. Hilbers, and S. S. Wijmenga "NMR Studies of RNA and DNA. Improved Structure Determination via Incorporation of Chemical Shift Restraints and Global Structure Information.", in: D.Ziessow, W.Lubitz, F.Lendzian (Eds.): Magnetic resonance and Related Phenomena; Proc. 29th Ampere-13th ISMAR; Berlin, August 2-7, 1998, pp. 132-133 (1998).