Nuclear magnetic resonance, heteronuclear protein

Clore GM, Gronenbom AM (1994) Multidimensional heteronuclear nuclear-magnetic-resonance of proteins. Nucl Magn Reson Pt C 239 349-363... 

Fig. 8. Heteronuclear single-quantum coherenc (HSQC) spectrum of the hypothetical protein of the flowering locus T protein produced in the cell-free system. The FT protein was synthesized in the same way as in Fig. 6 except that Ala, Leu, Gly, and Gin in both translation and substrate mixture were replaced with their -labeled forms (Isotec, Inc ). After incubation for 48 h, the reaction mixture (1 mL) was dialyzed against 10 mMphosphate buffer (pH 6.5) overnight, and then centrifuged at 30,000g for 10 min. The supernatant containing 30 xMof the protein was directly subjected to nuclear magnetic resonance spectroscopy. The spectrum was recorded on a Broker DMX-500 spectrometer at 25°C, and 2048 scans were averaged for the final H- WHSQC spectrum.

This chapter describes protocols for preparing 15N-labeled proteins (ubiquitin is used as an example) using Escherichia coli cells (with purification) and the wheat germ cell-free system (without purification). A comparison of I I-15N heteronuclear single-quantum coherence (HSQC) spectra of yeast ubiquitin prepared using each method indicates that this wheat germ cell-free system may be used for rapid nuclear magnetic resonance analyses of proteins without purification. 

Sattler M, Schleucher J, Griesinger C. Heteronuclear multidimensional NMR experiments for the structure determination of proteins in solution employing pulsed field gradients. Progress Nuclear Magnet. Reson. Spectros. 1999 34 93-158. 

Battiste JL, Wagner G (2000) Utilization of site-directed spin labeling and high-resolution heteronuclear nuclear magnetic resonance for global fold determination of large proteins with limited nuclear Overhauser effect data. Biochemistry 39(18) 5355-5365... 

Abstract Nuclear Magnetic Resonance (NMR) relaxation is a powerful technique that provides information about internal dynamics associated with configurational energetics in proteins, as well as site-specific information involved in conformational equilibria. In particular, N relaxation is a useful probe to characterize overall and internal backbone dynamics of proteins because the relaxation mainly reflects reorientational motion of the N-H bond vector. Over the past 20 years, experiments and protocols for analysis of N Ri, R2, and the heteronuclear N- NOE data have been well established. The development of these methods... 

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