Long-range 19F15N distance measurements in highly-13C, 15N-enriched solid proteins with 19F-dephased REDOR shift (FRESH) spectroscopy

Abstract

We present a novel rotational-echo double resonance (REDOR) method for detection of multiple ¹⁹F¹⁵N distances in solid proteins. The method is applicable to protein samples containing a single ¹⁹F label, in addition to high levels of ¹³C and ¹⁵N enrichment. REDOR dephasing pulses are applied on the ¹⁹F channel during an indirect constant time chemical shift evolution period on ¹⁵N, and polarization is then transferred to ¹³C for detection, with high-power ¹H decoupling throughout the sequence. This four-channel experiment reports site-specifically on ¹⁹F¹⁵N distances, with highly accurate determinations of ∼5 Å distances and detection of correlations arising from internuclear distances of at least 8 Å. We demonstrate the method on the well-characterized 56-residue model protein GB1, where the sole tryptophan residue (Trp-43) has been labeled with 5-¹⁹F-Trp, in a bacterial growth medium also including ¹³C-glucose and ¹⁵N ammonium chloride. In GB1, 11 distances are determined, all agreeing within 20% of the X-ray structure distances. We envision the experiment will be utilized to measure quantitative long-range distances for protein structure determination. Copyright © 2007 John Wiley & Sons, Ltd.