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

✍ Scribed by Daniel T. Graesser; Benjamin J. Wylie; Andrew J. Nieuwkoop; W. Trent Franks; Chad M. Rienstra

• Publisher: John Wiley and Sons
• Year: 2007
• Tongue: English
• Weight: 469 KB
• Volume: 45
• Category: Article
• ISSN: 0749-1581
• DOI: 10.1002/mrc.2126

✦ Synopsis

We present a novel rotational-echo double resonance (REDOR) method for detection of multiple 19 F-15 N distances in solid proteins. The method is applicable to protein samples containing a single 19 F label, in addition to high levels of 13 C and 15 N enrichment. REDOR dephasing pulses are applied on the 19 F channel during an indirect constant time chemical shift evolution period on 15 N, and polarization is then transferred to 13 C for detection, with high-power 1 H decoupling throughout the sequence. This four-channel experiment reports site-specifically on 19 F-15 N 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 5-19 F-Trp, in a bacterial growth medium also including 13 C-glucose and 15 N 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.