Acceleration of protein backbone NMR assignment by combinatorial labeling: Application to a small molecule binding study. Issue 5 (May 2017)

• Record Type: Journal Article
• Authors: Hein, Christopher; Löhr, Frank; Schwarz, Daniel; Dötsch, Volker
• Is Part Of: Biopolymers. Volume 107:Issue 5(2017)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2017-05
• Subjects: BEST‐TROSY -- cell‐free expression -- cyclophilin D -- ligand screening -- selective isotope labeling; Biopolymers -- Periodicals; Peptides -- Periodicals; Spectrum analysis -- Periodicals; 572.33
• DOI: 10.1002/bip.23013
• Languages: English
• ISSNs: 0006-3525

Abstract

Abstract: Selective labeling with stable isotopes has long been recognized as a valuable tool in protein NMR to alleviate signal overlap and sensitivity limitations. In this study, combinatorial 15 N‐, 13 C α ‐, and 13 C'‐selective labeling has been used during the backbone assignment of human cyclophilin D to explore binding of an inhibitor molecule. Using a cell‐free expression system, a scheme that involves 15 N, 1‐ 13 C, 2‐ 13 C, fully 15 N/ 13 C, and unlabeled amino acids was optimized to gain a maximum of assignment information from three samples. This scheme was combined with time‐shared triple‐resonance NMR experiments, which allows a fast and efficient backbone assignment by giving the unambiguous assignment of unique amino acid pairs in the protein, the identity of ambiguous pairs and information about all 19 non‐proline amino acid types. It is therefore well suited for binding studies where de novo assignments of amide 1 H and 15 N resonances need to be obtained, even in cases where sensitivity is the limiting factor.