A unified NMR strategy for high-throughput determination of backbone fold of small proteins

An efficient semi-automated strategy called PFBD (i.e. Protein Fold from Backbone Data only) has been presented for rapid backbone fold determination of small proteins. It makes use of NMR parameters involving backbone atoms only. These include chemical shifts, amide?Camide NOEs and H-bonds. The backbone chemical shifts are obtained in an automated manner from the orthogonal 2D projections of variants of HNN and HN(C)N experiments (Kumar et al., in Magn Reson Chem 50(5):357?C363, 2012) using AUTOBA (Borkar et al. in J Biomol NMR 50(3):285?C297, 2011); backbone H-bonds are manually derived from constant time long-range 2D-HnCO spectrum (Cordier and Grzesiek in J Am Chem Soc 121:1601?C1602, 1999); and amide?Camide NOEs are derived from 3D HNCO NOESY experiment which provides NOEs along the direct ¹H dimension that has maximum resolution (Lohr and Ruterjans in J Biomol NMR 9(1):371?C388, 1997). All the experiments needed for the execution of PFBD can be recorded and analyzed in about 24?C48?h depending upon the concentration of the protein and dispersion of amide cross-peaks in the ¹H?C¹⁵N correlation spectrum. Thus, we believe that the strategy, because of its speed and simplicity will be very valuable in Biomolecular NMR community for high-throughput structural proteomics of small folded proteins of MW?<?10?C12?kDa, the regime where NMR is generally preferred over X-ray crystallography. The strategy has been validated and demonstrated here on two small globular proteins: human ubiquitin (76 aa) and chicken SH3 domain (62 aa).