Journal of Structural and Functional Genomics

, Volume 13, Issue 4, pp 201–212 | Cite as

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–amide 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):357–363, 2012) using AUTOBA (Borkar et al. in J Biomol NMR 50(3):285–297, 2011); backbone H-bonds are manually derived from constant time long-range 2D-HnCO spectrum (Cordier and Grzesiek in J Am Chem Soc 121:1601–1602, 1999); and amide–amide 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):371–388, 1997). All the experiments needed for the execution of PFBD can be recorded and analyzed in about 24–48 h depending upon the concentration of the protein and dispersion of amide cross-peaks in the 1H–15N 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 < 10–12 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).


AUTOBA Automated backbone assignment Backbone fold Check points HNCO NOESY NMR PFBD Structural proteomics 



Automatic backbone assignment


Biological magnetic resonance bank


Computer aided resonance assigment (a software for NMR data analysis)


Heteronuclear single quantum correlation


Nuclear magnetic resonance


Nuclear overhauser effect spectroscopy


Protein data bank


Protein fold determination from backbone data



This work is being financially supported by the Department of Science and Technology under SERC Fast Track Scheme (Registration Number: SR/FT/LS-114/2011) for carrying out the research work. We gratefully acknowledge the High field NMR facility, at the Centre for Biomedical Magnetic Resonance (CBMR)—Lucknow, India where all the experiments were carried out. The basic 3D pulse sequences: HN(C)N, hNnH, hncoCANH, and hnCOcaNH and the software-program AUTOBA described here are freely accessible at:

Supplementary material

10969_2012_9144_MOESM1_ESM.doc (3 mb)
Plots for residue-wise dihedral angle constraints derived from backbone (1HN, 15N, 13Cα, and 13C′) and mainchain (1HN, 15N, 13Cα, 1Hα, 13Cβ and 13C′) resonances of ubiquitin have been shown in Figure S1. The reliability of 13Cα and 13C′ chemical shifts for estimating backbone dihedral angle (ϕ and ψ) constraints has also been evaluated in Appendix I by calculating the cumulative (13Cα and 13C′) secondary chemical shifts. Figure S2 displays residue-wise cumulative (13Cα and 13C′) secondary shifts both for human ubiquitin and chicken Sh3 domain. An illustrative stretch of HNCO-NOESY spectrum of chicken SH3 domain (for residues Ala11-Lys18) has been shown in Figure S3. An overlay of the long range constant time and normal 2D-HnCO spectra of Human Ubiquitin has been shown in Figure S4. An overlay of the long range constant time and normal 2D-HnCO spectra of chicken Sh3 domain has been shown in Figure S5. The 20 CYANA generated backbone structures of ubiquitin using backbone amide NOEs, backbone dihedral angles, and backbone RDCs data have been shown in supplementary material (Fig. S6). The coordinates of 20 best backbone structures by NMR and the used NMR restraints have been deposited in the Protein Data Bank under the accession codes: 2LD9 (for human ubiquitin) and 2LJ3 (for chicken SH3 domain). The various experiments recorded on two proteins and the corresponding acquisition parameters along with acquisition times in each case are given in Table S1. The structural statistics of ubiquitin NMR structure 2LD9 has been shown in Table S2. (DOC 3056 kb)


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Copyright information

© Springer Science+Business Media Dordrecht 2012

Authors and Affiliations

  1. 1.Centre of Biomedical Magnetic Resonance (CBMR)LucknowIndia
  2. 2.Department of Chemical SciencesTata Institute of Fundamental ResearchColaba, MumbaiIndia

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