title: Reduced dimensionality tailored HN(C)N experiments for facile backbone resonance assignment of proteins through unambiguous identification of sequential HSQC peaks.
abstract: Two novel reduced dimensionality (RD) tailored HN(C)N [S.C. Panchal, N.S. Bhavesh, R.V. Hosur, Improved 3D triple resonance experiments, HNN and HN(C)N, for HN and 15N sequential correlations in (13C, 15N) labeled proteins: application to unfolded proteins, J. Biomol. NMR 20 (2001) 135-147] experiments are proposed to facilitate the backbone resonance assignment of proteins both in terms of its accuracy and speed. These experiments - referred here as (4,3)D-hNCOcaNH and (4,3)D-hNcoCANH - exploit the linear combination of backbone (15)N and (13)C'/(13)C(α) chemical shifts simultaneously to achieve higher peak dispersion and randomness along their respective F1 dimensions. Simply, this has been achieved by modulating the backbone (15)N(i) chemical shifts with that of (13)C' (i-1)/(13)C(α) (i-1) spins following the established reduced dimensionality NMR approach [T. Szyperski, D.C. Yeh, D.K. Sukumaran, H.N. Moseley, G.T. Montelione, Reduced-dimensionality NMR spectroscopy for high-throughput protein resonance assignment, Proc. Natl. Acad. Sci. USA 99 (2002) 8009-8014]. Though the modification is simple it has resulted an ingenious improvement of HN(C)N both in terms of peak dispersion and easiness of establishing the sequential connectivities. The increased dispersion along F1 dimension solves two purposes here: (i) resolves the ambiguities arising because of degenerate (15)N chemical shifts and (ii) reduces the signal overlap in F2((15)N)-F3((1)H) planes (an important requisite in HN(C)N based assignment protocol for facile and unambiguous identification of sequentially connected HSQC peaks). The performance of both these experiments and the assignment protocol has been demonstrated using bovine apo Calbindin-d9k (75 aa) and urea denatured UNC60B (a 152 amino acid ADF/cofilin family protein of Caenorhabditis elegans), as representatives of folded and unfolded protein systems, respectively.
authors.affiliation: Centre of Biomedical Research (CBMR), Sanjay Gandhi Post-Graduate Institute of Medical Sciences Campus, Raibareli Road, Lucknow 226014, Uttar Pradesh, India. Electronic address: dineshcbmr@gmail.com.
authors.forename: Dinesh
authors.initials: D
authors.lastname: Kumar
mesh_terms: D000577:Amides / Q000737:chemistry; D002250:Carbon Radioisotopes; D004574:Electromagnetic Fields; D007202:Indicators and Reagents; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D050050:Protein Carbonylation; D011506:Proteins / Q000737:chemistry*; D015203:Reproducibility of Results
chemical_list: D000577:Amides; D002250:Carbon Radioisotopes; D007202:Indicators and Reagents; D011506:Proteins
keywords: (4,3)D-hNCOcaNH; (4,3)D-hNcoCANH; Backbone assignment; CARA; Computer Aided Resonance Assignment; HN(C)N; HSQC; Heteronuclear Single Quantum Correlation; NMR; Nuclear Magnetic Resonance; RD; Reduced dimensionality NMR; Sequential amide correlations; reduced dimensionality