Amide (HN) projection planes of 3D CBCANH experiment and its variants  for spectroscopic labeling of Glycines, Alanines, and Serines/Threonines in the <sup>1</sup>H–<sup>15</sup>N HSQC spectrum of uniformly (<sup>13</sup>C–<sup>15</sup>N) labeled proteins
Creators: name: Kumar, Dinesh
name: Jaiswal, Nancy
name: Guleria, Anupam
Publisher: name: F1000 Research Limited
Year: 2017

description: The complementary pairs of triple resonance amide resolved 3D NMR experiments -HNCA/HN(CO)CA, HN(CA)CO/HNCO, CBCA(CO)NH/CBCANH and HNCACB/HNCOCACB- are routinely used for backbone resonance assignment of small-medium sized proteins (molecular weight &lt;20 kDa).<sup>1-3</sup> The last two complementary pairs -which correlate amide protons via C<sup>alpha</sup> and C<sup>beta</sup> resonances- have the additional advantage over HNCA/HN(CO)CA and HN(CA)CO/HNCO, that they have cross signals to the C<sup>beta</sup> atoms of the amino acids providing additional information i.e. identification of residues corresponding to Glycines (G), Alanine (A) and Serine/Threonines (S/T) or residues following them in the sequence owing to specific patterns of Ca and Cb chemical shifts i.e. Gly(Ca@45ppm), Ala(Ca@53 &amp; Cb@19ppm), Ser(Ca@58 &amp; Cb@63ppm) and Thr(Ca@62 &amp; Cb@69ppm). The distinctive C<sup>a</sup>/C<sup>b</sup> chemical shifts of these amino acids have been exploited in the design of various NMR experiments providing spectroscopic labelling of G, A and ST or their successors in 2D <sup>1</sup>H-<sup>15</sup>N spectrum of <sup>13</sup>C/<sup>15</sup>N labelled proteins like variants of (HC)NH,<sup>4</sup> (HN)NH,<sup>5</sup> MUSIC experiments<sup>6</sup> and amide projection planes of HN(CN) suite of experiments.<sup>7-10</sup> Recently, an alternative approach to rapidly identify amino acid types in proteins using phase modulated 2D HNCACB or HN(CO)CACB experiments has also been demonstrated.<sup>11</sup> This method is based on the fact that <sup>13</sup>C<sup>β</sup> chemical shifts of different amino acid types fall in distinct spectral regions and by tuning the evolution time of <sup>13</sup>C chemical shifts in these experiments, the phase of the cross peaks of different amino acid residues can be modulated depending on their <sup>13</sup>C<sup>β</sup> shift values. The conceptual approach can also be applied for spectroscopic tailoring of CBCANH experiment for labelling various amino acids in the <sup>1</sup>H–<sup>15</sup>N HSQC spectrum of uniformly (<sup>13</sup>C–<sup>15</sup>N) labelled proteins. Here, even a more simple approach has been presented for spectroscopic labelling of G, A and ST amino acids in the <sup>1</sup>H–<sup>15</sup>N HSQC spectrum using amide (HN) projection planes of CBCANH experiments. The approach involves tailoring of C<sup>a</sup>-C<sup>b</sup> coupling evolution through varying the bandwidth of radiofrequency shaped pulse applied on <sup>13</sup>C<sup>ali</sup> carbons during INEPT based magnetization transfer from them to backbone amide 15N nuclei without changing the evolution time. The modified versions of CBCANH experiment providing spectroscopic labelling of G, A and ST amino acids have been referred here as cbcaNH-G, cbcaNH-A and cbcaNH-ST, respectively. The performance of these experiments has been demonstrated on a urea denatured NS2 protein of influenza-A virus H1N1 (Strain: Influenza-A/ India/ NIV 30784/ 2010 (H1N1)) containing 13 extra residues belonging to purification tag (for details see the poster presentation). NS2 (also known as Nuclear export protein) is a 119 residue long non-structural protein expressed <i>in vivo</i> by H1N1 virus which is known to mediates the nuclear export of encapsidated genomic RNAs (ribonucleoproteins, RNPs) through acting as an adapter between viral RNPs complexes and the nuclear export machinery of the cell (Ref: http://www.uniprot.org/uniprot/G0KFM2). In recent years, non-structural viral proteins including H1N1-NS2 have been recognized as potential therapeutic targets for developing next generation anti-viral drugs.<sup>12</sup> This derived our interest to study the mechanistic structural of H1N1-NS2 protein of therapeutic relevance and to further use the structural information in the rational design of inhibitors. The recombinant protein has purified and the presented NMR methodology has been used to verify the amino acid sequence through identifying the number of glycines, alanines, serines and theronines.  Further structural and functional studies on this protein are underway.<br> <br>References:<br>(1)     Cavanagh, J.; Fairbrother, W. J.; Palmer III, A. G.; Skelton, N. J. Protein NMR spectroscopy: principles and practice. 1995,<br>(2)     Grzesiek, S.; Bax, A. An efficient experiment for sequential backbone assignment of medium-sized isotopically enriched proteins. <i>Journal of Magnetic Resonance</i> 1992, 99, 201-207.<br>(3)     Kumar, D.; Hosur, R. V. hNCOcanH pulse sequence and a robust protocol for rapid and unambiguous assignment of backbone (1HN, 15N and 13C) resonances in 15 N/13C labeled proteins. <i>Magnetic Resonance in Chemistry</i> 2011, 49, 575-583.<br>(4)     Chugh, J.; Hosur, R. V. Spectroscopic labeling of A, S/T in the 1H-15N HSQC spectrum of uniformly (15N-13C) labeled proteins. <i>Journal of Magnetic Resonance</i> 2008, 194, 289-294.<br>(5)     Kumar, D.; Paul, S.; Hosur, R. V. BEST-HNN and 2D-(HN) NH experiments for rapid backbone assignment in proteins. <i>Journal of Magnetic Resonance</i> 2010, 204, 111-117.<br>(6)     Schubert, M.; Oschkinat, H.; Schmieder, P. MUSIC, selective pulses, and tuned delays: amino acid type-selective 1H-15N correlations, II. <i>Journal of Magnetic Resonance</i> 2001, 148, 61-72.<br>(7)     Panchal, S. C.; Bhavesh, N. S.; Hosur, R. V. 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. <i>Journal of biomolecular NMR</i> 2001, 20, 135-147.<br>(8)     Kumar, D.; Chugh, J.; Hosur, R. V. Generation of serine/threonine check points in HN (C) N spectra. <i>Journal of Chemical Sciences</i> 2009, 121, 955-964.<br>(9)     Kumar, D.; Hosur, R. V. An efficient high-throughput protocol based on 2D-HN (C) N for unambiguous H N and 15 N backbone assignment in small folded proteins in less than a day. <i>Current Science</i> 2010,  1581-1586.<br>(10)   Borkar, A.; Kumar, D.; Hosur, R. V. AUTOBA: automation of backbone assignment from HN (C) N suite of experiments. <i>Journal of biomolecular NMR</i> 2011, 50, 285-297.<br>(11)   Dubey, A.; Mondal, S.; Chandra, K.; Atreya, H. S. Rapid identification of amino acid types in proteins using phase modulated 2D HN(CACB) and 2D HN(COCACB). <i>Journal of Magnetic Resonance</i> 2016, 267, 22-29.<br>(12)   Darapaneni, V.; Prabhaker, V. K.; Kukol, A. Large-scale analysis of influenza A virus sequences reveals potential drug target sites of non-structural proteins. <i>J. Gen. Virol.</i> 2009, 90, 2124-2133.