Generation of serine/threonine check points in HN(C)N spectra (original) (raw)
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Alanine check points in HNN and HN(C)N spectra
Journal of Magnetic Resonance, 2006
Rapid resonance assignment is a key requirement in structural genomics research by NMR. In this context we present here two new pulse sequences, namely, HNN-A and HN(C)N-A that have been developed by simple modification of the previously described pulse sequences, HNN and HN(C)N [S.C. Panchal, N.S. Bhavesh, R.V. Hosur, Improved 3D triple resonance experiments, HNN and HN(C)N, for H N and 15 N sequential correlations in ( 13 C, 15 N) labeled proteins: application to unfolded proteins, J. Biomol. NMR, 20 (2001) 135-147]. These increase the number of start/check points in HNN and/or HN(C)N spectra and hence help in pacing up resonance assignment in proteins.
Assignment of protein backbone resonances is most routinely carried out using triple resonance three dimensional NMR experiments involving amide 1 H and 15 N resonances. However for intrinsically unstructured proteins, alpha-helical proteins or proteins containing several disordered fragments, the assignment becomes problematic because of high degree of backbone shift degeneracy. In this backdrop, a novel reduced dimensionality (RD) experiment -(5,3)D-hNCO-CANH-is presented to facilitate (and/or to validate) the sequential backbone resonance assignment in such proteins. The proposed 3D NMR experiment makes use of the modulated amide 15 N chemical shifts (resulting from the joint sampling along both its indirect dimensions) to resolve the ambiguity involved in connecting the neighboring amide resonances (i.e. HiNi and Hi-1Ni-1) for overlapping amide NH peaks. The experiment -encoding 5D spectral information-leads to a conventional 3D spectrum with significantly reduced spectral crowding and complexity. The improvisation is based on the fact that the linear combinations of intra-residue and inter-residue backbone chemical shifts along both the co-evolved indirect dimensions span a wider spectral range and produce better peak dispersion than the individual shifts themselves. Taken together, the experiment -in combination with routine triple resonance 3D NMR experiments involving backbone amide ( 1 H and 15 N) and carbon ( 13 C and 13 C') chemical shifts-will serve as a powerful complementary tool to achieve the nearly complete assignment of protein backbone resonances in a time efficient manner. The performance of the experiment and application of the method have been demonstrated here using a 15.4 kDa size folded protein and a 12 kDa size unfolded protein. 3 Introduction: Over the decades, NMR has emerged as a powerful technique for studying the structure and dynamics of proteins and their complexes in solution. Further, it is the technique of choice for studying conformational properties of intrinsically unstructured proteins (IDPs), and their interactions with their physiological binding partners in solution [1-3]. For various such studies on proteins by NMR, the very first and key requirement is the sequence specific assignment of backbone ( 1 H, 15 N, 13 C' and 13 C') resonances [4,5]. The well-established and most routinely used assignment strategies involve the use of 15 N, 1 H N resolved triple resonance experiments sequentially linking 13 C , 13 C' or 15 N shifts [6-22] and many proteins have been assigned this way (evident from the Biological Magnetic Resonance Bank: http://www.bmrb.wisc.edu). However for proteins exhibiting high degree of backbone amide and carbon shift degeneracy (e.g. -helical proteins or proteins containing disordered fragments including IDPs), getting this information in an unambiguous and time-efficient manner has always remained problematic and challenging. Therefore new or alternative NMR methods and strategies -for rapid and efficient assignment of backbone resonances in such proteins-are required.
Biochemical and Biophysical Research Communications, 2003
We recently described a triple resonance experiment, HN(C)N, for sequential correlation of H N and 15 N atoms in (15 N, 13 C) labeled proteins [J. Biomol. NMR. 20 (2001) 135]. Here, we describe an approach based on this experiment for estimation of one bond N-C a J-couplings in medium size labeled proteins, which seem to show good correlations with w torsion angles along the protein backbone. The approach uses the ratio of the intensities of the sequential and diagonal peaks in the F 2-F 3 planes of the HN(C)N spectrum. The reliability of the approach has been demonstrated using a short peptide wherein the coupling constants have been measured by the present method and also independently from peak splittings in HSQC spectra. The two results agree within 10%. The applicability of the procedure to proteins has been demonstrated using doubly labeled FK506 binding protein (FKBP, molecular mass $12 kDa). Coupling constant estimates have been obtained for 62 out of 100 non-proline residues and they show a correlation with w torsion angles, as has been reported before. This semi-quantitative application of HN(C)N extends the significance of the experiment especially, in the context of structural genomics, since the single experiment, not only provides a great enhancement in the speed of resonance assignment, but also provides quantitative structural information.
Analytical Chemistry, 2012
While nuclear magnetic resonance (NMR) has had commendable success in atomic-level investigation of folded proteins, intrinsically unfolded and partially folded proteins have always posed a great challenge, because of poor chemical shift dispersions. We present here a reduceddimensionality-based NMR triple resonance pulse sequence, (4,3)D-HN(C)NH, which not only helps to disperse the peaks further by combining 15 N and amide 1 H chemical shifts, but also directly establishes correlations between 1 H i N , 15 N i , 1 H i+1 N , and 15 N i+1 spins along the F 1 −F 3 planes. The F 2 −F 3 projection planes of this experiment provide unique identification of the check points in amide resonances. An assignment strategy derived by combining information along the F 1 −F 3 planes and in the F 2 −F 3 projection planes of the experiment has been presented and shown to be very useful for both intrinsically disordered/unfolded proteins and folded protein alike. The experiment and the protocol would be valuable for protein folding, proteomics, and drug discovery programs.
A selective experiment for the sequential protein backbone assignment from 3D heteronuclear spectra
2005
Two modifications of the triple-resonance CANCO sequence, designed for backbone assignment in proteins [Angew. Chem. Int. Ed. 43 (2004) 2257], are presented here. These two new sequences display the intra-residue Ca-CO correlation selectively, while in the original sequence both the inter-and the intra-residue correlations were present. In addition, one of the two variants benefits from an improved sensitivity. Both sequences are a useful complement to the CANCO sequence for facile sequence-specific protein assignment by protonless NMR.
Journal of biomolecular NMR, 2003
NMR experiments are presented which allow backbone resonance assignment, secondary structure identification, and in favorable cases also molecular fold topology determination from a series of two-dimensional 1H-15N HSQC-like spectra. The 1H-15N correlation peaks are frequency shifted by an amount +/- omegaX along the 15N dimension, where omegaX is the Calpha, Cbeta, or Halpha frequency of the same or the preceding residue. Because of the low dimensionality (2D) of the experiments, high-resolution spectra are obtained in a short overall experimental time. The whole series of seven experiments can be performed in typically less than one day. This approach significantly reduces experimental time when compared to the standard 3D-based methods. The here presented methodology is thus especially appealing in the context of high-throughput NMR studies of protein structure, dynamics or molecular interfaces.
Journal of Biomolecular NMR, 1993
We recently proposed a novel four-dimensional (4D) NMR strategy for the assignment of backbone nuclei in spectra of ~3C/~SN-labelled proteins (Boucher et al. (1992) J. Am. Chem. Soc., 114, 2262-2264. In this paper we extend this approach with a new constant time 4D HCC(CO)NNH experiment that also correlates the chemical shifts of the aliphatic sidechain (1H and ~3C) and backbone (~H, 13C~ and ~SN) nuclei. It separates the sidechain resonances, which may heavily overlap in spectra of proteins with large numbers of similar residues, according to the backbone nitrogen and amide proton chemical shifts. When used in conjunction with a 4D HCANNH or HNCAHA experiment it allows, in principle, complete assignment of aliphatic sidechain and backbone resonances with just two 4D NMR experiments.