A reduced dimensionality NMR pulse sequence and an efficient protocol for unambiguous assignment in intrinsically disordered proteins

Abstract

Resonance assignment in intrinsically disordered proteins poses a great challenge because of poor chemical shift dispersion in most of the nuclei that are commonly monitored. Reduced dimensionality (RD) experiments where more than one nuclei are co-evolved simultaneously along one of the time axes of a multi-dimensional NMR experiment help to resolve this problem partially, and one can conceive of different combinations of nuclei for co-evolution depending upon the magnetization transfer pathways and the desired information content in the spectrum. Here, we present a RD experiment, (4,3)D-hNCOCAnH, which uses a combination of CO and CA chemical shifts along one of the axes of the 3-dimensional spectrum, to improve spectral dispersion on one hand, and provide information on four backbone atoms of every residue-HN, N, CA and CO chemical shifts-from a single experiment, on the other. The experiment provides multiple unidirectional sequential (i → i - 1) amide (1)H correlations along different planes of the spectrum enabling easy assignment of most nuclei along the protein backbone. Occasional ambiguities that may arise due to degeneracy of amide proton chemical shifts are proposed to be resolved using the HNN experiment described previously (Panchal et al. in J Biomol NMR 20:135-147, 2001). Applications of the experiment and the assignment protocol have been demonstrated using intrinsically disordered α-synuclein (140 aa) protein.

Triphala inhibits alpha-synuclein fibrillization and their interaction study by NMR provides insights into the self-association of the protein

“…Chemical shi assignments were obtained by transfer from BMRB (accession number 16300) and other previous reports. 45,46 Chemical shi perturbations (CSP) were calculated Native-polyacrylamide gel electrophoresis (native-PAGE)…”

“…(A) 1 H-15 N HSQC spectrum of aSyn in the presence (blue) and absence (red) of 0.75 mg ml À1 Triphala. The peaks were assigned by transfer of assignments from BMRB (accession number 16300) and other previous reports 45,46. (B) Chemical shift perturbation (CSP) for 150 mM aSyn observed in 1 H-15 N HSQC spectra in the presence of 0.75 mg ml À1 Triphala relative to free aSyn.…”

An Approach to NMR Assignment of Intrinsically Disordered Proteins

“…Multidimensional experiments generating sequential amide correlations, such as (H)N(CA)NH, (H)N(COCA)NH and HN(CA)CONH, have been extensively applied to IDPs 4d. h, j, l Considering that [ 1 H, 15 N]‐resolved triple‐resonance experiments may not always lead to satisfactory results in the study of disordered systems, either due to poor dispersion of cross‐peaks in the 15 N– 1 H correlation spectra or due to fast exchange of the solvent‐exposed labile protons with water protons, a variety of alternative strategies involving 1 H α and 13 C direct detection have been reported for the study of IDPs 5. From these studies it is apparent that dispersion of inter‐residue cross‐correlation peaks observed in spectra such as 15 N– 13 C′ and 13 C′– 1 H N is typically better than the intra‐residue cross peaks′ dispersion in conventional [ 1 H, 15 N]‐correlation spectra of disordered proteins 4i.…”

A Set of Efficient nD NMR Protocols for Resonance Assignments of Intrinsically Disordered Proteins

“…Frequency switching in the 13 Ca nd 1 Hc hannels was carried out, where needed, whent he relevant magnetisation was along the z-axis. The uniformly ( 13 C, 15 N) labelled sample of a-synuclein (pH 6.2, 1.5 mm), an IDP that has been extensively studied via solution-state NMR, [1,2,22,42,56,57] was prepareda sp er established procedures. [58] The non-uniformly sampled multidimensional data sets were collectede mploying random sampling in the indirect dimensions and processed with the multidimensional decomposition processing protocol [59] availablei n the Bruker To pspin version 3.2 software.…”