GFT NMR Experiments for Polypeptide Backbone and¹³C^βChemical Shift Assignment

Abstract

(4,3)D, (5,3)D and (5,2)D GFT triple resonance NMR experiments are presented for polypeptide backbone and (13)C(beta) resonance assignment of (15)N/(13)C labeled proteins. The joint sampling of m = 2, 3 or 4 indirect chemical shift evolution periods of 4D and 5D NMR experiments yields the measurement of 2(m) - 1 linear combinations of shifts. To obtain sequential assignments, these are matched in corresponding experiments delineating either intra or interresidue correlations. Hence, an increased set of matches is registered when compared to conventional approaches, and the 4D or 5D information allows one to efficiently break chemical shift degeneracy. Moreover, comparison of single-quantum chemical shifts obtained after a least squares fit using either the intra or the interresidue data demonstrates that GFT NMR warrants highly accurate shift measurements. The new features of GFT NMR based resonance assignment strategies promise to be of particular value for establishing automated protocols.

NMR data collection and analysis protocol for high-throughput protein structure determination

“…In these cases, (3,2)D GFT NMR can provide within a few hours the information required for assignment (8)(9)(10)(11). However, when compared with the current protocol based on 4D and 5D information, such strategies would not offer similar robustness in high throughput.…”

Distant echoes of the accordion: Reduced dimensionality, GFT‐NMR, and projection‐reconstruction of multidimensional spectra

“…The methodology was extended to the more demanding problem of four-dimensional spectroscopy reduced to only two dimensions. Kim and Szyperski (40,41) described the general procedure for reducing the dimensionality of three-, four-, and five-dimensional spectra. This was followed by independent investigations by Kozminski and Zhukov (42) and a review by Simon and Sattler (43).…”

G-matrix Fourier transform NMR spectroscopy for complete protein resonance assignment

“…5 and 6), whereas the use of basic spectra requires that shifts are first calculated from the respective systems of equations ( Table 1). Sampling of (5,3)D HC-C-CH can be accelerated by 25% when central peaks in HC-type experiments are derived from 1 H magnetization (10). When using cryogenic probes, this may become a viable option even for larger systems: central peaks detected in 96 h from steady-state magnetization at 750 MHz and in 6 h from 1 H magnetization at 600 MHz by using a cryogenic probe exhibit quite comparable signal-to-noise ratios (Fig.…”

“…5c) indicate feasibility for systems of at least 20 kDa. If high-shift degeneracy is encountered, the core set can be complemented by GFT NMR experiments delineating 1 H ␣ and͞or 13 CЈ chemical shifts (10,23). Moreover, heteronuclear-resolved [ 1 H, 1 H]-nuclear Overhauser effect GFT NMR spectroscopy will allow one to also accelerate the collection of through-space nuclear Overhauser effect connectivities required for determination of 3D structures.…”

“…4), fast collection of multidimensional spectra has emerged as a subject of broader scientific interest in structural biology and pharmacology (5)(6)(7)(8). Recently, we introduced G-matrix Fourier transform (GFT) NMR spectroscopy to meet this demand (9,10).…”