Untitled

Abstract

This paper describes the use of single transition-to-single transition polarization transfer (ST2-PT) in transverse relaxation-optimized spectroscopy (TROSY), where it affords a [Formula: see text] sensitivity enhancement for kinetically stable amide 15N-1H groups in proteins. Additional, conventional improvements of [15N,1H]-TROSY include that signal loss for kinetically labile 15N-1H groups due to saturation transfer from the solvent water is suppressed with the 'water flip back' technique and that the number of phase steps is reduced to two, which is attractive for the use of [15N,1H]-TROSY as an element in more complex NMR schemes. Finally, we show that the impact of the inclusion of the 15N steady-state magnetization (Pervushin et al., 1998) on the signal-to-noise ratio achieved with [15N,1H]-TROSY exceeds by up to two-fold the gain expected from the gyromagnetic ratios of 1H and 15N.

Functional Expression of the PorAH Channel from Corynebacterium glutamicum in Cell-free Expression Systems

“…NMR Spectroscopy-Two-dimensional, 15 N, 1 H transverse relaxation optimized spectroscopy (TROSY) spectra (28,29) of PorA and T-PorH were obtained after preparative scale expression of the proteins in the presence of labeled amino acids. NMR samples (0.25 mM) were prepared in 20 mM Tris-Cl, pH 6.7, in LDAO (ϳ2%), and the spectra were recorded at 25°C.…”

In‐Cell NMR Spectroscopy

“…This increase by a factor of two of the rotational correlation time also increases the apparent molecular weight of the protein by a factor of two. Fortunately, the introduction of TROSY [44] and similar techniques [45,46] a couple of years ago has extended the applicability of NMR spectroscopy to large macromolecules with a molecular weight of 100 kDa and more. These technical advances, combined with the relatively low viscosity of the cellular cytoplasm, predict that the cytoplasmic viscosity is not a major limitation for the observation of proteins inside living cells.…”

qTROSY – a novel scheme for recovery of the anti-TROSY magnetisation

“…As in conventional TROSY (Pervushin et al, 1998), natural c N AEN z polarisation contributes jc N =c H j ¼ 10% additional intensity unless explicitly eliminated, e.g. by alternatingly inverting the INEPT module, or inserting an initial 90°( 15 N) excitation pulse followed by a dephasing z-spoil gradient: Figure 1.…”

“…The terminal ST2-PT module (Pervushin et al, 1998) then transfers both S 3 coherence and polarisation from 15 N to H N with preservation of spin states. Pulse imperfections, incomplete spinstate separation by the initial S 3 -CT module, and cross-talk relaxation (Meissner et al, 1998b), however, invariably cause some mixture of proton spin states for the desired N y ( 1 2 + H z ) coherence and N y ( 1 2 ) H z ) polarisation.…”

“…Spin state selection, however, inevitably comes at the cost of splitting the total polarisation equally into two (for 1/2-spins), only half of which is converted into the desired TROSY signal. The complementary anti-TROSY component has so far mostly been discarded through phase cycling (Pervushin et al, 1998) or pulsed field gradients (Palmer et al, 1991;Czisch and Boelens, 1998;Weigelt, 1998), except in a few schemes to retain anti-TROSY along with the TROSY coherence after its modulation by chemical shift and transverse relaxation (Andersson et al, 1998;SchulteHerbru¨ggen et al, 1999). Although to some extent compensated by reduced relaxation losses especially for larger biomolecules, the initial sacrifice of half the available polarisation thus remains a dissatisfactory aspect of TROSY spectroscopy.…”