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

Abstract

In TROSY experiments, spin state selection (S 3 ) retains only the single HSQC sub-spectrum with minimal T 2 relaxation and maximal resolution, yet at the cost of eliminating half of the available polarisation as undesired anti-TROSY component. We here introduce queued TROSY (qTROSY) as a novel scheme to partially recover and exploit this anti-TROSY polarisation in two concatenated scans. After initial orthogonal spin state separation (oS 3 ), anti-TROSY polarisation is explicitly stored while its TROSY counterpart follows the desired coherence pathway recorded in a first scan A. The immediately appended scan B then quantitatively converts the recovered anti-TROSY polarisation into a second TROSY spectrum, skipping the time-limiting long reequilibration delay. Both concatenated qTROSY scans thus ideally exploit the full initial polarisation within almost the same measurement time. In practice, T 2 relaxation losses accruing during the coupling evolution delays reduced anti-TROSY polarisation recovery below 40%, obviating sensitivity enhancement through addition of both qTROSY scans; yet, scan B retained a complete scan A spectrum with up to 75% intensity. We therefore propose to employ qTROSY asymmetrically, compacting two separate conventional into one queued TROSY-type experiment with significantly reduced measurement time, implying primarily the concatenation of different three-or higherdimensional experiments. Both anti-TROSY polarisation recoveries and possible time savings are largest for deuterated and smaller non-deuterated proteins, extending the rentability limit of the TROSY principle towards smaller molecular weights.Abbreviations: oS 3 -orthogonal spin state separation; S 3 -spin state selective; S 3 -CT -S 3 coherence transfer; S 3 E -S 3 excitation; ST2-PT -spin state to spin state selective polarisation transfer.

TROSY-based NMR experiments for NMR studies of large biomolecules

“…This deviation is due to the more efficient recovery of the 15 N magnetization, S z , during the inter-scan periods. Recently, another scheme, termed qTROSY, was proposed to enhance the sensitivity of the transfer by recovering some of the magnetization from the semi-TROSY peaks [78].…”

NMR in the SPINE Structural Proteomics project

“…Faster repetition of the same experiment can be achieved by accelerating the re-equilibration of the selected proton subset (Pervushin et al, 2002). Alternatively, two experiments can be compacted into the same measurement time (Diercks & Orekhov, 2005). Novel schemes for polarization recovery based on HSQCtype or TROSY-type (Pervushin et al, 1997) transfers have been developed by the Utrecht group: the latter is mostly employed for deuterated proteins.…”

“…Novel schemes for polarization recovery based on HSQCtype or TROSY-type (Pervushin et al, 1997) transfers have been developed by the Utrecht group: the latter is mostly employed for deuterated proteins. The use of recovered H N anti-TROSY polarization in queued TROSY (Diercks & Orekhov, 2005) has been introduced, affording up to 50% time saving for recording experiment pairs. In contrast, HSQC-type transfer is the method of choice for standard non-deuterated proteins.…”

“…NMR data-collection times have been further reduced by enhanced sensitivity obtained from unused proton polarization (Diercks & Orekhov, 2005;Diercks et al, 2006). The use of residual dipolar couplings (RDCs) combined with more conventional structural parameters provides an interesting possibility for semi-automated structure determination (Bax, 2003), as well as for obtaining structural information on systems sampling different conformational substates (Bertini, Del Bianco et al, 2004).…”

UTOPIA NMR: activating unexploited magnetization using interleaved low-gamma detection

“…However, as the complexity of the systems studied by NMR spectroscopy grows, the challenges to get the necessary information for accurate structural analysis also increase dramatically. Instrumental improvements, isotope labeling schemes (Lundstrom et al 2012) and developments in pulse sequences (Diercks and Orekhov 2005; Farmer 1991; Frueh et al 2009; Kay et al 1990; Kay et al 1992; Kern et al 2008; Lescop et al 2007; Parella and Nolis 2010; Perez-Trujillo et al 2007; Sattler et al 1995; Schanda and Brutscher 2005; Schanda et al 2007; Szyperski et al 1996) can shorten the time for data acquisition and improve the spectral quality, thus partially compensating the growing sample complexity. Several interesting concepts such as time-shared NMR (Farmer 1991; Kay et al 1990; Kay et al 1992; Parella and Nolis 2010; Perez-Trujillo et al 2007; Sattler et al 1995), BEST (Lescop et al 2007) and SOFAST (Kern et al 2008; Schanda and Brutscher 2005; Schanda et al 2007) techniques, have been developed to use the available magnetization more effectively.…”