Extended Flip-back Schemes for Sensitivity Enhancement in Multidimensional HSQC-type Out-and-back Experiments

Abstract

In many NMR experiments, only polarisation of a limited sub-set of all protons is converted into observable coherence. As recently shown by the ''longitudinal'' TROSY implementation (Pervushin et al. (2002) J. Am. Chem. Soc., 124, 12898-12902) and SOFAST-HMQC (Schanda and Brutscher (2005) J. Am. Chem. Soc., 127, 8014-8015), recovery of unused polarisation can be used indirectly and unspecifically to cool the proton lattice and, thus, accelerate re-equilibration for the selected proton subset. Here we illustrate transfer of this principle to HSQC-based multi-dimensional out-and-back experiments that exploit only polarisation of 15 N-bound protons. The presented modifications to the pulse sequences can be implemented broadly and easily, extending standard flip-back of water polarisation to a much larger pool of protons that may comprise all non) 15 N-bound protons. The underlying orthogonal separation of H N polarisation (selected by the main transfer path) from unused H u polarisation (flipped-back on the recovery path) is thereby achieved through positive or negative selection by J-coupling, or using band-selective pulses. In practice, H u polarisation recovery degrades mostly through cumulative pulse imperfections and transverse relaxation; we present, however, strategies to substantially minimise such losses particularly during interim proton decoupling. Depending on the protein's relaxation properties and the extended flipback scheme employed, we recovered up to 60% H u equilibrium polarisation. The concomitant cooling of the proton lattice afforded substantial gains of more than 40%, relative to the water-only flip-back version, in the fast pulsing regime with re-equilibration delays s much shorter than optimal ðs opt ¼ 1:25 Á T 1 ðH N ÞÞ. These would be typically employed if resolution requirements dominate the total measurement time. Contrarily, if sensitivity is limiting and optimal interscan delays s opt can be set (optimal pulsing regime), the best of the presented flip-back schemes may still afford up to ca. 10% absolute sensitivity enhancement.Abbreviations: bsfb -band-selective flip-back; cpd -continuous pulsing decoupling; efb -extended flip-back; H u -unselected proton magnetisation; H C fb -H C flip-back; ufb -universal flip-back; wfb -water flip-back

Similar Modes of Interaction Enable Trailer Hitch and EDC3 To Associate with DCP1 and Me31B in Distinct Protein Complexes

“…cDNAs encoding full-length Tral, DCP1, DCP2, EDC3, Me31B, HPat, CUP, LSm1, LSm4, and LSm7 proteins or protein domains were amplified with primers containing appropriate restriction sites, using a (dT) 15 -primed S2 cDNA library as a template. The amplified cDNAs were cloned into a vector allowing the expression of green fluorescent protein (GFP) or N-hemagglutinin (HA)-peptide fusions (pAc5.1B-EGFP or pAc5.1B-N-HA, respectively) as described previously (18,19,50).…”

“…The LSm domain of D. melanogaster Tral (UniProtKB entry Q9VTZ0; M1 to P84) was amplified from a (dT) 15 primed S2 cell cDNA library and cloned into the pETM60 vector (derived from pET24-d; Novagen). The protein was expressed in the E. coli strain BL21(DE3) Rosetta 2 at 20°C overnight.…”

“…The protein was expressed in the E. coli strain BL21(DE3) Rosetta 2 at 20°C overnight. To uniformly label Tral LSm domain with 15 N/ 13 C or 15 N, cells were grown in M9 minimal medium supplemented with 15 NH 4 Cl with or without 13 C 6 -labeled glucose. Cell lysates were purified by affinity chromatography using a nickel-nitrilotriacetic acid HiTrap chelating HP column (GE Healthcare), followed by cleavage of the tag with overnight exposure to TEV protease.…”

“…All spectra were recorded at 25°C on Bruker DMX600, DMX750, and AVANCE900 spectrometers. Backbone sequential assignments were completed by using standard triple-resonance experiments implemented using selective proton flipback techniques for fast pulsing (15). Aliphatic side chain assignments and aromatic assignments were completed as described before (9).…”

The structure of a folding intermediate provides insight into differences in immunoglobulin amyloidogenicity

“…For folding studies, 15 N-labeled unfolded CL in PBS containing 2 M GdmCl was diluted 10-fold by adding ice-cold PBS without GdmCl. Real-time 15 N-1 H HSQC spectra were recorded at 2°C every 14 min by using selective proton flip-back pulses (48). Identical processing of all of the spectra was performed by using the program TOPSPIN 1.3 (Bruker BioSpin).…”