Time-shared experiments for efficient assignment of triple-selectively labeled proteins
 

Time-shared experiments for efficient assignment of triple-selectively labeled proteins


Publication date: Available online 30 September 2014
Source:Journal of Magnetic Resonance</br>
Author(s): Frank Löhr , Aisha Laguerre , Christoph Bock , Sina Reckel , Peter J. Connolly , Norzehan Abdul-Manan , Franz Tumulka , Rupert Abele , Jonathan M. Moore , Volker Dötsch</br>
Combinatorial triple-selective labeling facilitates the NMR assignment process for proteins that are subject to signal overlap and insufficient signal-to-noise in standard triple-resonance experiments. Aiming at maximum amino-acid type and sequence-specific information, the method represents a trade-off between the number of selectively labeled samples that have to be prepared and the number of spectra to be recorded per sample. In order to address the demand of long measurement times, we here propose pulse sequences in which individual phase-shifted transients are stored separately and recombined later to produce several 2D HN(CX) type spectra that are usually acquired sequentially. Sign encoding by the phases of 13C 90° pulses allows to either select or discriminate against 13C’ or 13C? spins coupled to 15N. As a result, 1H-15N correlation maps of the various isotopomeric species present in triple-selectively labeled proteins are deconvoluted which in turn reduces problems due to spectral overlap. The new methods are demonstrated with four different membrane proteins with rotational correlation times ranging from 18 to 52 ns.
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Source: Journal of Magnetic Resonance