NMR Resonance Assignments of Sparsely Labeled Proteins: Amide Proton Exchange Correlations in Native and Denatured States

Abstract

Protein NMR assignments of large proteins using traditional triple resonance techniques depends on double or triple labeling of samples with 15 N, 13 C, and 2 H. This is not always practical with proteins that require expression in non-bacterial hosts. Labeling with isotopically labeled versions of single amino acids (sparse labeling) often is possible, however resonance assignment then requires a new strategy. Here a procedure for the assignment of cross peaks in 15 N-1 H correlation spectra of sparsely labeled proteins is presented. It relies on the correlation of proton-deuterium amide exchange rates in native and denatured spectra of the intact protein, followed by correlation of chemical shifts in the spectra of the denatured protein with chemical shifts of sequenced peptides derived from the protein.The procedure is successfully demonstrated on a sample of a protein, Galectin-3, selectively labeled with 15 N at all alanine residues.

Multi-dimensional NMR without coherence transfer: Minimizing losses in large systems

“…Deuteration is also not currently possible in mammalian host cells, removing much of the advantage of TROSY based sequences. New assignment strategies are making it possible to assign sparsely labeled spectra [29,30], and sparse constraints from long range paramagnetic relaxation effects and RDCs are making at least low resolution structure determination possible when supplemented with computational modeling [31–34]. Hence we anticipate an increased number of cases where large systems have relatively small numbers of peaks.…”

NH exchange in point mutants of human ubiquitin

“…All spectra were recorded at 298 K or 300 K on a Bruker Avance-700 instrument equipped with a cryo probe head. The resonance assignments of the human ubiquitin were available [10,11] and formed the starting basis. Backbone assignments of the protein were based on 1 H-15 N-HSQC, HNCACB, HNCA, HN(CO)CA, and HNCO.…”

“…The signal assignment in the four ubiquitin point mutants was straightforward, since the resonance assignment of the wild type is well known and documented in the literature [10,11]. From all new variants the necessary 2D-and 3D-NMR spectra have been recorded and compared with those of the wild type.…”

NMR assignments of sparsely labeled proteins using a genetic algorithm

“…For sparsely labeled systems there has been some prior effort at resonance assignment. We have attempted to use amide exchange rate correlations in NMR and MS data to achieve assignments of 15 N labeled sites (Feng et al 2007; Nkari and Prestegard 2009). There have also been approaches that label proteins with combinations of amino acids to make assignments based on connectivities similar to those seen in triple resonance experiments (Kato et al 2010; Lohr et al 2015; Maslennikov and Choe 2013), and of course, assignment in sparsely labeled proteins can be facilitated by mutating residues to remove crosspeaks from labeled sites one at a time (Tzakos et al 2006).…”