TROSY NMR with a 52 kDa sugar transport protein and the binding of a small-molecule inhibitor. (June 2014)

• Record Type: Journal Article
• Authors: Kalverda, Arnout P.; Gowdy, James; Thompson, Gary S.; Homans, Steve W.; Henderson, Peter J. F.; Patching, Simon G.
• Is Part Of: Molecular membrane biology. Volume 31:Number 4(2014)
• Page Start: 131
• Page End: 140
• Publication Date: 2014-06
• Subjects: Membranes (Biology) -- Periodicals; Biochemistry -- Periodicals; Cell membranes -- Periodicals; Molecular biology -- Periodicals; 571.64
• DOI: 10.3109/09687688.2014.911980
• Languages: English
• ISSNs: 0968-7688

Abstract

<abstract> <title>Abstract</title> <p>Using the sugar transport protein, GalP, from <italic>Escherichia coli</italic>, which is a homologue of human GLUT transporters, we have overcome the challenges for achieving high-resolution [<sup>15</sup>N-<sup>1</sup>H]- and [<sup>13</sup>C-<sup>1</sup>H]-methyl-TROSY NMR spectra with a 52 kDa membrane protein that putatively has 12 transmembrane-spanning α-helices and used the spectra to detect inhibitor binding. The protein reconstituted in DDM detergent micelles retained structural and functional integrity for at least 48 h at a temperature of 25 °C as demonstrated by circular dichroism spectroscopy and fluorescence measurements of ligand binding, respectively. Selective labelling of tryptophan residues reproducibly gave 12 resolved signals for tryptophan <sup>15</sup>N backbone positions and also resolved signals for <sup>15</sup>N side-chain positions. For improved sensitivity isoleucine, leucine and valine (ILV) methyl-labelled protein was prepared, which produced unexpectedly well resolved [<sup>13</sup>C-<sup>1</sup>H]-methyl-TROSY spectra showing clear signals for the majority of methyl groups. The GalP/GLUT inhibitor forskolin was added to the ILV-labelled sample inducing a pronounced chemical shift change in one Ile residue and more subtle changes in other methyl groups. This work demonstrates that high-resolution TROSY NMR spectra can be achieved with large complex α-helical membrane proteins without the use of elevated temperatures. This is a prerequisite to applying further labelling strategies and NMR experiments for measurement of dynamics, structure elucidation and use of the spectra to screen ligand binding.</p> </abstract>