Selective NMR observation of inhibitor and sugar binding to the galactose-H+ symport protein GalP, of Escherichia coli

The binding of the transport inhibitor forskolin, synthetically labelled wi th C-13, to th, galactose-H+ symport protein GalP, overexpressed in its nat ive inner membranes from Escherichia coli, was studied using cross-polariza tion magic angle spinning C-13 NMR. C-13-Labelled D-galactose and D-glucose were displaced from GalP with the singly labelled [7-(OCOCH3)-C-13]forskol in and were not bound to any alternative site within the protein, demonstra ting that any multiple sugar binding sites are not simultaneously accessibl e to these sugars and the inhibitor within GalP. The observation of singly (13)-labelled forskolin was hampered by interference from natural abundance C-13 in the membranes and so the effectiveness of double-quantum filtratio n was assessed for the exclusive detection of C-13 spin pairs in sugar (D-[ 1,2-C-13(2)]glucose) and inhibitor ([7-(OCOCH3)-C-13-C-13]forskolin) bound to the GalP protein. The solid state NMR methodology was not effective in c reating double-quantum selection of ligand bound with membranes in the 'flu id' state (approx. 2 degreesC) but could be applied in a straightforward wa y to systems that were kept frozen. At -35 degreesC, double-quantum filtrat ion detected unbound sugar that was incorporated into ice structure within the sample, and was not distinguished from protein-bound sugar. However, th e method detected doubly labelled forskolin that is selectively bound only to the transport system under these conditions and provided very effective suppression of interference from natural abundance C-13 background. These r esults indicate that solid state NMR methods can be used to resolve selecti vely the interactions of more hydrophobic ligands in the binding sites of t arget proteins. (C) 2000 Elsevier Science B.V. All rights reserved.