NMR OBSERVATION OF SUBSTRATE IN THE BINDING-SITE OF AN ACTIVE SUGAR-H+ SYMPORT PROTEIN IN NATIVE MEMBRANES

NMR methods have been adopted to observe directly the characteristics of substrate binding to the galactose-H+ symport protein GalP, in its native environment, the inner membranes of Escherichia coli. Sedimente d inner-membrane vesicles containing the GalP protein, overexpressed t o levels above 50% of total protein, were analyzed by C-13 magic-angle spinning NMR, when in their normal ''fluid'' state and with incorpora ted D-[1-C-13]glucose. Using conditions of cross-polarization intended to discriminate bound substrate alone, it was possible to detect as l ittle as 250 nmol of substrate added to the membranes containing about 0.5 mu mol (approximate to 26 mg) of GalP protein. Such high measurin g sensitivity was possible from the fluid membranes by virtue of their motional contributions to rapid relaxation recovery of the observed n uclei and due to a high-resolution response that approached the static field inhomogeneity in these experiments. This good spectral resoluti on showed that the native state of the membranes presents a substrate binding environment with high structural homogeneity. Inhibitors of th e GalP protein, cytochalasin B and forskolin, which are specific, and D-galactose, but not L-galactose, prevent or suppress detection of the C-13-labeled glucose substrate, confirming that the observed signal w as due to specific interactions with the GalP protein. This specific s ubstrate binding exhibits a preference for the beta-anomer of D-glucos e and substrate translocation is determined to be slow, on the 10(-2) s time scale. The work describes a straightforward NMR approach, which achieves high sensitivity, selectivity, and resolution for nuclei ass ociated with complex membrane proteins and which may be combined with other NMR meth odologies to yield additional structural information on the binding site for the current transport system without isolating i t from its native membrane environment.