NMR observation of substrate in the binding site of an active sugar-H+ symport protein in native membranes.

Abstract

NMR methods have been adopted to observe directly the characteristics of substrate binding to the galactose-H+ symport protein GaIP, in its native environment, the inner membranes of Escherichia coli. Sedimented innermembrane vesicles containing the GaiP protein, overexpressed to levels above 50% of total protein, were analyzed by 13C magic-angle spinning NMR, when in their normal "fluid" state and with incorporated D-[1-'3C]glucose. Using conditions of cross-polarization intended to discriminate bound substrate alone, it was possible to detect as little as 250 nmol of substrate added to the membranes containing about 0.5 Amol (=26 mg) of GalP protein. Such-high measuring sensitivity was possible from the fluid membranes by virtue of their motional contributions to rapid relaxation recovery of the observed nuclei and due to a high-resolution response that approached the static field inhomogeneity in these experiments. This good spectral resolution showed that the native state of the membranes presents a substrate binding environment with high structural homogeneity. Inhibitors of the GaIP protein, cytochalasin B and forskolin, which are specific, and D-galactose, but not L-galactose, prevent or suppress detection of the 13C-labeled glucose substrate, confirming that the observed signal was due to specific interactions with the GalP protein. This specific substrate binding exhibits a preference for the j-anomer of D-glucose 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 associated with complex membrane proteins and which may be combined with other NMR methodologies to yield additional structural information on the binding site for the current transport system without isolating it from its native membrane environment.

Investigating transport proteins by solid state NMR

“…Highly amplified expression levels were achieved for some transporters (GalP, NucP, LacS, FucP) allowing substrate binding studies by ssNMR of these proteins in their natural membranes (Patching et al 2004a;Spooner et al 1999Spooner et al , 1998Spooner et al , 1994a. Nevertheless, in most cases proteins have to be solubilised from membranes, purified and reconstituted into phospholipids.…”

“…This approach has been attempted on doubly labelled glucose and forskolin. Unfortunately, it was found that in fluid membranes at 2°C no substrate detection was possible, but freezing samples at Spooner et al (1994a) −35°C allowed an efficient double-quantum filtering and hence substrate detection, most likely due to restrictions of molecular motion. At this temperature, both bound and non-bound substrates start to become immobilised which makes dynamic filtering difficult (Appleyard et al 2000).…”

“…13 C]glucose have been determined from NMR relaxation measurements (Spooner et al 1994a). The proton spin-lattice relaxation in the laboratory frame T 1Z is influenced by high frequency motions (10 −9 s) and was found to be much faster for bound substrate compared to its crystalline state.…”

“…The approach presented by Spooner et al (1994a) relies on the effect of molecular dynamics on cross polarisation (CP) (Schaefer et al 1977). Cross polarisation is usually used to transfer polarisation via dipolar couplings from protons to less sensitive nuclei such as The timescales of the molecular dynamics of bound D-[1-…”

“…Solid state NMR studies on transporters So far, only a limited number of solid state NMR studies on transporters have been reported (Appleyard et al 2000;Glaubitz et al 2000;Mason et al 2004;Patching et al 2004aPatching et al ,b, 2005Spooner et al 1993Spooner et al , 1994aSpooner et al ,b, 1998Spooner et al , 1999. Applications cover in principle three areas: interactions of substrates with the membrane, detection of substrates bound to transporters, and methodological studies describing how to prepare isotope labelled transporters.…”