Weak Substrate Binding to Transport Proteins Studied by NMR

Abstract

The weak binding of sugar substrates fails to induce any quantifiable physical changes in the L-fucose-H+ symport protein, FucP, from Escherichia coli, and this protein lacks any strongly binding ligands for competitive binding assays. Access to substrate binding behavior is however possible using NMR methods which rely on substrate immobiliza-tion for detection. Cross-polarization from proton to carbon spins could detect the portion of 13C-labeled substrate associated with 0.2 micromol of the functional transport system overexpressed in the native membranes. The detected substrate was shown to be in the FucP binding site because its signal was diminished by the unlabeled substrates L-fucose and L-galactose but was unaffected by a three- to fivefold molar excess of the non-transportable stereoisomer D-fucose. FucP appeared to bind both anomers of its substrates equally well. An NMR method, designed to measure the rate of substrate exchange, could show that substrate exchanged slowly with the carrier center (>10(-1) s), although its dynamics are not necessarily coupled strongly to this site within the protein. Relaxation measurements support this view that fluctuations in the interaction with substrate would be confined to the binding site in this transport system.

Solid‐state NMR for the analysis of high‐affinity ligand/receptor interactions

“…CP has been used as a filter for bound ligands in a wide variety of integral membrane proteins (112-114) including a large range of bacterial transporters (115)(116)(117)(118). In the case of sugar transport proteins, the sugar is sufficiently immobilized during the transport process for efficient CP to occur and thus selectively observed (115) (Fig.…”

“…For ligand exchange, which occurs on the timescale of longitudinal relaxation of membrane systems (typically >100 ms), methods such as dipolar-dephased CP allow the characterization of the exchange process (113,115,116). This technique relies on the differences in transverse relaxation between bound and free ligand to create two distinct ''spectroscopic'' pools.…”

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.…”

“…High expression levels, FucP constitutes 20% total inner membrane protein, enabled ssNMR studies directly on native membranes (Spooner et al 1998).…”

“…This has triggered a number of further studies aimed at obtaining structural information concerning the position of the binding site (Spooner et al 1998), as well as the ligand/protein association constant (Patching et al 2004a). Recently, it has also been shown that isotope labelled transporters can be prepared at a quantity and purity suitable for ssNMR (Mason et al 2004).…”