Investigating transport proteins by solid state NMR

Abstract

Transporters form an interesting and complex class of membrane proteins. Many of them are potential drug targets due to their role in translocation of ions, small molecules and peptides across the membrane or due to their role in multidrug resistance. Hence elucidating their structure and mechanism is of great importance and may lead to a host of new drugs and methods to alter or inhibit their function. Solid state NMR is an emerging technique for investigating transport proteins. Along with other biochemical and biophysical techniques solid state NMR can provide data on drug binding, protein dynamics and structure at the interface between structural biology and functional analysis. Here, we review solid state NMR applications to primary active and secondary transporters involved in translocation of small molecules. We discuss current experimental limitations and give an overall perspective on how the technique may be used to address some pertinent questions relevant to transporters.

Solid-State NMR Spectroscopy Detects Interactions between Tryptophan Residues of the E. coli Sugar Transporter GalP and the α-Anomer of the d-Glucose Substrate

“…Cross-polarization magic-angle spinning (CP-MAS) solidstate NMR has been used to provide quantitative information about ligand binding affinities for proteins embedded in natural lipid membranes. [11][12][13] The method exploits differences in molecular dynamics of the ligand in the free and bound environmentssrapid and isotropic in the free state and slow and anisotropic in the bound statesto detect signals only from 13 C-ligand associated with the membrane components. With appropriate control experiments using competitive ligands it is possible to assign peaks in the NMR spectrum to ligands or substrates bound to a specific protein.…”

Activity assay of membrane transport proteins

“…NMR spectroscopy depends on the splitting of nuclear energy levels in a magnetic field and the transition induced between the levels. Interaction between many transporters and substrates has been assayed using this technique [6]. For example, Patching et al [55] assayed the interaction between methyl-β-D-glucuronide with glucuronide transporter GusB and revealed the dissociation constant K D is higher than the Michaelis-Menten constant K m for energized transport.…”

A multi-faceted world of transporters

“…Each review covers a specific transport system, such as the ABC-transporters in bacteria (Oswald et al 2006), sodium-coupled transporters in bacteria and in tissues with hormonal activities, respectively (Alexeej and Müller 2006;Geyer et al 2006), organic anion transporters in the liver, kidney, intestine, and brain (König et al 2006) and peptide transporters associated with putative roles in antigen processing in dendritic cells (Zhao et al 2006). One review deals with solid-state NMR techniques targeting the identification of dynamic transporter structures during the transport cycles (Blasting et al 2006), and another review describes how drug transporters are embedded in the pharmacokinetic process of drug elimination by the liver .…”

“…Blasting et al (2006) describe one of these approaches for small multidrug resistance proteins of bacterial origin. They also depict the requirements for long-range structure analysis based on the labelling of disulfide-linked cysteines of the carrier protein by 19F-trifluoroacetone.…”