FTIR spectroscopy of secondary-structure reorientation of melibiose permease modulated by substrate binding.
ABSTRACT Analysis of infrared polarized absorbance spectra and linear dichroism spectra of reconstituted melibiose permease from Escherichia coli shows that the oriented structures correspond mainly to tilted transmembrane alpha-helices, forming an average angle of approximately 26 degrees with the membrane normal in substrate-free medium. Examination of the deconvoluted linear dichroism spectra in H(2)O and D(2)O makes apparent two populations of alpha-helices differing by their tilt angle (helix types I and II). Moreover, the average helical tilt angle significantly varies upon substrate binding: it is increased upon Na(+) binding, whereas it decreases upon subsequent melibiose binding in the presence of Na(+). In contrast, melibiose binding in the presence of H(+) causes virtually no change in the average tilt angle. The data also suggest that the two helix populations change their tilting and H/D exchange level in different ways depending on the bound substrate(s). Notably, cation binding essentially influences type I helices, whereas melibiose binding modifies the tilting of both helix populations.
- SourceAvailable from: Víctor A Lórenz-Fonfría[Show abstract] [Hide abstract]
ABSTRACT: The melibiose carrier from Escherichia coli (MelB) couples the accumulation of the disaccharide melibiose to the downhill entry of H(+), Na(+), or Li(+). In this work, substrate-induced FTIR difference spectroscopy was used in combination with fluorescence spectroscopy to quantitatively compare the conformational properties of MelB mutants, implicated previously in sodium binding, with those of a fully functional Cys-less MelB permease. The results first suggest that Asp55 and Asp59 are essential ligands for Na(+) binding. Secondly, though Asp124 is not essential for Na(+) binding, this acidic residue may play a critical role, possibly by its interaction with the bound cation, in the full Na(+)-induced conformational changes required for efficient coupling between the ion- and sugar-binding sites; this residue may also be a sugar ligand. Thirdly, Asp19 does not participate in Na(+) binding but it is a melibiose ligand. The location of these residues in two independent threading models of MelB is consistent with their proposed role.Proceedings of the National Academy of Sciences 12/2010; 107(51):22078-83. · 9.81 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: The trimeric Na(+)-coupled betaine symporter BetP from Corynebactrium glutamicum adjusts transport activity according to the external osmolality. BetP senses the increasing internal K(+) concentration, which is an immediate consequence of osmotic upshift in C. glutamicum. It is assumed that BetP specifically binds potassium to yet unidentified binding sites, thereby inducing conformational changes resulting in activation. Atomic structures of BetP were obtained in the absence of potassium allowing only a speculative glimpse on a putative mechanism of K(+)-induced transport activation. The structural data suggest that activation in BetP is crucially linked to its trimeric state involving an interaction network between several arginines and glutamates and aspartates. Here, we describe the effect of K(+)-induced activation on the specific ionic interaction sites in terminal domains and loops and on the protomer-protomer interactions within the trimer studied by ATR-FTIR spectroscopy. We suggest that arginine and aspartate and/or glutamate residues at the trimeric interface rearrange upon K(+)-induced activation, although they remain assembled in an interaction network. Our data propose a two-step mechanism comprising first a change in solvent exposure of charged residues and second a modification of their interaction sites in a partner-switching manner. FTIR reveals a higher α-helical content than expected from the X-ray structures that we attribute to the structurally unresolved N-terminal domain modulating regulation. In situ(1)H/(2)H exchange studies point towards an altered exposure of backbone regions to buffer solution upon activation, most likely due to conformational changes in both terminal domains, which further affects ionic interactions within the trimer.Biochimica et Biophysica Acta 01/2013; · 4.66 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: In this review we discuss recent insights obtained from well-characterized model systems into the factors that determine the orientation and tilt angles of transmembrane peptides in lipid bilayers. We will compare tilt angles of synthetic peptides with those of natural peptides and proteins, and we will discuss how tilt can be modulated by hydrophobic mismatch between the thickness of the bilayer and the length of the membrane spanning part of the peptide or protein. In particular, we will focus on results obtained on tryptophan-flanked model peptides (WALP peptides) as a case study to illustrate possible consequences of hydrophobic mismatch in molecular detail and to highlight the importance of peptide dynamics for the experimental determination of tilt angles. We will conclude with discussing some future prospects and challenges concerning the use of simple peptide/lipid model systems as a tool to understand membrane structure and function.Biophysics of Structure and Mechanism 12/2009; 39(4):609-21. · 2.44 Impact Factor