FTIR Spectroscopy of Secondary-Structure Reorientation of Melibiose Permease Modulated by Substrate Binding

Unitat de Biofísica, Departament de Bioquímica i de Biologia Molecular, Facultat de Medicina, and Centre d'Estudis en Biofísica, Universitat Autònoma de Barcelona, Barcelona, Spain.
Biophysical Journal (Impact Factor: 3.97). 06/2008; 94(9):3659-70. DOI: 10.1529/biophysj.107.115550
Source: PubMed


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.

Download full-text


Available from: Víctor A Lórenz-Fonfría, Jan 23, 2015
  • Source
    • "Therefore we considered the second derivative profiles of the fit envelope in comparison to the original spectra as well (Fig. 3B and D). The band located at 1655 cm −1 is attributed to the α-helical content [20] [37], while the bands at 1634 and 1639 cm −1 are assigned to unordered structure, and/or loops in hydrophilic regions outside the membrane as well as solvated short helices and/or 3 10 helices [20] [38] [39]. The peak at 1681 and 1674 cm −1 can be attributed to hydrogen bonded turns or closed loops buried in the hydrophobic region, located most likely in the membrane, thus unaffected from 2 H 2 O-buffer. "
    [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.
    Full-text · Article · Jan 2013 · Biochimica et Biophysica Acta
  • [Show abstract] [Hide abstract]
    ABSTRACT: This essay shows how Fourier Transform infrared spectroscopy (FTIR) can be applied to study membrane phase behavior of cells that are relevant for biomedical applications such as red blood cells and platelets. FTIR studies are minimally invasive and do not require labeling. FTIR can give unique information on conformation and stability of membranes in cells that are exposed to heating, freezing or dehydration stress. By combining in situ FTIR techniques with cell viability studies, cell damage can be correlated with membrane phase changes. Understanding the complex behavior of biomembranes during heating, freezing and drying is directly relevant for thermal processing of cells such as is done in cryopreservation and cryosurgery.
    No preview · Article · Jan 2009
  • [Show abstract] [Hide abstract]
    ABSTRACT: Atomic force microscopy (AFM) and FTIR spectroscopy techniques have been exploited to investigate the inverted hexagonal phase (H(II)) of cardiolipin obtained by dehydration of a phospholipid water dispersion on a solid support. The characteristic cylinders of the H(II) phase have been imaged by AFM and the effects of different preparation conditions (temperature and the presence of chemicals) on the structural parameters and on the presence of local nanoscale defects have been studied. It has been found that the measured repeat spacing of the H(II) cylinders decreases upon increase of temperature and addition of pentachlorophenol (PCP), a chemical which is known to affect the structure and function of lipid bilayers. It has been shown that AFM can help in revealing some features of the mechanism of the inverted hexagonal phase formation, corroborating the results of a recent molecular dynamics study on the H(II) phase formation from multilamellar phospholipid structures.
    No preview · Article · Mar 2009 · The Journal of Physical Chemistry B
Show more