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Biophys J, August 2000, p. 747-755, Vol. 79, No. 2

Secondary Structure Components and Properties of the Melibiose Permease from Escherichia coli: A Fourier Transform Infrared Spectroscopy Analysis

Natàlia Dave,* Agnès Troullier,dagger Isabelle Mus-Veteau,Dagger Mireia Duñach,* Gérard Leblanc,Dagger and Esteve Padrós*

 *Unitat de Biofísica, Departament de Bioquímica i de Biologia Molecular, Facultat de Medicina, Universitat Autònoma de Barcelona, Bellaterra, Barcelona 08193, Spain;  dagger Laboratoire de Biophysique Moléculaire et Cellulaire, Unité Mixte de Recherche, Centre National de la Recherche Scientifique, Département de Biophysique Moléculaire et Structurale, CEA-Grenoble, 38054 Grenoble 09, France; and  Dagger Laboratoire de Physiologie des Membranes Cellulaires, Laboratoire de Recherche Correspondant du Commissariat à l'Energie Atomique 16V, Université de Nice Sophia-Antipolis, and Centre National de la Recherche Scientifique (ERS 1253), 06238 Villefranche sur Mer, France

The structure of the melibiose permease from Escherichia coli has been investigated by Fourier transform infrared spectroscopy, using the purified transporter either in the solubilized state or reconstituted in E. coli lipids. In both instances, the spectra suggest that the permease secondary structure is dominated by alpha -helical components (up to 50%) and contains beta -structure (20%) and additional components assigned to turns, 310 helix, and nonordered structures (30%). Two distinct and strong absorption bands are recorded at 1660 and 1653 cm-1, i.e., in the usual range of absorption of helices of membrane proteins. Moreover, conditions that preserve the transporter functionality (reconstitution in liposomes or solubilization with dodecyl maltoside) make possible the detection of two separate alpha -helical bands of comparable intensity. In contrast, a single intense band, centered at ~1656 cm-1, is recorded from the inactive permease in Triton X-100, or a merged and broader signal is recorded after the solubilized protein is heated in dodecyl maltoside. It is suggested that in the functional permease, distinct signals at 1660 and 1653 cm-1 arise from two different populations of alpha -helical domains. Furthermore, the sodium- and/or melibiose-induced changes in amide I line shape, and in particular, in the relative amplitudes of the 1660 and 1653 cm-1 bands, indicate that the secondary structure is modified during the early step of sugar transport. Finally, the observation that ~80% of the backbone amide protons can be exchanged suggests high conformational flexibility and/or a large accessibility of the membrane domains to the aqueous solvent.

Biophys J, August 2000, p. 747-755, Vol. 79, No. 2
© 2000 by the Biophysical Society   0006-3495/00/08/747/09  $2.00


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