Changes in Secondary Structures and Acidic Side Chains of Melibiose Permease Upon Cosubstrates Binding

¹ Univ. Autònoma de Barcelona
² Commissariat à l'Energie Atomique Saclay

^* To whom correspondence should be addressed. E-mail: esteve.padros{at}uab.es.

Submitted on May 31, 2006
Revised on July 10, 2006
Accepted on 12 September 2006

	Abstract

Infrared difference spectroscopy analysis of the purified melibiose permease (MelB) of Escherichia coli reconstituted into liposomes was carried out as a function of the presence of the two symporter substrates (Na⁺, melibiose) in either H₂O or in D₂O media. Essentially, the data first show that addition of Na⁺ induces appearance of peaks assigned to changes in the environment and/or orientation of -helical domains of MelB. Likewise, melibiose addition in the presence of Na⁺ produces peaks corresponding to additional changes of -helix environment or tilt. In addition to these changes, a pair of peaks (1599 (+) cm^-1 / 1576 (-) cm^-1) appearing in the Na⁺-induced difference spectrum is assigned to the antisymmetric stretching of COO^- groups, since they show practically no shift upon H/D exchange. It is proposed that these acidic groups participate in Na⁺ coordination. A corresponding pair of peaks, again fairly insensitive to H/D substitution (1591 (-) cm^-1/ 1567 (+) cm^-1), appears in the melibiose-induced difference spectra, and may again be assigned to COO^- groups. The latter carboxyl groups may correspond to part or all of the acidic residues interacting with Lys or Arg in the resting state that become free upon melibiose binding.

Key Words: ATR-FTIR, carboxylic side chains, conformational changes, difference spectra, infrared spectroscopy, secondary structure

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