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J. Biol. Chem., Vol. 280, Issue 42, 35148-35156, October 21, 2005

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Substrate-induced Conformational Changes in the Membrane-embedded IIC^mtl-domain of the Mannitol Permease from Escherichia coli, EnzymeII^mtl, Probed by Tryptophan Phosphorescence Spectroscopy^*

Gertjan Veldhuis1, Edi Gabellieri, Erwin P. P. Vos, Bert Poolman, Giovanni B. Strambini2, and Jaap Broos3

From the Department of Biochemistry and Biophysical Chemistry, Groningen Biomolecular Science and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands and the Istituto di Biofisica, Consiglio Nazionale delle Ricerche, Area della Ricerca, Via Moruzzi 1, 56124 Pisa, Italy

Membrane-bound transport proteins are expected to proceed via different conformational states during the translocation of a solute across the membrane. Tryptophan phosphorescence spectroscopy is one of the most sensitive methods used for detecting conformational changes in proteins. We employed this technique to study substrate-induced conformational changes in the mannitol permease, EnzymeII^mtl, of the phosphoenolpyruvate-dependent phosphotransferase system from Escherichia coli. Ten mutants containing a single tryptophan were engineered in the membrane-embedded IIC^mtl-domain, harboring the mannitol translocation pathway. The mutants were characterized with respect to steady-state and time-resolved phosphorescence, yielding detailed, site-specific information of the Trp microenvironment and protein conformational homogeneity. The study revealed that the Trp environments vary from apolar, unstructured, and flexible sites to buried, highly homogeneous, rigid peptide cores. The most remarkable example of the latter was observed for position 97, because its long sub-second phosphorescence lifetime and highly structured spectra in both glassy and fluid media imply a well defined and rigid core around the probe that is typical of -sheet-rich structural motifs. The addition of mannitol had a large impact on most of the Trp positions studied. In the case of position 97, mannitol binding induced partial unfolding of the rigid protein core. On the contrary, for residue positions 126, 133, and 147, both steady-state and time-resolved data showed that mannitol binding induces a more ordered and homogeneous structure around these residues. The observations are discussed in context of the current mechanistic and structural model of EII^mtl.

Received for publication, June 28, 2005 , and in revised form, August 4, 2005.

^* This work was supported in part by the Netherlands Organization of Scientific Research (NWO-CW) (Grant JC 99-535) and the Italian National Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Received a Netherlands Organization of Scientific Research (NWO-CW) travel grant.

² To whom correspondence may be addressed. Tel.: 39-050-315-3046; Fax: 39-050-315-2760; E-mail: giovanni.strambini{at}ib.pi.cnr.it. ³ To whom correspondence may be addressed. Tel.: 31-(0)50–363-4277; Fax: 31-(0)50–363-4800; E-mail: j.broos{at}rug.nl.

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