Substrate-induced Conformational Changes in the Membrane-embedded IICmtl-domain of the Mannitol Permease from Escherichia coli, EnzymeIImtl, Probed by Tryptophan Phosphorescence Spectroscopy

Gertjan Veldhuis,Edi Gabellieri,Erwin P.P Vos,Bert Poolman,Giovanni B Strambini,Jaap Broos

doi:10.1074/jbc.m507061200

Gertjan Veldhuis, Edi Gabellieri + Show 4 more

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https://doi.org/10.1074/jbc.m507061200

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Abstract

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 beta-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).

Highlights

A phoA fusion study and hydropathy analysis of the IICmtl-domain resulted in a topology model with three small periplasmic loops, two large cytoplasmic loops, and six putative membrane-spanning helices [6]
The observation of multiple ␶P values for a single Trp position reflects the presence of different protein conformational states, which do not rapidly interchange on the time scale of ␶P
The present study extends this approach with ten single-Trp mutants, containing tryptophans in either putative transmembrane helices or cytoplasmic loops of the IICmtl-domain (Fig. 1)

Summary

Introduction

A phoA fusion study and hydropathy analysis of the IICmtl-domain resulted in a topology model with three small periplasmic loops, two large cytoplasmic loops, and six putative membrane-spanning helices [6]. Of the available spectroscopic techniques, Trp phosphorescence spectroscopy is one of the most sensitive approaches used to study changes in protein conformation, due to the extremely slow (radiative) de-excitation rate of the triplet excited state (ϳ0.2 sϪ1). This makes Trp phosphorescence 108 times more sensitive for quenching processes than Trp fluorescence. The present study extends this approach with ten single-Trp mutants, containing tryptophans in either putative transmembrane helices or cytoplasmic loops of the IICmtl-domain (Fig. 1). The positions in the proposed topology model are helix II (Trp66), a loop protruding the membrane (Trp-97), the following cytoplasmic loop (Trp-114 and Trp-126), helix III (Trp-133 and Trp-147), helix IV (Trp-167), and the following loop (Trp-188 and Trp-198)

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