PMID:10978174
| Citation |
Broos, J, Strambini, GB, Gonnelli, M, Vos, EP, Koolhof, M and Robillard, GT (2000) Sensitive monitoring of the dynamics of a membrane-bound transport protein by tryptophan phosphorescence spectroscopy. Biochemistry 39:10877-83 |
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| Abstract |
This paper presents a tryptophan phosphorescence spectroscopy study on the membrane-bound mannitol transporter, EII(mtl), from E. coli. The protein contains four tryptophans at positions 30, 42, 109, and 117. Phosphorescence decays in buffer at 1 degrees C revealed large variations of the triplet lifetimes of the wild-type protein and four single-tryptophan-containing mutants. They ranged from <70 microseconds for the tryptophan at position 109 to 55 ms for the residue at position 30, attesting to widely different flexibilities of the tryptophan microenvironments. The decay of all tryptophans is multiexponential, reflecting multiple stable conformations of the protein. Both mannitol binding and enzyme phosphorylation had large effects on the triplet lifetimes. Mannitol binding induces a more ordered structure near the mannitol binding site, and the decay becomes significantly more homogeneous. In contrast, enzyme phosphorylation induces a large relaxation of the protein structure at the reporter sites. The implications of these structural changes on the coupling mechanism between the transport and the phosphorylation activity of EII(mtl) are discussed. Taken as a whole, our data show that tryptophan phosphorescence spectroscopy is a very sensitive technique to explore conformational dynamics in membrane proteins. |
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| Keywords |
Binding Sites/genetics; Biological Transport/genetics; Carrier Proteins/chemistry; Carrier Proteins/genetics; Carrier Proteins/metabolism; Cysteine/chemistry; Cysteine/metabolism; Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli Proteins; Freezing; Glass; Histidine/chemistry; Histidine/metabolism; Luminescent Measurements; Mannitol/chemistry; Mannitol/metabolism; Membrane Proteins/chemistry; Membrane Proteins/genetics; Membrane Proteins/metabolism; Monosaccharide Transport Proteins; Mutagenesis, Site-Directed; Phosphoenolpyruvate Sugar Phosphotransferase System/chemistry; Phosphoenolpyruvate Sugar Phosphotransferase System/genetics; Phosphoenolpyruvate Sugar Phosphotransferase System/metabolism; Phosphorylation; Solutions; Spectrometry, Fluorescence/methods; Thermodynamics; Tryptophan/chemistry; Tryptophan/genetics |
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