Rapid activation of the melibiose permease MelB immobilized on a solid-supported membrane.
Rapid solution exchange on a solid-supported membrane (SSM) is investigated using fluidic structures and a solid-supported membrane of 1 mm diameter in wall jet geometry. The flow is analyzed with a new technique based on specific ion interactions with the surface combined with an electrical measurement. The critical parameters affecting the time course of the solution exchange and the transfer function describing the time resolution of the SSM system are determined. The experimental data indicate that solution transport represents an intermediate situation between the plug flow and the Hagen-Poiseuille laminar flow regime. However, to a good approximation the rise of the surface concentration can be described by Hagen-Poiseuille flow with ideal mixing at the surface of the SSM. Using an improved cuvette design, solution exchange as fast as 2 ms was achieved at the surface of a solid-supported membrane. As an application of the technique, the rate constant of a fast electrogenic reaction in the melibiose permease MelB, a bacterial ( Escherichia coli) sugar transporter, is determined. For comparison, the kinetics of a conformational transition of the same transporter was measured using stopped-flow tryptophan fluorescence spectroscopy. The relaxation time constant obtained for the charge displacement agrees with that determined in the stopped-flow experiments. This demonstrates that upon sugar binding MelB undergoes an electrogenic conformational transition with a rate constant of k approximately 250 s (-1).
Department of Biophysical Chemistry, Max Planck Institute of Biophysics, D-60438 Frankfurt, Germany.
Juan J
JJ
Garcia-Celma
Benjamin
B
Dueck
Martin
M
Stein
Michela
M
Schlueter
Kerstin
K
Meyer-Lipp
Gerard
G
Leblanc
Klaus
K
Fendler
D004566:Electrodes; D004789:Enzyme Activation; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D027981:Symporters / Q000737:chemistry / Q000378:metabolism*; D013997:Time Factors
D004800:Enzymes, Immobilized; D027981:Symporters; C021548:melibiose permease