High-throughput screening of bacteriorhodopsin mutants in whole cell pastes.
A high-throughput screening method has been developed which enables functional analysis of bacteriorhodpsin in whole cell pastes. Reflectance spectra, from as little as 5 ml of Halobacterium salinarum cells, show close correspondence to that obtained from the purified purple membrane (PM), containing bacteriorhodopsin (BR) as the sole protein component. We demonstrate accurate quantification of BR accumulation by ratiometric analysis of BR (A(max) 568) and a membrane-bound cytochrome (A(max) 410). In addition, ground-state light- and dark-adapted (LA and DA, respectively) spectral differences were determined with high accuracy and precision. Using cells expressing the BR mutant D85N, we monitored transitions between intermediate-state homologues of the reprotonation phase of the light-activated proton pumping mechanism. We demonstrate that phenotypes of three mutants (D85N/T170C, D85N/D96N, and D85N/R82Q) previously characterized for their effect on photocycle transitions are reproduced in the whole cell samples. D85N/T170C stabilizes accumulation of the N state while D85N/D96N accumulates no N state. D85N/R82Q was found to have perturbed the pK(a) of M accumulation. These studies illustrate the correspondence between pH-dependent ground-state transitions accessed by D85N and the transitions accessed by the wild-type protein following photoexcitation. We demonstrate that whole cell reflectance spectroscopy can be used to efficiently characterize the large numbers of mutants generated by engineering strategies that exploit saturation mutagenesis.
Martinez
Lynell C
LC
Department of Physiology and Biophysics and the Neurosciences Program, The University of Miami School of Medicine, Miami, FL 33101, USA.
Turner
George J
GJ
D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D019617:Halobacterium salinarum / Q000737:chemistry* / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D018717:Purple Membrane / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013053:Spectrophotometry
D011994:Recombinant Proteins; D001436:Bacteriorhodopsins