Abstract
The light-driven proton pump bacteriorhodopsin (BR) from the extreme halophilic archaeon Halobacterium salinarum is a retinal-binding protein, which forms highly ordered and thermally stable 2D crystals in native membranes (termed purple membranes). BR and purple membranes (PMs) have been and are still being intensively studied by numerous researchers from different scientific disciplines. Furthermore, PMs are being successfully used in new, emerging technologies such as bioelectronics and bionanotechnology. Most published studies used the wild-type form of BR, because of the intrinsic difficulty to produce genetically modified versions in purple membranes homologously. However, modification and engineering is crucial for studies in basic research and, in particular, to tailor BR for specific applications in applied sciences. We present an extensive and detailed protocol ranging from the genetic modification and cultivation of H. salinarum to the isolation, and biochemical, biophysical and functional characterization of BR and purple membranes. Pitfalls and problems of the homologous expression of BR versions in H. salinarum are discussed and possible solutions presented. The protocol is intended to facilitate the access to genetically modified BR versions for researchers of different scientific disciplines, thus increasing the application of this versatile biomaterial.
Highlights
The use of BR to study proton transport across membranes in different time scales using time-resolved serial femtosecond crystallography at X-ray free electron lasers [17,18,19,20] and as an alignment tool for NMR studies [21] highlights its significant contribution to emerging technologies
BR natively occurs in patches in the membrane of H. salinarum, called purple membranes (PMs)
A proton is transported from the intra- to the extracellular side of the protein, establishing an electric current. This photoelectric effect is applied in photovoltaic devices [28], optical switches [29], optical logic gates [30], light sensors [31], single electron [31]- and field effect transistors [32], chemical sensors [33,34], optical microcavities [35] and may even lead to protein-based retinal implants [36]
Summary
Since its discovery in 1971 [1], the light-driven proton pump bacteriorhodopsin (BR) from the extreme halophilic archaeon Halobacterium salinarum (previously known as Halobacterium halobium) has become an intensively studied model membrane protein for proton translocation across biological membranes [2,3,4,5], photochemical events in proteins [6,7,8,9], photophosphorylation [10,11,12] and structural studies [13,14,15,16]. Upon illumination with light at 568 nm, the covalently bound retinal chromophore of BR undergoes isomerization by absorbing a photon This induces a photocycle, involving a series of conformational changes and protonation states through different, consecutive states. A proton is transported from the intra- to the extracellular side of the protein, establishing an electric current (photocurrent) This photoelectric effect is applied in photovoltaic devices [28], optical switches [29], optical logic gates [30], light sensors [31], single electron [31]- and field effect transistors [32], chemical sensors [33,34], optical microcavities [35] and may even lead to protein-based retinal implants [36]. Methods for the cultivation of H. salinarum [61,62] and the isolation of purple membranes [63] were established early in BR research and were adapted in many scientific publications, including this protocol
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