RESUMEN
The Q photoproduct of bacteriorhodopsin (BR) is the basis of several biophotonic technologies that employ BR as the photoactive element. Several blue BR (bBR) mutants, generated by using directed evolution, were investigated with respect to the photochemical formation of the Q state. We report here a new bBR mutant, D85E/D96Q, which is capable of efficiently converting the entire sample to and from the Q photoproduct. At pH 8.5, where Q formation is optimal, the Q photoproduct requires 65 kJ mol(-1) of amber light irradiation (590 nm) for formation and 5 kJ mol(-1) of blue light (450 nm) for reversion, respectively. The melting temperature of the resting state and Q photoproduct, measured via differential scanning calorimetry, is observed at 100 °C and 89 °C at pH 8.5 or 91 °C and 82 °C at pH 9.5, respectively. We hypothesize that the protein stability of D85E/D96Q compared to other blue mutants is associated with a rapid equilibrium between the blue form E85(H) and the purple form E85(-) of the protein, the latter providing enhanced structural stability. Additionally, the protein is shown to be stable and functional when suspended in an acrylamide matrix at alkaline pH. Real-time photoconversion to and from the Q state is also demonstrated with the immobilized protein. Finally, the holographic efficiency of an ideal thin film using the Q state of D85E/D96Q is calculated to be 16.7%, which is significantly better than that provided by native BR (6-8%) and presents the highest efficiency of any BR mutant to date.
Asunto(s)
Bacteriorodopsinas/fisiología , Bacteriorodopsinas/química , Bacteriorodopsinas/genética , Rastreo Diferencial de Calorimetría , Calor , Concentración de Iones de Hidrógeno , Espectrofotometría UltravioletaRESUMEN
Functional analysis of the proteome is an essential part of genomic research. To facilitate different proteomic approaches, a MORF (moveable ORF) library of 5854 yeast expression plasmids was constructed, each expressing a sequence-verified ORF as a C-terminal ORF fusion protein, under regulated control. Analysis of 5573 MORFs demonstrates that nearly all verified ORFs are expressed, suggests the authenticity of 48 ORFs characterized as dubious, and implicates specific processes including cytoskeletal organization and transcriptional control in growth inhibition caused by overexpression. Global analysis of glycosylated proteins identifies 109 new confirmed N-linked and 345 candidate glycoproteins, nearly doubling the known yeast glycome.
Asunto(s)
Glicoproteínas/genética , Sistemas de Lectura Abierta , Proteoma , Levaduras/genética , Procesos de Crecimiento Celular/genética , Proteínas Fúngicas/análisis , Proteínas Fúngicas/genética , Biblioteca de Genes , Glicoproteínas/análisis , Proteómica/métodosAsunto(s)
Bacteriorodopsinas/genética , Evolución Molecular Dirigida/métodos , Sustitución de Aminoácidos , Bacteriorodopsinas/química , Bacteriorodopsinas/metabolismo , Clonación Molecular/métodos , Modelos Moleculares , Mutagénesis , Mutagénesis Sitio-Dirigida , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Mapeo Restrictivo , TermodinámicaRESUMEN
Bacteriorhodopsin (BR) is the photoactive proton pump found in the purple membrane of the salt marsh archaeon Halobacterium salinarum. Evolution has optimized this protein for high photochemical efficiency, thermal stability and cyclicity, as the organism must be able to function in a hot, stagnant and resource-limited environment. Photonic materials generated via organic chemistry have yet to surpass the native protein in terms of quantum efficiency or cyclicity. However, the native protein still lacks the overall efficiency necessary for commercial viability and virtually all successful photonic devices using bacteriorhodopsin are based on chemical or genetic variants of the native protein. We show that genetic engineering can provide significant improvement in the device capabilities of proteins and, in the case of bacteriorhodopsin, a 700-fold improvement has been realized in volumetric data storage. We conclude that semi-random mutagenesis and directed evolution will play a prominent role in future efforts in bioelectronic optimization.
