RESUMEN
Light-sensing protein domains that link an exogenous light signal to the activity of an enzyme have attracted much attention for the engineering of new regulatory mechanisms into proteins and for studying the dynamic behavior of intracellular reactions and reaction cascades. Light-oxygen-voltage (LOV) photoreceptors are blue-light-sensing modules that have been intensely characterized for this purpose and linked to several proteins of interest. For the successful application of these tools, it is crucial to identify appropriate fusion strategies for combining sensor and enzyme domains that sustain activity and light-induced responsivity. Terminal fusion of LOV domains is the natural strategy; however, this is not transferrable to T7 RNA polymerase because both of its termini are involved in catalysis. It is shown herein that it is possible to covalently insert LOV domains into the polymerase protein, while preserving its activity and generating new light-responsive allosteric coupling.
Asunto(s)
Bacteriófago T7/enzimología , ARN Polimerasas Dirigidas por ADN/química , Fotorreceptores de Plantas/química , Proteínas Recombinantes de Fusión/química , Transcripción Genética/efectos de la radiación , Proteínas Virales/química , Secuencia de Aminoácidos , Avena/química , ARN Polimerasas Dirigidas por ADN/genética , Luz , Simulación de Dinámica Molecular , Fotorreceptores de Plantas/genética , Fotorreceptores de Plantas/efectos de la radiación , Dominios Proteicos/efectos de la radiación , Ingeniería de Proteínas , ARN/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/efectos de la radiación , Proteínas Virales/genéticaRESUMEN
In nature, a multitude of mechanisms have emerged for regulating biological processes and, specifically, protein activity. Light as a natural regulatory element is of outstanding interest for studying and modulating protein activity because it can be precisely applied with regard to a site of action, instant of time, or intensity. Naturally occurring photoresponsive proteins, predominantly those containing a light-oxygen-voltage (LOV) domain, have been characterized structurally and mechanistically and also conjugated to various proteins of interest. Immediate advantages of these new photoresponsive proteins such as genetic encoding, no requirement of chemical modification, and reversibility are paid for by difficulties in predicting the envisaged activity or type and site of domain fusion. In this article, we summarize recent advances and give a survey on currently available design concepts for engineering photoswitchable proteins.