Abstract:Optogenetics, the use of genetically encoded tools to control protein function
with light, can generate localized changes in signaling within living cells and animals.
For years it has been focused on channel proteins for neurobiology, but has recently
expanded to cover many different types of proteins, using a broad array of different
protein engineering approaches. These methods have largely been directed at proteins
involved in motility, cytoskeletal regulation and gene expression. This review provides a
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“…In addition to small molecule activation of protein function, tools for optical activation of protein function have received significant interest in recent years. 17,18 Small molecule control and light control complement each other and can be synergistically employed in a powerful fashion. 19,20 …”
Using small molecules to control the function of proteins in live cells with complete specificity is highly desirable, but challenging. Here we report a small molecule switch that can be used to control protein activity. The approach uses a phosphine-mediated Staudinger reduction to activate protein function. Genetic encoding of an ortho-azidobenzyloxycarbonyl amino acid using a pyrrolysyl tRNA synthetase/tRNACUA pair in mammalian cells enables the site-specific introduction of a small molecule-removable protecting group into the protein of interest. Strategic placement of this group renders the protein inactive until deprotection through a bioorthogonal Staudinger reduction delivers the active, wild-type protein. This developed methodology was applied to the conditional control of several cellular processes, including bioluminescence (luciferase), fluorescence (EGFP), protein translocation (nuclear localization sequence), DNA recombination (Cre), and gene editing (Cas9).
“…In addition to small molecule activation of protein function, tools for optical activation of protein function have received significant interest in recent years. 17,18 Small molecule control and light control complement each other and can be synergistically employed in a powerful fashion. 19,20 …”
Using small molecules to control the function of proteins in live cells with complete specificity is highly desirable, but challenging. Here we report a small molecule switch that can be used to control protein activity. The approach uses a phosphine-mediated Staudinger reduction to activate protein function. Genetic encoding of an ortho-azidobenzyloxycarbonyl amino acid using a pyrrolysyl tRNA synthetase/tRNACUA pair in mammalian cells enables the site-specific introduction of a small molecule-removable protecting group into the protein of interest. Strategic placement of this group renders the protein inactive until deprotection through a bioorthogonal Staudinger reduction delivers the active, wild-type protein. This developed methodology was applied to the conditional control of several cellular processes, including bioluminescence (luciferase), fluorescence (EGFP), protein translocation (nuclear localization sequence), DNA recombination (Cre), and gene editing (Cas9).
“…[39] Light-gated ion channels, notably channelrhodopsins, open to allow the flow of ions in response to illumination. Non-channel classes of light actuators and their application to cell motility have been recently reviewed[40]. …”
Section: Controlled Activation Of Protein Activitymentioning
“…One such protein is phytochrome B (PhyB) which undergoes a conformational change upon exposure to light of visible red wavelengths (âŒ650â670 nm) to heterodimerize with the transcription factor phytochrome-interacting factor 3 (PIF3). Unlike other photosensitive proteins, this dimerization can be reverted through exposure to longer wavelengths of light (âŒ700â750 nm), which induces monoisomerization of PhyB and releases PIF3, allowing for very precise control of protein activity [32,36]. …”
A major objective in biological research is to understand spatial and temporal requirements for any given gene, especially in dynamic processes acting over short periods, such as catalytically driven reactions, subcellular transport, cell division, cell rearrangement and cell migration. The interrogation of such processes requires the use of rapid and flexible methods of interfering with gene function. However, many of the most widely used interventional approaches, such as RNAi or CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated 9), operate at the level of the gene or its transcripts, meaning that the effects of gene perturbation are exhibited over longer time frames than the process under investigation. There has been much activity over the last few years to address this fundamental problem. In the present review, we describe recent advances in disruption technologies acting at the level of the expressed protein, involving inducible methods of protein cleavage, (in)activation, protein sequestration or degradation. Drawing on examples from model organisms we illustrate the utility of fast-acting techniques and discuss how different components of the molecular toolkit can be employed to dissect previously intractable biochemical processes and cellular behaviours.
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