Optical modulation of proteins provides superior spatiotemporal resolution for understanding biological processes, and photoswitches built on light-sensitive proteins have been significantly ...advancing neuronal and cellular studies. Small molecule photoswitches could complement protein-based switches by mitigating potential interference and affording high specificity for modulation sites. However, genetic encodability and responsiveness to nonultraviolet light, two desired properties possessed by protein photoswitches, are challenging to be engineered into small molecule photoswitches. Here we developed a small molecule photoswitch that can be genetically installed onto proteins in situ and controlled by visible light. A pentafluoro azobenzene-based photoswitchable click amino acid (F-PSCaa) was designed to isomerize in response to visible light. After genetic incorporation into proteins via the expansion of the genetic code, F-PSCaa reacts with a nearby cysteine within the protein generating an azo bridge in situ. The resultant bridge is switchable by visible light and allows conformation and binding of CaM to be regulated by such light. This photoswitch should prove valuable in optobiology for its minimal interference, site flexibility, genetic encodability, and response to the more biocompatible visible light.
Chemical reactivity is essential for functional modification of biomolecules with small molecules and the development of covalent drugs. The reactivity between a chemical functional group of a small ...molecule and that of a large biomolecule cannot be reliably predicted from the reactivity of the corresponding functional groups separately installed on two small molecules, because the proximity effect on reactivity resulting from the binding of the small molecule to the biomolecule is challenging to achieve by mixing two small molecules. Here we present a new strategy to determine the chemical reactivity of two functional groups in the context of close proximity afforded by proteins. The functional groups to be tested were separately installed at the interface of two interacting proteins in the format of amino acid side chains via the expansion of the genetic code. Reaction of the two functional groups resulted in covalent cross-linking of interacting proteins, readily detectable by gel electrophoresis. Using this strategy, we evolved new synthetases to genetically encode N ε-fluoroacetyllysine (FAcK), an isosteric fluorine analogue of acetyllysine. We demonstrated that fluoroacetamide installed on FAcK, previously thought inert to biological functional groups, actually reacted with the thiol group of cysteine when in proximity. This strategy should be valuable for accurately evaluating chemical reactivity of small molecules toward large biomolecules, which will help avoid undesired side reactions of drugs and expand the repertoire of functional groups to covalently target biomolecules.
Access to phosphoproteins with stoichiometric and site-specific phosphorylation status is key to understanding the role of protein phosphorylation. Here we report an efficient method to generate ...pure, active phosphotyrosine-containing proteins by genetically encoding a stable phosphotyrosine analog that is convertible to native phosphotyrosine. We demonstrate its general compatibility with proteins of various sizes, phosphotyrosine sites and functions, and reveal a possible role of tyrosine phosphorylation in negative regulation of ubiquitination.
The ability to reversibly control protein structure and function with light would offer high spatiotemporal resolution for investigating biological processes. To confer photoresponsiveness on general ...proteins, we genetically incorporated a set of photoswitchable click amino acids (PSCaas), which contain both a reversible photoswitch and an additional click functional group for further modifications. Orthogonal tRNA‐synthetases were evolved to genetically encode PSCaas bearing azobenzene with an alkene, keto, or benzyl chloride group in E. coli and in mammalian cells. After incorporation into calmodulin, the benzyl chloride PSCaa spontaneously generated a covalent protein bridge by reacting with a nearby cysteine residue through proximity‐enabled bioreactivity. The resultant azobenzene bridge isomerized in response to light, thereby changing the conformation of calmodulin. These genetically encodable PSCaas will prove valuable for engineering photoswitchable bridges into proteins for reversible optogenetic regulation.
Click and switch: Photoswitchable click amino acids (PSCaas) contain a photoisomerizable azobenzene and an additional click functional group. After being incorporated into proteins, the PSCaa spontaneously generates a protein bridge with a nearby cysteine residue through proximity‐enabled bioreactivity. The bridge isomerizes in response to light, thereby altering protein conformation. These PSCaas may enable optogenetic control of proteins in situ.
Engineering light-sensitivity into proteins has wide ranging applications in molecular studies and neuroscience. Commonly used tethered photoswitchable ligands, however, require solvent-accessible ...protein labeling, face structural constrains, and are bulky. Here, we designed a set of optocontrollable NMDA receptors by directly incorporating single photoswitchable amino acids (PSAAs) providing genetic encodability, reversibility, and site tolerance. We identified several positions within the multi-domain receptor endowing robust photomodulation. PSAA photoisomerization at the GluN1 clamshell hinge is sufficient to control glycine sensitivity and activation efficacy. Strikingly, in the pore domain, flipping of a M3 residue within a conserved transmembrane cavity impacts both gating and permeation properties. Our study demonstrates the first detection of molecular rearrangements in real-time due to the reversible light-switching of single amino acid side-chains, adding a dynamic dimension to protein site-directed mutagenesis. This novel approach to interrogate neuronal protein function has general applicability in the fast expanding field of optopharmacology.
Fluorescence labelling of an intracellular biomolecule in native living cells is a powerful strategy to achieve in-depth understanding of the biomolecule's roles and functions. Besides being nontoxic ...and specific, desirable labelling probes should be highly cell permeable without nonspecific interactions with other cellular components to warrant high signal-to-noise ratio. While it is critical, rational design for such probes is tricky. Here we report the first predictive model for cell permeable background-free probe development through optimized lipophilicity, water solubility and charged van der Waals surface area. The model was developed by utilizing high-throughput screening in combination with cheminformatics. We demonstrate its reliability by developing CO-1 and AzG-1, a cyclooctyne- and azide-containing BODIPY probe, respectively, which specifically label intracellular target organelles and engineered proteins with minimum background. The results provide an efficient strategy for development of background-free probes, referred to as 'tame' probes, and novel tools for live cell intracellular imaging.
Continued interest in protein therapeutics has motivated the development of improved bioanalytical tools to support development programs. LC-MS offers specificity, sensitivity, and multiplexing ...capabilities without the need for target-specific reagents, making it a valuable alternative to ligand binding assays. Immunoaffinity purification (IP) and enzymatic digestion are critical, yet extensive and time-consuming components of the "gold standard" bottom-up approach to LC-MS-based protein quantitation. In the present work, commercially available technology, based on membrane-immobilized reagents in spin column and plate format, is applied to reduce IP and digestion times from hours to minutes. For a standard monoclonal antibody, the lower limit of quantitation was 0.1 ng μL
−1
compared to 0.05 ng μL
−1
for the standard method. A pharmacokinetics (PK) study dosing Herceptin in rat was analyzed by both the membrane and the standard method with a total sample processing time of 4 h and 20 h, respectively. The calculated concentrations at each time point agreed within 8% between both methods, and PK values including area under the curve (AUC), half-life (T1/2), mean residence time (MRT), clearance (CL), and volume of distribution (Vdss) agreed within 6% underscoring the utility of the membrane methodology for quantitative bioanalysis workflows.
Rapid spin membrane technology decreases the time for IP and digestion of therapeutic proteins.
Protein-protein interactions (PPIs) play central roles in orchestrating biological processes. While some PPIs are stable, many important ones are transient and hard to detect with conventional ...approaches. We developed ReBiL, a recombinase enhanced bimolecular luciferase complementation platform, to enable detection of weak PPIs in living cells. ReBiL readily identified challenging transient interactions between an E3 ubiquitin ligase and an E2 ubiquitin-conjugating enzyme. ReBiL's ability to rapidly interrogate PPIs in diverse conditions revealed that some stapled α-helical peptides, a class of PPI antagonists, induce target-independent cytosolic leakage and cytotoxicity that is antagonized by serum. These results explain the requirement for serum-free conditions to detect stapled peptide activity, and define a required parameter to evaluate for peptide antagonist approaches. ReBiL's ability to expedite PPI analysis, assess target specificity and cell permeability, and reveal off-target effects of PPI modifiers should facilitate the development of effective, cell-permeable PPI therapeutics and the elaboration of diverse biological mechanisms.
Click the switch: By using a photoswitchable click amino acid (PSCaa) a light‐induced intramolecular thiol‐ene click reaction with a neighboring cysteine under very mild conditions results in an ...azobenzene bridge (see figure). By expanding the genetic code for PSCaa the specific incorporation of photoswitch units into proteins in living cells can result in an exciting approach for studying light‐controllable activity, in vivo.
Over the past decade, photoswitchable molecules have been emerging as attractive tools for investigating biological processes with spatiotemporal resolution in a minimally invasive fashion. ...Photoswitches built on light-sensitive proteins or domains have significantly advanced neuronal and cellular studies. To install photosensitivity to general proteins and to enable high specificity for modulation, photoswitchable click amino acids (PSCaas) based on azobenzene have been developed and recently genetically incorporated into proteins via the expansion of the genetic code. PSCaas allow targeting selected sites in a protein for high specificity and are generally applicable to various proteins. In addition, PSCaas contain a click functional group, which selectively reacts with an appropriately positioned cysteine forming a photocontrollable bridge on the protein in situ. The photocontrollable bridge enables reversible modulation of the secondary structure of the spanned region and thus the function of the protein. In this chapter we describe the design and genetic encoding of PSCaa. Protocols are presented for incorporating PSCaa into a model protein calmodulin to build the bridge followed by photocontrol of calmodulin's conformation and binding function.
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