A general method for chemogenetic control of peptide function.

Natural or engineered peptides serve important biological functions. A general approach to achieve chemical-dependent activation of short peptides will be valuable for spatial and temporal control of cellular processes. Here we present a pair of chemically activated protein domains (CAPs) for controlling the accessibility of both the N- and C-terminal portion of a peptide. CAPs were developed through directed evolution of an FK506-binding protein. By fusing a peptide to one or both CAPs, the function of the peptide is blocked until a small molecule displaces them from the FK506-binding protein ligand-binding site. We demonstrate that CAPs are generally applicable to a range of short peptides, including a protease cleavage site, a dimerization-inducing heptapeptide, a nuclear localization signal peptide, and an opioid peptide, with a chemical dependence up to 156-fold. We show that the CAPs system can be utilized in cell cultures and multiple organs in living animals.

Measurement of α-Synuclein Dynamics In Vivo Using Microdialysis with a Novel Homogeneous Immunoassay.

Understanding the regulation of α-synuclein release could be important in better understanding Parkinson's disease development, progression, and treatment. Advances in such studies are hindered by technical challenges that limit the ability to monitor α-synuclein concentration in vivo. We developed a novel α-synuclein microdialysis method coupled with a specific and sensitive immunoassay that requires a small sample volume (1 µL). Using this method, basal α-synuclein level was estimated at 254 ± 78 pM in the striatum of freely moving mice. Additionally, we observed that potassium (75 mM) and nicotine (0.5 mg/kg) administration significantly increased α-synuclein in dialysates. These results provide evidence that the methods we report here can be useful to investigate the physiological roles of α-synuclein and support the idea that α-synuclein is secreted to the extracellular space in a neuronal activity-dependent manner.

SPARK: A Transcriptional Assay for Recording Protein-Protein Interactions in a Defined Time Window.

Protein-protein interactions (PPIs) are ubiquitously involved in cellular processes such as gene expression, enzymatic catalysis, and signal transduction. To study dynamic PPIs, real-time methods such as Förster resonance energy transfer and bioluminescence resonance energy transfer can provide high temporal resolution, but they only allow PPI detection in a limited area at a time and do not permit post-PPI analysis or manipulation of the cells. Integration methods such as the yeast two-hybrid system and split protein systems integrate PPI signals over time and allow subsequent analysis, but they lose information on dynamics. To address some of these limitations, an assay named SPARK (Specific Protein Association tool giving transcriptional Readout with rapid Kinetics) has recently been published. Similar to many existing integrators, SPARK converts PPIs into a transcriptional signal. SPARK, however, also adds blue light as a co-stimulus to achieve temporal gating; SPARK only records PPIs during light stimulation. Here, we describe the procedures for using SPARK assays to study a dynamic PPI of interest, including designing DNA constructs and optimization in HEK293T/17 cell cultures. These protocols are generally applicable to various PPI partners and can be used in different biological contexts. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Designing DNA constructs for SPARK Basic Protocol 2: Performing the SPARK assay in HEK293T/17 cell cultures Support Protocol 1: Lentivirus preparation Support Protocol 2: Immunostaining of SPARK components.

Circularly permuted AsLOV2 as an optogenetic module for engineering photoswitchable peptides.

We re-engineered a commonly-used light-sensing protein, AsLOV2, using a circular permutation strategy to allow photoswitchable control of the C-terminus of a peptide. We demonstrate that the circularly permuted AsLOV2 can be used on its own or together with the original AsLOV2 for enhanced caging. In summary, circularly permuted AsLOV2 could expand the engineering capabilities of optogenetic tools.