Spatiotemporal Control of Protein Activity through Optogenetic Allosteric Regulation

Summary

This protocol describes the application of an engineered blue-light-activated allosteric switch (LightR) domain for reversible spatiotemporal control of protein activity. Utilizing Src tyrosine kinase as a model, this study offers an elaborate protocol for developing and characterizing light-regulated Src (LightR-Src). It demonstrates the versatility of this approach across enzyme classes.

Abstract

Optogenetics offers the potential for mimicking complex spatiotemporal control of enzyme activity down to a subcellular resolution. However, most optogenetic approaches often face significant challenges in integrating multiple capabilities in a single tool applicable to a wide range of target proteins. Achieving precise control over ON/OFF kinetics, ensuring minimum leakiness in the dark, and demonstrating efficient performance in mammalian cells with subcellular precision are some of the most common challenges faced in this field. A promising solution lies in the application of rationally designed light-sensitive domains to allosterically control protein activity. Using that strategy, we generated an optogenetic method combining all the desired features. The approach involves the incorporation of the Light-regulated allosteric switch module (LightR) in the target protein to regulate enzyme activity using blue (465 nm) light. The LightR domain is generated by linking two Vivid (VVD) photoreceptor domains, creating a light-sensitive clamp that can be incorporated into a small flexible loop within the catalytic domain of an enzyme. In its dark state, LightR clamp is open, thus distorting the enzyme's catalytic domain and inactivating it. Upon exposure to blue light, the LightR domain closes and restores the catalytic domain's structure and enzyme activity. In this manuscript, we discuss design strategies to generate a light-regulated protein kinase and demonstrate its control by blue light, reversibility, kinetics, and precise regulation at the subcellular level, enabling tight spatiotemporal precision. Utilizing Src tyrosine kinase as a model, we showcase a protocol for effectively regulating LightR-Src kinase activity. We also demonstrate LightR applicability across different enzyme classes, expanding the utility of the tool system in addressing mechanistic questions of signaling pathways in different diseases.

Introduction

The ability of the cell to interpret external signals and convert them into specific responses in physiological or pathological contexts is directed by dedicated groups of proteins. The contribution of any protein to such complex responses is often defined by its subcellular location, level of expression, and the timing of transient, sustained, or oscillatory activation. Dissecting the role of these individual parameters in the regulation of signaling demands methods capable of replicating intricate spatiotemporal control of protein activity at the subcellular level. Traditional techniques such as genetic manipulation and small molecule inhibitors fall short in this r....

Protocol

1. Design and development of LightR ( Figure 1)

1. Strategic planning
   NOTE: The LightR domain is comprised of two tandemly connected Vivid (VVD) photoreceptor domains from Neurospora crassa^13,14,15,16. VVDs homodimerize in the presence of light, and such dimerization involves a conformational change that brings.......

Discussion

Our study presents an optogenetic approach for the investigation of diverse signaling pathways and demonstrates its wide applicability in addressing different biological questions. The LightR tool system provides several essential advantages: (1) Allosteric regulation of protein activity, (2) Tight temporal control of activity that can be tuned to achieve different kinetics of activation and inactivation, (3) Spatial resolution of activity at the subcellular level, (4) Specificity of signaling modulation and biological a.......

Materials

Name	Company	Catalog Number	Comments
#1.5 Glass Coverslips 25 mm Round	Warner Instruments	64-0715
1.5 mL Tubes	USA Scientific	cc7682-3394
2x Laemmli Buffer			For 500 mL: 5.18 g of  Tris-HCL, 131.5 mL of glycerol, 52.5 mL of 20% SDS, 0.5 g of bromophenol blue, final pH 6.8
4-20% Mini-PROTEAN TGX Precast Gel	Biorad	4561096
5x Phusion Plus Buffer	Thermo Scientific	F538L
60 LED Microscope Ring Light	Boli Optics	ML46241324	Blue LED, 60 mm diameter, 5 W
Agarose	GoldBiotech	A-201
Anti-Erk 1/2 Antibody	Cell Signaling	9102
Anti-GAPDH Antibody	invitrogen	AM4300
Anti-GFP	Clontech	632380
Anti-mCherry Antibody	invitrogen	M11217
Anti-MEK	Cell Signaling	9122
Anti-p130Cas	BD Biosciences	610271
Anti-paxillin	Cell Signaling	2542
Anti-phospho-Erk 1/2 T202/Y204 Antibody	Cell Signaling	9101
Anti-phospho-pY249 p130Cas	Cell Signaling	4014
Anti-phospho-Y118 Paxillin	Cell Signaling	2541
Anto-phospho-S217/221 MEK	Cell Signaling	9121
Arduino Compatable Power Supply	Corporate Computer	LJH -186
Arduino Uno Rev3	Arduino	‎A000066
Attofluor Cell Chamber	invitrogen	A7816
Benchmark Fetal Bovine Serum (FBS)	Gemini Bio-products	100-106	Heat Inactivated Triple 0.1 µm sterile-filtered
bRaf-V600E-Venus			Gift from Dr. John O'Bryan, MUSC
BSA	GoldBiotech	A-420
Carbenicillin (Disodium)	Gold Biotechnology	C-103-25
CellMask Deep Red plasma membrane dye	invitrogen	c10046
Colony Screen MasterMix	Genesee	42-138
DH5a competent cells	NEB	C2987H
DMEM	Corning	15-013-CV
DNA Ladder	GoldBio	D010-500
dNTPs	NEB	N04475
Dpn1 Enzyme	NEB	R01765
DTT	GoldBio	DTT10	DL-Dithiothreitol, Cleland's Reagents
EGTA	Acros	409910250
FastLightR-bRaf-mVenus	Addgene	#162155
Fibronectin from bovine plasma	Sigma	F1141
FuGENE(R) 6 Transfection Reagent	Promega	E2692	Transfection reagent
Gel Green Nucleic Acid Stain	GoldBio	G-740-500
Gel Loading Dye Purple 6x	NEB	B7024A
GeneJET Gel extraction Kit	Thermo Scientific	K0692	Gel Extraction Kit
GeneJET Plasmid Miniprep Kit	Thermo Scientific	K0502
Glutamax	Gibco	35050-061	GlutaMAX-l (100x) 100 mL
HEK 293T Cells	ATCC	CRL-11268
HeLa Cells	ATCC	CRM-CCL-2
HEPES	Fischer	BP310-500
Iot Relay	Digital Loggers	DLI 705020645490	AC/DC control relay for illumination
Kanamycin Monosulfate	Gold Biotechnology	K-120-25
KCl	Sigma	P-4504
L-15 1x	Corning	10-045-CV
LB Agar	Fisher	BP1425-2
LED Grow Light System	HQRP	884667106091218	LED panel lamp system
LightR-bRaf-mVenus	Addgene	#162154
LightR-iCre-miRFP670	Addgene	#162158
MATLAB	Mathworks	R2024a	Software for running CellGEO Scripts
Metamorph	Molecular Devices		Imaging Analysis Software
MgCl2	Fisher Chemical	M33-500
Mineral Oil	Sigma	M5310
MiniPrep Kit	Gene Choice	96-308
Mini-PROTEAN TGX Precast Gels 12 well	Bio-Rad	4561085
Molecular Biology Grade Water	Corning	46-000-CV
Multiband Polychroic Mirror	89903BS	Chroma
NaCl	Fisher Chemical	S271-3
PBS w/o Ca and Mg	Corning	21-031-CV
pCAG-iCre	Addgene	#89573
pcDNA3.1_Floxed-STOP mCherry	Addgene	#122963
pCEFL-ERK2			Gift from Dr. Channing Der's Lab, UNC
PCR Tubes	labForce	1149Z65	0.2 mL 8-Strip Tubes and Caps, Rigid Strip Individually Attached Dome Caps
Phusion Plus DNA Polymerase	Thermo Scientific	F630S
pmiRFP670-N1	Addgene	#79987
Polygon 400 Patterned Illuminator	Mightex	DSI-G-00C
Primers	IDT
PVDF Membranes	BioRad	1620219	Immun-Blot PVDF/Filter Paper Sandwiches
T0.25% Trypsin, 2.21 mM, eDTA, 1x [-] sodium	Corning	25-053-CI
Tris-Acetate-EDTA (TAE) 50x	Fischer	BP1332-1	For electrophoresis
UPlanSApo 40x Microscope Objective	Olympus	1-U2B828
USB TTL Box	National Instruments	6501	For TTL interface
β-Mercaptoethanol	Fisher Chemical	O3446I-100