Mechanical and Optical Response of Proteins to Force Studied with Single Molecule Magnetic Tweezers
Mentor 1
Ionel Popa
Location
Union Wisconsin Room
Start Date
5-4-2019 1:30 PM
End Date
5-4-2019 3:30 PM
Description
The objective of this project is to establish a system that combines single molecule magnetic tweezers with two-photon FRET micro-spectroscopy. We use a polyprotein that has been successfully engineered and expressed, containing the following domains from N to C: HaloTag-(I27)-(I27)-SpyTag-(I27)-(I27)-AviTag as well as another protein, eGFP-SpyCatcher. The HaloTag and AviTag are used to tether this molecule using magnetic tweezers, and the four I27 domains (from muscle protein titin) produce a mechanical fingerprint. SpyTag-SpyCatcher form a covalent isopeptide bond and allow the attachment of fluorescent proteins sideways, such that they cannot experience the force placed on the tethered protein. This type of attachment is crucial, as fluorescent proteins simply placed in series would unfold and bleach. We also implemented a system based on the binding between fluorescently tagged anitbodies and bacterial protein L. In this case, the mechanical fingerprint comes from the unfolding of protein L domains while the fluorescent signal comes from eGFP. We anticipate that this new technology will foster an entirely new approach – 3D force spectroscopy, which will evolve into an invaluable tool in the study of biological processes such as cellular mechano-transduction, sensing, and communication. Additionally, it will provide key insights into the reduced dimensionality of ligand binding and enzymatic reactions under normal physiological conditions and in disease. The immediate outcome will be to establish the parameters needed to operate this new instrument and to demonstrate the new technique on a simple protein construct.
Mechanical and Optical Response of Proteins to Force Studied with Single Molecule Magnetic Tweezers
Union Wisconsin Room
The objective of this project is to establish a system that combines single molecule magnetic tweezers with two-photon FRET micro-spectroscopy. We use a polyprotein that has been successfully engineered and expressed, containing the following domains from N to C: HaloTag-(I27)-(I27)-SpyTag-(I27)-(I27)-AviTag as well as another protein, eGFP-SpyCatcher. The HaloTag and AviTag are used to tether this molecule using magnetic tweezers, and the four I27 domains (from muscle protein titin) produce a mechanical fingerprint. SpyTag-SpyCatcher form a covalent isopeptide bond and allow the attachment of fluorescent proteins sideways, such that they cannot experience the force placed on the tethered protein. This type of attachment is crucial, as fluorescent proteins simply placed in series would unfold and bleach. We also implemented a system based on the binding between fluorescently tagged anitbodies and bacterial protein L. In this case, the mechanical fingerprint comes from the unfolding of protein L domains while the fluorescent signal comes from eGFP. We anticipate that this new technology will foster an entirely new approach – 3D force spectroscopy, which will evolve into an invaluable tool in the study of biological processes such as cellular mechano-transduction, sensing, and communication. Additionally, it will provide key insights into the reduced dimensionality of ligand binding and enzymatic reactions under normal physiological conditions and in disease. The immediate outcome will be to establish the parameters needed to operate this new instrument and to demonstrate the new technique on a simple protein construct.
