high-speed-magnetic-tweezers-for-nanomechanical-measurements-on-force

High-Speed Magnetic Tweezers for Nanomechanical Measurements on Force-Sensitive Elements

Single-molecule magnetic tweezers (MTs) have served as powerful tools to forcefully interrogate biomolecules, such as nucleic acids and proteins, and are ...

Seoul National University

Single-molecule magnetic tweezers deliver magnetic force and torque to single target molecules, permitting the study of dynamic changes in biomolecular structures and their interactions. Because the magnetic tweezer setups can generate magnetic fields that vary slowly over tens of millimeters-far larger than the nanometer scale of the single molecule events being observed-this technique can maintain essentially constant force levels during biochemical experiments while generating a biologically meaningful force on the order of 1-100 pN. When using bead-tether constructs to pull on single molecules, smaller magnetic beads and shorter submicrometer tethers improve dynamic response times and measurement precision. In addition, employing high-speed cameras, stronger light sources, and a graphics programming unit permits true high-resolution single-molecule magnetic tweezers that can track nanometer changes in target molecules on a millisecond or even submillisecond time scale. The unique force-clamping capacity of the magnetic tweezer technique provides a way to conduct measurements under near-equilibrium conditions and directly map the energy landscapes underlying various molecular phenomena. High-resolution single-molecule magnetic tweezerscan thus be used to monitor crucial conformational changes in single-protein molecules, including those involved in mechanotransduction and protein folding.

High-Resolution Single-Molecule Magnetic Tweezers.

Despite advances in resolving the structures of multi-pass membrane proteins, little is known about the native folding pathways of these complex structures. Using single-molecule magnetic tweezers, we here report a folding pathway of purified human glucose transporter 3 (GLUT3) reconstituted within synthetic lipid bilayers. The N-terminal major facilitator superfamily (MFS) fold strictly forms first, serving as a structural template for its C-terminal counterpart. We found polar residues comprising the conduit for glucose molecules present major folding challenges. The endoplasmic reticulum membrane protein complex facilitates insertion of these hydrophilic transmembrane helices, thrusting GLUT3's microstate sampling toward folded structures. Final assembly between the N- and C-terminal MFS folds depends on specific lipids that ease desolvation of the lipid shells surrounding the domain interfaces. Sequence analysis suggests that this asymmetric folding propensity across the N- and C-terminal MFS folds prevails for metazoan sugar porters, revealing evolutionary conflicts between foldability and functionality faced by many multi-pass membrane proteins.

Article	Year
High-Resolution Single-Molecule Magnetic Tweezers. Annual review of biochemistry\| PubMed ID: 35287472	2022
Evolutionary balance between foldability and functionality of a glucose transporter. Nature chemical biology\| PubMed ID: 35484435	2022

Hyun Gyu Kim

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