Revealing Conformational Variants of Solution‐Phase Intrinsically Disordered Tau Protein at the Single‐Molecule Level

Abstract

Intrinsically disordered proteins,such as tau protein, adopt av ariety of conformations in solution, complicating solution-phase structural studies.W ee mployed an anti-Brownian electrokinetic (ABEL) trap to prolong measurements of single tau proteins in solution. Once trapped, we recorded the fluorescence anisotropyt oi nvestigate the diversity of conformations sampled by the single molecules.Adistribution of anisotropyv alues obtained from trapped tau protein is conspicuously bimodal while those obtained by trapping ag lobular protein or individual fluorophores are not. Timeresolved fluorescence anisotropym easurements were used to providea ne xplanation of the bimodal distribution as originating from as hift in the compaction of the two different families of conformations.

Conformational Dynamics of Intrinsically Disordered Proteins Regulate Biomolecular Condensate Chemistry

“…reported the presence of two conformations of full-length tau protein (htau40), stable on time scales of multiple seconds, based on a difference in their steady-state fluorescence anisotropies. 189 htau40 has two naturally occurring cysteines, C291 and C322, which were mutated to serine, and tyrosine Y310 was instead mutated to a cysteine, to which the fluorescent ATTO647N dye was attached. To be able to collect enough photons for statistics and discern multiple populations, htau40 molecules had to be observed for several seconds without perturbative immobilization.…”

Interferometric Scattering Enables Fluorescence-Free Electrokinetic Trapping of Single Nanoparticles in Free Solution

“…33 These steps must be implemented quickly enough to overcome the diffusive motion of the particle, and significant recent progress has been made toward optimizing this control loop to enable trapping of individual small organic fluorophores. 28,34 Trap implementations that utilize either intrinsic or label-based fluorescence to track emissive particles have been employed to characterize time-varying photophysical states, [35][36][37][38][39][40] molecular dynamics and kinetics, [41][42][43][44][45][46][47] and more. [48][49][50] However, the trapping duration, and therefore data collection, in anti-Brownian traps is typically limited by photobleaching or by blinking because dark particles cannot be tracked and are quickly lost.…”

Observing monomer: dimer transitions of neurotensin receptors 1 in single SMALPs by homoFRET and in an ABELtrap

“…Longer observation times of individual SMALPs were required to provide stronger evidence for a fraction of NTSR1 dimers in the absence of neurotensin. The ABELtrap was invented by A. E. Cohen and W. E. Moerner and is the method of choice to hold single molecules and nanoparticles in solution in place and to record all fluorescence parameters 18,19,[46][47][48][49] . Here we utilizes an ABELtrap with cw laser excitation at 561 nm to determine the brightness levels of NTSR1-mRuby3 in SMALPs.…”