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

Intrinsically disordered proteins, such as tau protein, adopt a variety of conformations in solution, complicating solution‐phase structural studies. We employed an anti‐Brownian electrokinetic (ABEL) trap to prolong measurements of single tau proteins in solution. Once trapped, we recorded the fluorescence anisotropy to investigate the diversity of conformations sampled by the single molecules. A distribution of anisotropy values obtained from trapped tau protein is conspicuously bimodal while those obtained by trapping a globular protein or individual fluorophores are not. Time‐resolved fluorescence anisotropy measurements were used to provide an explanation of the bimodal distribution as originating from a shift in the compaction of the two different families of conformations.     

Quantifying the Initial Unfolding of Bacteriorhodopsin Reveals Retinal Stabilization

The forces that stabilize membrane proteins remain elusive to precise quantification. Particularly important, but poorly resolved, are the forces present during the initial unfolding of a membrane protein, where the most native set of interactions is present. A high‐precision, atomic force microscopy assay was developed to study the initial unfolding of bacteriorhodopsin. A rapid near‐equilibrium folding between the first three unfolding states was discovered, the two transitions corresponded to the unfolding of five and three amino acids, respectively, when using a cantilever optimized for 2 μs resolution. The third of these states was retinal‐stabilized and previously undetected, despite being the most mechanically stable state in the whole unfolding pathway, supporting 150 pN for more than 1 min. This ability to measure the dynamics of the initial unfolding of bacteriorhodopsin provides a platform for quantifying the energetics of membrane proteins under native‐like conditions.     

Single‐Molecule Monitoring of the Structural Switching Dynamics of Nucleic Acids through Controlling Fluorescence Blinking

Single‐molecule fluorescence resonance energy transfer (smFRET) is a powerful tool to investigate the dynamics of biomolecular events in real time. However, it requires two fluorophores and can be applied only to dynamics that accompany large changes in distance between the molecules. Herein, we introduce a method for kinetic analysis based on control of fluorescence blinking (KACB), a general approach to investigate the dynamics of biomolecules by using a single fluorophore. By controlling the kinetics of the redox reaction the blinking kinetics or pattern can be controlled to be affected by microenvironmental changes around a fluorophore (rKACB), thereby enabling real‐time single‐molecule measurement of the structure‐changing dynamics of nucleic acids.     

Towards Unveiling the Exact Molecular Structure of Amorphous Red Phosphorus by Single‐Molecule Studies

Since the discovery of amorphous red phosphorus (a‐red P) in 1847, many possible structures have been proposed. However, the exact molecular structure has not yet been determined because of its amorphous nature. Herein several methods are used to investigate basic properties of a‐red P. Data from scanning tunneling microscopy (STM) and gel permeation chromatography (GPC) confirm that a‐red P is a linear inorganic polymer with a broad molecular weight distribution. The theoretical single‐molecule elasticities of the possible a‐red P structures are obtained by quantum mechanical (QM) calculations. The experimental single‐molecule elasticity of a‐red P measured by single‐molecule AFM matches with the theoretical result of the zig‐zag ladder structure, indicating that a‐red P may adopt this structure. Although this conclusion needs further validation, this fundamental study represents progress towards solving the structure of a‐red P. It is expected that the strategy utilized in this work can be applied to study other inorganic polymers.