A Time-Resolved Diffusion Technique for Detection of the Conformational Changes and Molecular Assembly/Disassembly Processes of Biomolecules

Abstract

Biological liquid–liquid phase separation (LLPS) is driven by dynamic and multivalent interactions, which involves conformational changes and intermolecular assembly/disassembly processes of various biomolecules. To understand the molecular mechanisms of LLPS, kinetic measurements of the intra- and intermolecular reactions are essential. In this review, a time-resolved diffusion technique which has a potential to detect molecular events associated with LLPS is presented. This technique can detect changes in protein conformation and intermolecular interaction (oligomer formation, protein-DNA interaction, and protein-lipid interaction) in time domain, which are difficult to obtain by other methods. After the principle and methods for signal analyses are described in detail, studies on photoreactive molecules (intermolecular interaction between light sensor proteins and its target DNA) and a non-photoreactive molecule (binding and folding reaction of α-synuclein upon mixing with SDS micelle) are presented as typical examples of applications of this unique technique.

Taylor Dispersion Analysis and Atomic Force Microscopy Provide a Quantitative Insight into the Aggregation Kinetics of Aβ (1–40)/Aβ (1–42) Amyloid Peptide Mixtures

“…Further, Figure shows that this oligomeric population reaches a maximum in concentration at around 30 min for the Aβ (1–42) sample, while this maximum is shifted to higher times with the decrease in Aβ (1–42) proportion [1.3, 1.6, 5.5, and 16 h for 25, 50, 75, and 100% Aβ (1–40) mixtures, respectively]. Recent studies suggested that amyloid peptides can undergo liquid–liquid phase separation before the formation of amyloid fibrils. − The “high mass oligomers” population with R h ranging from 5 to 50 nm found in this work might correspond to high-density protein condensates. The size increase of the species over time can be explained by Ostwald ripening.…”

Time-resolved detection of light-induced conformational changes of heliorhodopsin

“…2(b)), the signal is attributed to the molecular diffusion (diffusion signal). As described previously, 20–22 if D is a constant within the observation time range, the diffusion signal should be expressed by the following bi-exponential function:δ n spe ( t ) = δ n P exp(− D P q 2 t ) − δ n R exp(− D R q 2 t )where D R and D P are the diffusion coefficients of the reactant and product, respectively. Below, we confirm that D is almost time-independent in a slow time region, >10 ms.…”

“…The fourth term of eqn ( 1 signal is attributed to the molecular diffusion (diffusion signal). As described previously, [20][21][22] if D is a constant within the observation time range, the diffusion signal should be expressed by the following bi-exponential function:…”

“…The experimental setup for the TG measurement was similar to that reported previously. [20][21][22] A laser pulse from the second harmonic of an Nd:YAG laser (GCR-170-10, Spectra-Physics, CA, USA) was used as an excitation beam. A diode laser (785 nm, L785P090, Thorlabs, NJ, USA) was used as the probe beam.…”

“…The transient grating (TG) method measures a diffraction of a probe beam (TG signal) by a periodic refractive index change in a protein solution associated with the photoreaction in the time domain. [20][21][22] The decay rate of the TG signal due to chemical species reflects the translational diffusion coefficient (D) of a protein, and time-dependent D can be determined by changing the grating wavenumber (q). If there is a structural change affecting the interaction between the protein and solvent, D of the product can be different from that of the reactant and even can alter over time during the photoreaction.…”