In Vitro FRET- and Fluorescence-Based Assays to Study Protein Conformation and Protein-Protein Interactions in Mitosis

Abstract

Proper cell division and the equal segregation of genetic material are essential for life. Cell division is mediated by the mitotic spindle, which is composed of microtubules (MTs) and MT-associated proteins that help align and segregate the chromosomes. The localization and characterization of many spindle proteins have been greatly aided by using GFP-tagged proteins in vivo, but these tools typically do not allow for understanding how their activity is regulated biochemically. With the recent explosion of the pallet of GFP-derived fluorescent proteins, fluorescence-based biosensors are becoming useful tools for the quantitative analysis of protein activity and protein-protein interactions. Here, we describe solution-based Förster resonance energy transfer (FRET) and fluorescence assays that can be used to quantify protein-protein interactions and to characterize protein conformations of MT-associated proteins involved in mitosis.

Importin α/β Promote Kif18B Microtubule Association to Spatially Control Microtubule Destabilization

“…The interactions between human Kif18B and human importin α/β or EB1 were reconstituted in vitro by using the FRET biosensors, His 6 -importin α-CyPet, His 6 -EB1-CyPet, and YPet-Kif18B-His 6 tail domains in solution as described previously (Ems-McClung and Walczak, 2020). To determine saturable binding, 100 nM of His 6 -importin α-CyPet together with 400 nM His 6 -S-importin β or 100 nM of His 6 -EB1-CyPet were mixed with 100 nM-1.0 µM YPet tagged Kif18B tail fragments in Reaction Buffer (10 mM HEPES pH 7.2, 55 mM ionic strength, 50 mM sucrose, 0.1 mM EDTA, 0.1 mM EGTA, 0.1 mg/ml casein).…”

“…To determine saturable binding, 100 nM of His 6 -importin α-CyPet together with 400 nM His 6 -S-importin β or 100 nM of His 6 -EB1-CyPet were mixed with 100 nM-1.0 µM YPet tagged Kif18B tail fragments in Reaction Buffer (10 mM HEPES pH 7.2, 55 mM ionic strength, 50 mM sucrose, 0.1 mM EDTA, 0.1 mM EGTA, 0.1 mg/ml casein). Where indicated, RanQ69L was added to a final concentration of 10 μM to the YPet-Kif18B tail + importin α-CyPet/ His 6 -S-importin β reactions after an initial FRET measurement, incubated for 10 min at RT, and FRET measured again (Ems-McClung and Walczak, 2020). To achieve a final ionic strength of 55 mM, XB dialysis buffer was added to the reactions accounting for the KCl and NaCl contributed by the different proteins.…”

“…A 50 µl aliquot of each reaction mix was transferred to a Costar #3964 96-well ½ area black plate and incubated at room temperature for 10 min before performing a spectral scan in a Synergy H1 plate reader (Bio-Tek). The spectral scan was performed by exciting His 6 -importin α-CyPet or His 6 -EB1-CyPet at 405 nm and recording the emission from 440 to 600 nm with 5 nm steps (Ems-McClung and Walczak, 2020). The recorded data was processed in Excel by subtracting the background YPet emission in control reactions without CyPet protein from YPet emission in the presence of CyPet protein, and the net emission was then normalized to the CyPet emission at 460 nm to obtain FRET ratios at 530 nm.…”

Fluorescence Quantum Yields in Complex Environments from QM-MM TDDFT Simulations: The Case of Indole in Different Solvents

“…Among these systems, proteins stand out. − There are only a few amino acids in nature presenting fluorescence: tyrosine, phenylalanine, and tryptophan. The fluorescence spectra of these chromophores are used as indicators of structural changes, such as folding/unfolding, , binding with a substrate or a ligand, , protein–protein interaction, , etc. The most widely used is undoubtedly tryptophan , because its optical properties present a greater sensitivity to the microenvironment in which it is found.…”

Fluorescence enables high throughput screening of polyelectrolyte–protein binding affinities

“…The GOx concentration during the measurement was 0.25 mg mL −1 (1.6 µM) although the measurement could be conducted at a much lower concentration if needed due to the high sensitivity of FRET technique. 39,40 Polymers composed from neutral monomers AAm or HEA and 0, 10, and 20% DMAPAm all showed very little FRET with GOx when compared to those having 50, 60, and 100% positively charged monomer. It is interesting to note that the polymer with 60% of its monomer units charged resulted in roughly the same FRET ratio as the polymer with all of its monomer units charged, suggesting that more charge is not necessarily better when designing polyelectrolytes for PIC particles formation with proteins.…”