Determination of oligomerization state of Drp1 protein in living cells at nanomolar concentrations

Abstract

Biochemistry in living cells is an emerging field of science. Current quantitative bioassays are performed ex vivo , thus equilibrium constants and reaction rates of reactions occurring in human cells are still unknown. To address this issue, we present a non-invasive method to quantitatively characterize interactions (equilibrium constants, K D ) directly within the cytosol of living cells. We reveal that cytosolic hydrodynamic drag depends exponentially on a probe’s size, and provide a model for its determination for different protein sizes (1–70 nm). We analysed oligomerization of dynamin-related protein 1 (Drp1, wild type and mutants: K668E, G363D, C505A) in HeLa cells. We detected the coexistence of wt-Drp1 dimers and tetramers in cytosol, and determined that K D for tetramers was 0.7 ± 0.5 μM. Drp1 kinetics was modelled by independent simulations, giving computational results which matched experimental data. This robust method can be applied to in vivo determination of K D for other protein-protein complexes, or drug-target interactions.

Analysis of Brightness of a Single Fluorophore for Quantitative Characterization of Biochemical Reactions

“…Having proofed the brightness approach to determine the equilibrium constant when unlabeled oligonucleotides are used, we also anticipate that this method could be used to study reactions in living cells. It can be especially useful, where other techniques like FCS 3 or FRET 51 cannot be used due to several reasons: not significant differences in diffusion coefficients or low FRET efficiency upon complex formation as well as difficulties in attaching fluorophores to biomolecules of interest. In contrast to FRET, TCCD, and FCCS, in our approach, only one of the substrates needs to be fluorescent.…”

Nanoscale Viscosity of Cytoplasm Is Conserved in Human Cell Lines

“… 10 − 12 Additionally, according to our research, scale-dependent heterogeneity of cytoplasmic viscosity is even more pronounced. 13 − 15 We found that objects of different sizes can experience different viscosity: the viscosity increases with the increasing size of the object. 13 It is an outcome of the complex composition of cytoplasm—various components provide obstacles at different length-scales: the only obstacle of similar or smaller size can hinder the diffusion of a probe (see Figure 1 : I).…”

“…We further presented applicability of this model to complex biological fluids, like cytosol of prokaryotic and eukaryotic cells, 5 , 13 and we experimentally proved and applied this model for determination of oligomerization state of proteins in living cells. 15 , 21 …”

In-cell destabilization of a homo-dimeric protein complex detected by DEER spectroscopy

“…In-cell FRET (Förster energy transfer) is effective for monitoring protein-protein interactions, determining dissociation constants and identifying conformational changes of appropriately fluorophore-labeled target proteins (23)(24)(25). In addition, fluorescence correlation spectroscopy (FCS) can be used determine dissociation constants of protein oligomers in cells (26).…”

“…Nonetheless, many proteins display poor in-cell NMR signal qualities (line broadening) due to restricted motions and transient interactions with cellular components (3,32,33). In-cell studies using NMR and fluorescence techniques reported on protein folding and stability in cells (17,24,(34)(35)(36)(37)(38)(39)(40), structural changes of disordered proteins (41)(42)(43) and, to some extent, on protein association e and the stabilization of oligomeric protein forms (23,25,26). EPR (electron-paramagnetic resonance)-based double electron-electron resonance (DEER, also called PELDOR) spectroscopy has been suggested as an attractive alternative method to interrogate protein structures in cells (21,42,(44)(45)(46)(47)(48)(49)(50)(51)(52)(53)(54)(55).…”