Formation of protein complexes in crowded environments – From in vitro to in vivo

Abstract

a b s t r a c tTraditionally, biochemical studies are performed in dilute homogenous solutions, which are very different from the dense mixture of molecules found in cells. Thus, the physiological relevance of these studies is in question. This recognition motivated scientists to formulate the effect of crowded solutions in general, and excluded volume in particular, on biochemical processes. Using polymers or proteins as crowders, it was shown that while crowding tends to significantly enhance the formation of complexes containing many subunits, dimerizations are only mildly affected. Computer simulations, together with experimental evidence, indicate soft interactions and diffusion as critical factors that operate in a concerted manner with excluded volume to modulate protein binding. Yet, these approaches do not truly mimic the cellular environment. In vivo studies may overcome this shortfall. The few studies conducted thus far suggest that in cells, binding and folding occur at rates close to those determined in dilute solutions. Obtaining quantitative biochemical information on reactions inside living cells is currently a main challenge of the field, as the complexity of the intracellular milieu was what motivated crowding research to begin with.

Impact of reconstituted cytosol on protein stability

“…Nonspecific repulsions increase ΔG o′ D because they make the crowder seem even larger. Nonspecific attractions decrease ΔG o′ D because more favorable interactions are able to form as the protein unfolds (2)(3)(4)(5)(6)(7)(8)(9).…”

Transcription factors in eukaryotic cells can functionally regulate gene expression by acting in oligomeric assemblies formed from an intrinsically disordered protein phase transition enabled by molecular crowding

“…In vitro Slimfield imaging of purified Mig1-GFP under identical imaging conditions for live cells similarly indicated monomeric Mig1-GFP foci in addition to a small fraction of brighter foci which were consistent with chance overlap of monomer GFP images. However, addition of a molecular crowding reagent in the form of low molecular weight polyethylene glycol (PEG) at a concentration known to correspond to small molecule “depletion” forces in cells [70] resulted in significant numbers of oligomers (Figure 1(j)), suggesting that Mig1 clusters present in live cells regardless of glucose may be stabilized by depletion components that are lost during biochemical purification.…”

Molecular simulations of cellular processes

“…The most challenging is the difficulty to simulate large systems represented at full atomistic resolution for timescales longer than a few μs. Simulations of slower processes occurring in a cell, such as protein (mis)folding, macromolecular (dis)assembly, chromosome (de)condensation, and co-translational events, are, therefore, unfeasible with today's resources (Bhattacharya et al 2013;Phillip and Schreiber 2013;Redler et al 2014;Perilla et al 2015;Nilsson et al 2015;Korolev et al 2016;Zheng and Wen 2017), unless coarser-thanatomistic representations are employed (Takada 2012;Trovato and Tozzini 2012).…”