One of the grand challenges of biophysical chemistry is to understand the principles that govern protein aggregation leading to amyloid fibrils, which is a highly complex and sensitive process. This review provides a comprehensive overview of how amyloid aggregation is affected by the various in vivo constituents and conditions.
Biomolecules evolve and function in densely crowded and highly heterogeneous cellular environments. Such conditions are often mimicked in the test tube by the addition of artificial macromolecular crowding agents. Still, it is unclear if such cosolutes indeed reflect the physicochemical properties of the cellular environment as the in-cell crowding effect has not yet been quantified. We have developed a macromolecular crowding sensor based on a FRET-labeled polymer to probe the macromolecular crowding effect inside single living cells. Surprisingly, we find that excluded-volume effects, although observed in the presence of artificial crowding agents, do not lead to a compression of the sensor in the cell. The average conformation of the sensor is similar to that in aqueous buffer solution and cell lysate. However, the in-cell crowding effect is distributed heterogeneously and changes significantly upon cell stress. We present a tool to systematically study the in-cell crowding effect as a modulator of biomolecular reactions.
In cells, proteins are embedded in
a crowded environment that controls
their properties via manifold avenues including weak protein–macromolecule
interactions. A molecular level understanding of these quinary interactions
and their contribution to protein stability, function, and localization
in the cell is central to modern structural biology. Using a mutational
analysis to quantify the energetic contributions of single amino acids
to the stability of the ALS related protein superoxide dismutase I
(SOD1) in mammalian cells, we show that quinary interactions destabilize
SOD1 by a similar energetic offset for most of the mutants, but there
are notable exceptions: Mutants that alter its surface properties
can even lead to a stabilization of the protein in the cell as compared
to the test tube. In conclusion, quinary interactions can amplify
and even reverse the mutational response of proteins, being a key
aspect in pathogenic protein misfolding and aggregation.
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