Protein shape modulates crowding effects

Abstract

Protein−protein interactions are usually studied in dilute buffered solutions with macromolecule concentrations of <10 g/L. In cells, however, the macromolecule concentration can exceed 300 g/L, resulting in nonspecific interactions between macromolecules. These interactions can be divided into hard-core steric repulsions and "soft" chemical interactions. Here, we test a hypothesis from scaled particle theory; the influence of hard-core repulsions on a protein dimer depends on its shape. We tested the idea using a side-by-side dumbbell-shaped dimer and a domain-swapped ellipsoidal dimer. Both dimers are variants of the B1 domain of protein G and differ by only three residues. The results from the relatively inert synthetic polymer crowding molecules, Ficoll and PEG, support the hypothesis, indicating that the domain-swapped dimer is stabilized by hard-core repulsions while the side-by-side dimer shows little to no stabilization. We also show that protein cosolutes, which interact primarily through nonspecific chemical interactions, have the same small effect on both dimers. Our results suggest that the shape of the protein dimer determines the influence of hard-core repulsions, providing cells with a mechanism for regulating protein−protein interactions. macromolecular crowding | protein−protein interactions | scaled particle theory P rotein−protein interactions are essential for maintaining cellular homeostasis (1). Details of their equilibria under thermodynamically ideal conditions have provided a trove of information. Ideality in this sense refers to dilute solutions, where each monomer contacts only solvent or another monomer, conditions far removed from those in cells where protein− protein interactions evolved. In the cytoplasm, and other cellular compartments and biological fluids, macromolecules can occupy up to 30% of the volume, and their concentrations often exceed 300 g/L (2).Protein molecules take part in more-complex interactions under nonideal conditions. The surrounding macromolecules influence proteins in two ways, neither of which is significant in dilute solution. Hard-core repulsions arise from high volume occupancy, because two molecules cannot occupy the same space at the same time. This volume exclusion favors the most compact state of a protein (3). Chemical interactions comprise transient contacts between protein surfaces arising from the diverse chemical landscapes of proteins (4). When repulsive (i.e., like charges), they favor the state that maximizes the distance between charges, adding to the hard-core repulsions and stabilizing the native state. Attractive chemical interactions (e.g., opposite charges, hydrogen bonds) are destabilizing. We are beginning to understand how hard-core repulsions and chemical interactions affect protein stability (5), but there are few studies about crowding effects on protein−protein interactions (6-9).Given the existential roles of both protein−protein interactions and crowding in biology (10), we are undertaking efforts to determine the effect of cro...

KRAS Ubiquitination at Lysine 104 Retains Exchange Factor Regulation by Dynamically Modulating the Conformation of the Interface

“…To provide further experimental support, we investigated whether osmolytes can similarly restore SOS activity from disruption of the H2/H3 interface by point mutation. We employed the osmolyte, TMAO, as this cosolute can stabilize proteins in solution by enhancing its compact folded conformation even under various denaturing stresses ( Guseman et al., 2018 ; Su et al., 2017 ). KRAS mutations at position 104, including K104Q, K104C, K104R, and K104A, impair SOS upregulation of RAS activity by disrupting the H2/H3 interface ( Baker et al., 2013b ; Yin et al., 2017 ).…”

Controlling and quantifying protein concentration in Escherichia coli

“…Aliquots of the extracted lysates (75 μL aliquots) were flash frozen and lyophilized for 12 h (Labconco Freezone). GB1 standards were expressed and purified as described …”

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

“…By the same token, protein dimers, the shapes of which deviate from spherical shapes are expected to display reduced stabilities in crowded environments and can even be destablized (85). Such a shape-dependent behavior was recently confirmed experimentally in-vitro for two engineered dimers of the Streptococcal protein G B1 domain (GB1) which have different eccentricity (86). In the presence of Ficoll and PEG, a domain-swapped GB1 dimer with a lower eccentricity was significantly stabilized, whereas for the side-by-side dimer of GB1 with a higher eccentricity the effect in Ficoll was marginal and destabilization was observed in PEG solutions.…”

“…The estimated destabilization free energy determined from the dissociation constants amounts to ∆∆G 0 =1.24-1.31 Kcal/mol, for a temperature range of 260-273 K. A range is given owing to the uncertainty of the freezing temperature. In the following paragraphs, we discuss these results in the context of emerging concepts of how crowding and dimer shapes (85,86), as well as surface charges (87)(88)(89)(90), contribute to dimer cellular stabilities. According to excluded volume theory (91,92), protein association is favored under conditions of macromolecular crowding with concomitant increases in complex stabilities (7,85).…”