Surface Charge Modulates Protein–Protein Interactions in Physiologically Relevant Environments

Guseman, Alex J.; Speer, Shannon L.; Goncalves, Gerardo M. Perez; Pielak, Gary J.

doi:10.1021/acs.biochem.8b00061

Cited by 62 publications

(61 citation statements)

References 43 publications

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“…At pH 7.5, BSA is predicted to have a charge of −19, while both GB1 dimers are predicted to have a charge of −4. The more-than-predicted stabilization can be explained by electrostatic repulsions between BSA and acidic patches on the surface of each dimer (29).…”

Section: Domain-swappedmentioning

confidence: 96%

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Protein shape modulates crowding effects

Guseman

Goncalves

Speer

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Section: Domain-swappedmentioning

confidence: 96%

“…The properties of protein surfaces determine the chemical interactions with cosolutes (29)(30)(31)(32)(33). An advantageous aspect of the GB1 system is that the interactions are similar for both dimers because their primary structures, and hence their surfaces, are highly similar ( Fig.…”

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confidence: 99%

Protein shape modulates crowding effects

Guseman

Goncalves

Speer

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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“…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).…”

Section: Discussionmentioning

confidence: 99%

“…Taking into account only excluded volume effects on in-cell dimer stability is, however, insufficient. Computer simulations and experimental work have shown that soft interactions as well as diffusion can modulate proteinprotein interactions (10,87,93). Contributions can also come from interaction with cellular metabolites and other physiological, small-molecule compounds (94).…”

Section: Discussionmentioning

confidence: 99%

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

Yang

Chen

Yang

et al. 2020

Preprint

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The complexity of the cellular medium can affect proteins' properties and therefore in-cell characterization of proteins is essential. We explored the stability and conformation of BIR1, the first baculoviral IAP repeat domain of X-chromosome-linked inhibitor of apoptosis (XIAP), as a model for a homo-dimer protein in human HeLa cells.We employed double electron-electron resonance (DEER) spectroscopy and labeling with redox stable and rigid Gd 3+ spin labels at three protein residues, C12 (flexible region), E22C and N28C (part of helical residues 26-31) in the N-terminal region. In contrast to predictions by excluded volume crowding theory, the dimer-monomer dissociation constant KD was markedly higher in cells than in solution and dilute cell lysate. As expected, this increase was recapitulated under conditions of high salt concentrations given that a conserved salt bridge at the dimer interface is critically required for association. Unexpectedly, however, also the addition of a crowding agent such as Ficoll destabilized the dimer, suggesting that Ficoll forms specific interactions with the monomeric protein. Changes in DEER distance distributions were observed for the E22C site, which displayed reduced conformational freedom in cells. Although overall DEER behaviors at E22C and N28C were compatible with a predicted compaction of disordered protein regions by excluded volume effects, we were unable to reproduce E22C properties in artificially crowded solutions. These results highlight the importance of in-cell DEER measurements to appreciate the complexities of cellular in vivo effects on protein structures and functions.

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Protein folding and quinary interactions: creating cellular organisation through functional disorder

2018

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The marginal stability of globular proteins in the cell is determined by the balance between excluded volume effect and soft interactions. Quinary interactions are a type of soft interactions involved in intracellular organisation and known to have stabilising or destabilising effects on globular proteins. Recent studies suggest that globular proteins have structural flexibility, exhibiting more than one functional state. Here, we propose that the quinary-induced destabilisation can be sufficient to produce functional partially unfolded states of globular proteins. The biological relevance of this mechanism is explored, involving intracellular phase separation and regulatory stress response mechanisms.

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Surface Charge Modulates Protein–Protein Interactions in Physiologically Relevant Environments

Cited by 62 publications

References 43 publications

Protein shape modulates crowding effects

Protein shape modulates crowding effects

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

Protein folding and quinary interactions: creating cellular organisation through functional disorder

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