Molecular evolution, intracellular organization, and the quinary structure of proteins.

107

178

Significance Proteins function in a sea of macromolecules within cells, but are traditionally studied under ideal conditions in vitro . The more details we amass from experiments performed in cells, the closer we will get to understanding fundamental aspects of protein chemistry in the cellular environment. In addition to furthering our essential knowledge of biochemistry, advancements in the field of macromolecular crowding will drive efforts to stabilize protein-based therapeutics. Here, we show that protein stability can be measured at the residue level in living cells without adding destabilizing cosolutes or heat.

mentioning

confidence: 99%

Residue level quantification of protein stability in living cells

Monteith¹,

Pielak

2014

107

178

“…These interactions are probably of functional importance in cells. For instance, it has been hypothesized that weak protein-protein interactions give rise to cellular organization (58), as observed by centrifugation of whole cells (59) and in protein clusters (60). Such clustering leads to the formation of "supercrowded" regions surrounded by zones of dilute cytoplasm, which help direct cellular metabolism (61).…”

Section: Resultsmentioning

confidence: 99%

Impact of reconstituted cytosol on protein stability

Sarkar¹,

Smith²,

Pielak

2013

174

217

Protein stability is usually studied in simple buffered solutions, but most proteins function inside cells, where the heterogeneous and crowded environment presents a complex, nonideal system. Proteins are expected to behave differently under cellular crowding owing to two types of contacts: hard-core repulsions and weak, chemical interactions. The effect of hard-core repulsions is purely entropic, resulting in volume exclusion owing to the mere presence of the crowders. The weak interactions can be repulsive or attractive, thus enhancing or diminishing the excluded volume, respectively. We used a reductionist approach to assess the effects of intracellular crowding. Escherichia coli cytoplasm was dialyzed, lyophilized, and resuspended at two concentrations. NMRdetected amide proton exchange was then used to quantify the stability of the globular protein chymotrypsin inhibitor 2 (CI2) in these crowded solutions. The cytosol destabilizes CI2, and the destabilization increases with increasing cytosol concentration. This observation shows that the cytoplasm interacts favorably, but nonspecifically, with CI2, and these interactions overcome the stabilizing hard-core repulsions. The effects of the cytosol are even stronger than those of homogeneous protein crowders, reinforcing the biological significance of weak, nonspecific interactions.M acromolecules in Escherichia coli reach concentrations of 300-400 g/L and occupy up to 40% of the cellular volume (1), but proteins are normally studied in buffer alone. The effects of crowding arise from two phenomena, hard-core repulsions and nonspecific chemical (soft) interactions (2-9). Hard-core repulsions limit the volume available to biological macromolecules for the simple reason that two molecules cannot be in the same place at the same time. This press for space favors compact states over expanded states. The second phenomenon arises because crowders not only exclude volume, but also participate in chemical interactions. Even though individually weak, the high concentration of macromolecules can lead to a large net effect. Repulsive nonspecific interactions reinforce the hardcore repulsions, whereas attractive nonspecific interactions oppose them. We use the term "nonspecific attractive interactions" to distinguish these from specific chemical interactions, such as ligand binding.Our aim is to understand how the crowded and heterogeneous, intracellular environment affects the equilibrium thermodynamic stability of globular proteins. Globular proteins are marginally stable in buffer at room temperature (10), with Gibbs free energy differences of 5-15 kcal/mol between the efficiently packed native (N) state and the ensemble of higher-energy denatured (D) states ðΔG o′ D Þ (11). Crowding effects arise from entropic and enthalpic contributions; hard-core repulsions are entropic, whereas the consequent nonspecific chemical interactions are also enthalpic. Hard-core repulsions always increase ΔG o′ D for globular proteins because D occupies more space than N (12-14). However,...

“…The reaction order and K d can be estimated from χ green and χ red as a function of cell-volume change (as shown in SI Appendix, section S3). Thus, our method is particularly useful for detection of weak quinary interactions between proteins inside cells (5,14). For the association between AcGFP1 and mCherry (Fig.…”

Section: Analysis Of Quinary Interactions Using a Quantitative Volumementioning

confidence: 99%

“…Another type of protein complexes relies on weak, transient interactions, collectively termed quinary interactions (5)(6)(7). Such interactions, with a K d in the micromolar to millimolar range, are emerging as important components of the cell's signaling, regulatory, and stress adaptation mechanisms (6,8,9).…”

mentioning

confidence: 99%

Weak protein–protein interactions in live cells are quantified by cell-volume modulation

Sukenik

Ren

Gruebele

2017

123

Weakly bound protein complexes play a crucial role in metabolic, regulatory, and signaling pathways, due in part to the high tunability of their bound and unbound populations. This tunability makes weak binding (micromolar to millimolar dissociation constants) difficult to quantify under biologically relevant conditions. Here, we use rapid perturbation of cell volume to modulate the concentration of weakly bound protein complexes, allowing us to detect their dissociation constant and stoichiometry directly inside the cell. We control cell volume by modulating media osmotic pressure and observe the resulting complex association and dissociation by FRET microscopy. We quantitatively examine the interaction between GAPDH and PGK, two sequential enzymes in the glycolysis catalytic cycle. GAPDH and PGK have been shown to interact weakly, but the interaction has not been quantified in vivo. A quantitative model fits our experimental results with log K d = −9.7 ± 0.3 and a 2:1 prevalent stoichiometry of the GAPDH:PGK complex. Cellular volume perturbation is a widely applicable tool to detect transient protein interactions and other biomolecular interactions in situ. Our results also suggest that cells could use volume change (e.g., as occurs upon entry to mitosis) to regulate function by altering biomolecular complex concentrations.quinary interactions | protein-protein interactions | cell volume | FRET | live-cell microscopy F rom forming active enzymatic complexes to facilitating signal transduction in regulatory networks, protein interactions are pivotal to cell function. Strong interactions, with dissociation constants (K d ) of nanomolar and lower (1, 2), can be measured accurately both in vitro and in vivo (3, 4). These interactions are ideal in cases where the complex should form with high fidelity and persist. The downside of strong binding is that its regulation in the cellular environment is limited: Once a complex is formed, it seldom dissociates.Another type of protein complexes relies on weak, transient interactions, collectively termed quinary interactions (5-7). Such interactions, with a K d in the micromolar to millimolar range, are emerging as important components of the cell's signaling, regulatory, and stress adaptation mechanisms (6,8,9). Their transient nature is key to their function: Unlike tightly bound complexes, quinary interactions are highly sensitive to variations in their environment and respond rapidly to changes in temperature, pressure, pH, or the local concentration of surrounding molecules. The sensitivity of quinary interactions makes them important in fine-tuning cellular processes. For example, it has been proposed that sequential metabolic enzymes could associate to improve substrate transfer between catalytic enzymes (10), that weak protein association can mediate cytokine release (11), or that phase-separated protein droplets held together by quinary interactions could serve for cellular storage or stress response (12, 13).Despite growing interest, quantification of quinary inte...