Protein Crowding Tunes Protein Stability

Miklos, Andrew C.; Sarkar, Mohona; Wang, Yaqiang; Pielak, Gary J.

doi:10.1021/ja200067p

Cited by 269 publications

(357 citation statements)

References 38 publications

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“…Based on our rationale for in-cell stabilization, we expected stabilization of GB1 in 100 g/L BSA (pI = 4.7) compared with buffer alone because both GB1 and BSA have anionic surfaces. However, this destabilization is in agreement with our observations (24,26,56,70) for chymotrypsin inhibitor 2 (CI2, pI = 6.0), supporting the hypothesis that nonspecific, attractive backbone interactions can overcome charge-charge effects and hard-core repulsions. Moreover, the fact that cells are not crowded with only one protein complicates such simplistic comparisons.…”

Section: Discussionsupporting

confidence: 91%

“…Increased attention to transient, nonspecific protein-protein interactions (12)(13)(14)(15) has led both experimentalists (16)(17)(18)(19) and theoreticians (20)(21)(22) to recognize the effects of chemical interactions between crowder and test protein when assessing the net effect of macromolecular crowding. These weak, nonspecific interactions can reinforce or oppose the effect of hard-core repulsions, resulting in increased or decreased stability depending on the chemical nature of the test protein and crowder (23)(24)(25)(26).…”

mentioning

confidence: 99%

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Residue level quantification of protein stability in living cells

Monteith¹,

Pielak

2014

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

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108

178

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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.

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Section: Discussionsupporting

confidence: 91%

mentioning

confidence: 99%

Residue level quantification of protein stability in living cells

Monteith¹,

Pielak

2014

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

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108

178

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“…In vitro effects on stability: crowding by proteins Miklos et al (2011) assessed CI2 stability in solutions crowded by BSA and lysozyme. In contrast to the stabilization observed with synthetic polymers, the protein crowders destabilized CI2.…”

Section: Crowding and Assemblymentioning

confidence: 99%

Soft interactions and crowding

2013

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The intracellular milieu is complex, heterogeneous and crowded-an environment vastly different from dilute solutions in which most biophysical studies are performed. The crowded cytoplasm excludes about a third of the volume available to macromolecules in dilute solution. This excluded volume is the sum of two parts: steric repulsions and chemical interactions, also called soft interactions. Until recently, most efforts to understand crowding have focused on steric repulsions. Here, we summarize the results and conclusions from recent studies on macromolecular crowding, emphasizing the contribution of soft interactions to the equilibrium thermodynamics of protein stability. Despite their non-specific and weak nature, the large number of soft interactions present under many crowded conditions can sometimes overcome the stabilizing steric, excluded volume effect.

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“…Protein crowders are better mimics because cells contain large amounts of protein. Protein crowders can be destabilizing because they interact with proteins via attractive, weak, nonspecific contacts with the backbone (8,16,17). Nevertheless, cells are not crowded with one particular protein.…”

mentioning

confidence: 99%

“…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).…”

mentioning

confidence: 99%

Impact of reconstituted cytosol on protein stability

Sarkar¹,

Smith²,

Pielak

2013

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

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175

217

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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,...

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Protein Crowding Tunes Protein Stability

Cited by 269 publications

References 38 publications

Residue level quantification of protein stability in living cells

Residue level quantification of protein stability in living cells

Soft interactions and crowding

Impact of reconstituted cytosol on protein stability

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