Influence of crowded cellular environments on protein folding, binding, and oligomerization: Biological consequences and potentials of atomistic modeling

Zhou, Huan‐Xiang

doi:10.1016/j.febslet.2013.01.064

Cited by 155 publications

(160 citation statements)

References 88 publications

(155 reference statements)

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“…4 and 5). Recent advances in both the experimental (5-9, 13, 14, 16-19, 24, 26, 56, 63) and theoretical (20)(21)(22)25) aspects of macromolecular crowding allow this stabilization to be rationalized in terms of the properties of GB1 and the E. coli cytoplasm.…”

Section: Discussionmentioning

confidence: 99%

“…The expectation was that crowding enhances stability by favoring the compact native state over the ensemble of denatured states. 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).…”

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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.

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: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Residue level quantification of protein stability in living cells

Monteith¹,

Pielak

2014

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

108

178

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“…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.

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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“…We have shown previously that the constitutively active procaspase-3 variant, V266E, results in robust cell death. 31 Under the conditions used here, the constitutively active procaspase-3 results in apoptosis levels of 30 All proteins were in the background of procaspase-8(D 2 A).…”

Section: Increased Dimerization In Vitro Is Not Sufficient To Triggermentioning

confidence: 98%

“…The cellular environment cannot provide the high kosmotrope conditions of the in vitro studies, although molecular crowding in the cytoplasm may facilitate dimerization. 30 Thus, it was not clear that increased dimerization in vitro, resulting in a lower requirement for citrate, would manifest as constitutive activation in cellulo.…”

Section: Increased Dimerization In Vitro Is Not Sufficient To Triggermentioning

confidence: 99%

Redesigning the procaspase‐8 dimer interface for improved dimerization

MacKenzie

Clark

2014

Protein Science

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Caspase-8 is a cysteine directed aspartate-specific protease that is activated at the cytosolic face of the cell membrane upon receptor ligation. A key step in the activation of caspase-8 depends on adaptor-induced dimerization of procaspase-8 monomers. Dimerization is followed by limited autoproteolysis within the intersubunit linker (IL), which separates the large and small subunits of the catalytic domain. Although cleavage of the IL stabilizes the dimer, the uncleaved procaspase-8 dimer is sufficiently active to initiate apoptosis, so dimerization of the zymogen is an important mechanism to control apoptosis. In contrast, the effector caspase-3 is a stable dimer under physiological conditions but exhibits little enzymatic activity. The catalytic domains of caspases are structurally similar, but it is not known why procaspase-8 is a monomer while procaspase-3 is a dimer. To define the role of the dimer interface in assembly and activation of procaspase-8, we generated mutants that mimic the dimer interface of effector caspases. We show that procaspase-8 with a mutated dimer interface more readily forms dimers. Time course studies of refolding also show that the mutations accelerate dimerization. Transfection of HEK293A cells with the procaspase-8 variants, however, did not result in a significant increase in apoptosis, indicating that other factors are required in vivo. Overall, we show that redesigning the interface of procaspase-8 to remove negative design elements results in increased dimerization and activity in vitro, but increased dimerization, by itself, is not sufficient for robust activation of apoptosis.

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Influence of crowded cellular environments on protein folding, binding, and oligomerization: Biological consequences and potentials of atomistic modeling

Cited by 155 publications

References 88 publications

Residue level quantification of protein stability in living cells

Residue level quantification of protein stability in living cells

Impact of reconstituted cytosol on protein stability

Redesigning the procaspase‐8 dimer interface for improved dimerization

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