A Difference between <i>In Vitro</i> and In-Cell Protein Dimer Formation

Chu, I-Te; Stewart, Claire J.; Speer, Shannon L.; Pielak, Gary J.

doi:10.1021/acs.biochem.1c00780

Cited by 6 publications

(6 citation statements)

References 25 publications

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“…These discrepancies between the highly crowded cellular environment and dilute protein solutions that are used in most experiments can result in different protein behavior (Speer et al, 2022). Specifically, species observed in buffer are not necessarily observed in cells (Chu et al, 2022; Cohen & Pielak, 2017) and disordered proteins can exhibit different ensembles in cells than they do in buffer (Dedmon et al, 2002). Surprisingly, how crowding impacts protein folding and folded state stability in concentrated environments is yet to be fully resolved.…”

Section: Introductionmentioning

confidence: 99%

Resolving the enthalpy of protein stabilization by macromolecular crowding

et al. 2023

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Proteins in the cellular milieu reside in environments crowded by macromolecules and other solutes. Although crowding can significantly impact the protein folded state stability, most experiments are conducted in dilute buffered solutions. To resolve the effect of crowding on protein stability, we use 19 F nuclear magnetic resonance spectroscopy to follow the reversible, two-state unfolding thermodynamics of the N-terminal Src homology 3 domain of the Drosophila signal transduction protein drk in the presence of polyethylene glycols (PEGs) of various molecular weights and concentrations. Contrary to most current theories of crowding that emphasize steric protein-crowder interactions as the main driving force for entropically favored stabilization, our experiments show that PEG stabilization is accompanied by significant heat release, and entropy disfavors folding. Using our newly developed model, we find that stabilization by ethylene glycol and small PEGs is driven by favorable binding to the folded state. In contrast, for larger PEGs, chemical or soft PEG-protein interactions do not play a significant role. Instead, folding is favored by excluded volume PEG-protein interactions and an exothermic nonideal mixing contribution from release of confined PEG and water upon folding. Our results indicate that crowding acts through molecular interactions subtler than previously assumed and that interactions between solution components with both the folded and unfolded states must be carefully considered.

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Section: Introductionmentioning

confidence: 99%

Resolving the enthalpy of protein stabilization by macromolecular crowding

et al. 2023

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“…All biological functions are driven by interactions. The protein interactions with cellular environment have been shown to affect protein conformation (9,23,(27)(28)(29), folding and stability (11,12,14,16,40), and proteinprotein binding (18,(20)(21)(22) in cells. Here, we showed that the interactions also affect protein loop dynamics.…”

Section: Discussionmentioning

confidence: 99%

“…The interior of a cell is crowded with ions, metabolites, and macromolecules. Through excluded volume effect (1) or direct weak interactions (such as electrostatics, H-bonding, and stacking), which drive proteins to form quinary structures (2)(3)(4)(5), the cellular environment can change protein diffusion (6)(7)(8)(9), protein folding (10)(11)(12)(13)(14)(15)(16)(17), and protein ligand binding (18)(19)(20)(21)(22)(23)(24). For intrinsically disordered proteins (IDPs), the excluded volume effect limits their conformational space so that they tend to adopt more compact conformations (9,25).…”

Section: Introductionmentioning

confidence: 99%

Intracellular environment can change protein conformational dynamics in cells through weak interactions

et al. 2023

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Conformational dynamics is important for protein functions, many of which are performed in cells. How the intracellular environment may affect protein conformational dynamics is largely unknown. Here, loop conformational dynamics is studied for a model protein in Escherichia coli cells by using nuclear magnetic resonance (NMR) spectroscopy. The weak interactions between the protein and surrounding macromolecules in cells hinder the protein rotational diffusion, which extends the dynamic detection timescale up to microseconds by the NMR spin relaxation method. The loop picosecond to microsecond dynamics is confirmed by nanoparticle-assisted spin relaxation and residual dipolar coupling methods. The loop interactions with the intracellular environment are perturbed through point mutation of the loop sequence. For the sequence of the protein that interacts stronger with surrounding macromolecules, the loop becomes more rigid in cells. In contrast, the mutational effect on the loop dynamics in vitro is small. This study provides direct evidence that the intracellular environment can modify protein loop conformational dynamics through weak interactions.

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“…15 On the other hand, the reaction of the GB1 domain-swapped dimer 16,17 changes from an equilibrium between a folded dimer and a molten globule monomer in buffer to one between the dimer and unfolded monomers in Escherichia coli cells, suggesting that attractive chemical interactions in cells unfold the protein. 18 Although not directly physiologically relevant, the crowding effects of synthetic polymers are essential to the pharmaceutical and chemical industries where they are used as excipients to protect biological drugs, vaccines, and commercial enzymes. 19−21 Polyethylene glycols (PEGs), which are used to formulate pharmaceutically and industrially important proteins and vaccines, 21 are the most commonly employed synthetic polymers for crowding experiments because of their high solubility, low polydispersity, and availability in a range of molecular weights.…”

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confidence: 99%

“…For example, the stability of the side-by-side dimer made from the B1 domain of protein G (GB1) increases in both prokaryotic and eukaryotic cells compared to buffer, probably because of repulsive chemical interactions between the GB1 monomer and intracellular macromolecules . On the other hand, the reaction of the GB1 domain-swapped dimer , changes from an equilibrium between a folded dimer and a molten globule monomer in buffer to one between the dimer and unfolded monomers in Escherichia coli cells, suggesting that attractive chemical interactions in cells unfold the protein …”

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confidence: 99%

Macromolecular Crowding by Polyethylene Glycol Reduces Protein Breathing

Chu

Hutcheson

Malsch

et al. 2023

J. Phys. Chem. Lett.

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Most efforts to understand macromolecular crowding focus on global (i.e., complete) unfolding, but smaller excursions, often called breathing, promote aggregation, which is associated with several diseases and the bane of pharmaceutical and commercial protein production. We used NMR to assess the effects of ethylene glycol (EG) and polyethylene glycols (PEGs) on the structure and stability of the B1 domain of protein G (GB1). Our data show that EG and PEGs stabilize GB1 differently. EG interacts with GB1 more strongly than PEGs, but neither affects the structure of the folded state. EG and 12000 g/mol PEG stabilize GB1 more than PEGs of intermediate size, but EG and smaller PEGs stabilize GB1 enthalpically while the largest PEG acts entropically. Our key finding is that PEGs turn local unfolding into global unfolding, and meta-analysis of published data supports this conclusion. These efforts provide knowledge that can be applied to improve biological drugs and commercial enzymes.

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A Difference between In Vitro and In-Cell Protein Dimer Formation

Cited by 6 publications

References 25 publications

Resolving the enthalpy of protein stabilization by macromolecular crowding

Resolving the enthalpy of protein stabilization by macromolecular crowding

Intracellular environment can change protein conformational dynamics in cells through weak interactions

Macromolecular Crowding by Polyethylene Glycol Reduces Protein Breathing

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