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Coupled protein diffusion and folding in the cell.

ABSTRACT: When a protein unfolds in the cell, its diffusion coefficient is affected by its increased hydrodynamic radius and by interactions of exposed hydrophobic residues with the cytoplasmic matrix, including chaperones. We characterize protein diffusion by photobleaching whole cells at a single point, and imaging the concentration change of fluorescent-labeled protein throughout the cell as a function of time. As a folded reference protein we use green fluorescent protein. The resulting region-dependent anomalous diffusion is well characterized by 2-D or 3-D diffusion equations coupled to a clustering algorithm that accounts for position-dependent diffusion. Then we study diffusion of a destabilized mutant of the enzyme phosphoglycerate kinase (PGK) and of its stable control inside the cell. Unlike the green fluorescent protein control's diffusion coefficient, PGK's diffusion coefficient is a non-monotonic function of temperature, signaling 'sticking' of the protein in the cytosol as it begins to unfold. The temperature-dependent increase and subsequent decrease of the PGK diffusion coefficient in the cytosol is greater than a simple size-scaling model suggests. Chaperone binding of the unfolding protein inside the cell is one plausible candidate for even slower diffusion of PGK, and we test the plausibility of this hypothesis experimentally, although we do not rule out other candidates.

SUBMITTER: Guo M

PROVIDER: S-EPMC4249841 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Coupled protein diffusion and folding in the cell.

Guo Minghao M Gelman Hannah H Gruebele Martin M

PloS one 20141201 12

When a protein unfolds in the cell, its diffusion coefficient is affected by its increased hydrodynamic radius and by interactions of exposed hydrophobic residues with the cytoplasmic matrix, including chaperones. We characterize protein diffusion by photobleaching whole cells at a single point, and imaging the concentration change of fluorescent-labeled protein throughout the cell as a function of time. As a folded reference protein we use green fluorescent protein. The resulting region-depende ...[more]

PMID: 25436502

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Project description:Successful extensions of protein-folding energy landscape theory to intrinsically disordered proteins' (IDPs') binding-coupled-folding transition can enormously simplify this biomolecular process into diffusion along a limited number of reaction coordinates, and the dynamics subsequently is described by Kramers' rate theory. As the critical pre-factor, the diffusion coefficient D has direct implications on the binding kinetics. Here, we employ a structure-based model (SBM) to calculate D in the binding-folding of an IDP prototype. We identify a strong position-dependent D during binding by applying a reaction coordinate that directly measures the fluctuations in a Cartesian configuration space. Using the malleability of the SBM, we modulate the degree of conformational disorder in an isolated IDP and determine complex effects of intrinsic disorder on D varying for different binding stages. Here, D tends to increase with disorder during initial binding but shows a non-monotonic relationship with disorder in terms of a decrease-followed-by-increase in D during the late binding stage. The salt concentration, which correlates with electrostatic interactions via Debye-Hückel theory in our SBM, also modulates D in a stepwise way. The speeding up of diffusion by electrostatic interactions is observed during the formation of the encounter complex at the beginning of binding, while the last diffusive binding dynamics is hindered by non-native salt bridges. Because D describes the diffusive speed locally, which implicitly reflects the roughness of the energy landscape, we are eventually able to portray the binding energy landscape, including that from IDPs' binding, then to binding with partial folding, and finally to rigid docking, as well as that under different environmental salt concentrations. Our theoretical results provide key mechanistic insights into IDPs' binding-folding, which is internally conformation- and externally salt-controlled with respect to diffusion.

Dataset Information

Coupled protein diffusion and folding in the cell.

Publications

Coupled protein diffusion and folding in the cell.

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