Protein-binding dynamics imaged in a living cell

Phillip, Yael; Kiss, Vladimir; Schreiber, Gideon

doi:10.1073/pnas.1112171109

Cited by 108 publications

(131 citation statements)

References 39 publications

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“…4b; the population of nonspecific complexes (dRMS >5 Å) is enhanced as the proteincrowder attraction strength increases. These findings are also consistent with recent experimental results (Phillip et al 2012). …”

Section: Effects Of Mixed Macromolecular Crowdingsupporting

confidence: 94%

Protein–protein interactions in a crowded environment

2013

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Protein-protein interactions are important in many essential biological functions, such as transcription, translation, and signal transduction. Much progress has been made in understanding protein-protein association in dilute solution via experimentation and simulation. Cells, however, contain various macromolecules, such as DNA, RNA, proteins, among many others, and a myriad of non-specific interactions (usually weak) are present between these cellular constituents. In this review article, we describe the important developments in recent years that have furthered our understanding and even allowed prediction of the consequences of macromolecular crowding on protein-protein interactions. We outline the development of our crowding theory that can predict the change in binding free energy due to crowding quantitatively for both repulsive and attractive protein-crowder interactions. One of the most important findings from our recent work is that weak attractive interactions between crowders and proteins can actually destabilize protein complex formation as opposed to the commonly assumed stabilizing effect predicted based on traditional crowding theories that only account for the entropicexcluded volume effects. We also discuss the implications of macromolecular crowding on the population of encounter versus specific native complex.

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Section: Effects Of Mixed Macromolecular Crowdingsupporting

confidence: 94%

Protein–protein interactions in a crowded environment

2013

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“…We want to emphasize that there are other crowding-agent studies not reported on here, for example on the role of crowding on enzymatic activity (Vopel and Makhatadze 2012), protein-protein association (Phillip et al 2012), and mixtures of crowding agents (Batra et al 2009). Our in vitro/in silico studies, in combination with work by others, underscore that macromolecular crowding results in: (1) more stable proteins, (2) faster and more homogeneous folding kinetics, and (3) population shifts of conformational ensembles.…”

Section: Discussionmentioning

confidence: 96%

Effects of macromolecular crowding agents on protein folding in vitro and in silico

et al. 2013

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Proteins fold and function inside cells which are environments very different from that of dilute buffer solutions most often used in traditional experiments. The crowded milieu results in excluded-volume effects, increased bulk viscosity and amplified chances for intermolecular interactions. These environmental factors have not been accounted for in most mechanistic studies of protein folding executed during the last decades. The question thus arises as to how these effects-present when polypeptides normally fold in vivo-modulate protein biophysics. To address excluded volume effects, we use synthetic macromolecular crowding agents, which take up significant volume but do not interact with proteins, in combination with strategically selected proteins and a range of equilibrium and time-resolved biophysical (spectroscopic and computational) methods. In this review, we describe key observations on macromolecular crowding effects on protein stability, folding and structure drawn from combined in vitro and in silico studies. As expected based on Minton's early predictions, many proteins (apoflavodoxin, VlsE, cytochrome c, and S16) became more thermodynamically stable (magnitude depends inversely on protein stability in buffer) and, unexpectedly, for apoflavodoxin and VlsE, the folded states changed both secondary structure content and, for VlsE, overall shape in the presence of macromolecular crowding. For apoflavodoxin and cytochrome c, which have complex kinetic folding mechanisms, excluded volume effects made the folding energy landscapes smoother (i.e., less misfolding and/or kinetic heterogeneity) than in buffer.

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“…Flory-Huggins theories as used here might thus provide novel insights into the demixing of multicomponent polymeric systems (41). An interesting next step will be a direct comparison of experiments in vitro with intracellular measurements (14,26), and the required quantitative tools are beginning to emerge (54)(55)(56).…”

Section: Discussionmentioning

confidence: 99%

Single-molecule spectroscopy reveals polymer effects of disordered proteins in crowded environments

Soranno

Koenig

Borgia

et al. 2014

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

228

307

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Intrinsically disordered proteins (IDPs) are involved in a wide range of regulatory processes in the cell. Owing to their flexibility, their conformations are expected to be particularly sensitive to the crowded cellular environment. Here we use single-molecule Förster resonance energy transfer to quantify the effect of crowding as mimicked by commonly used biocompatible polymers. We observe a compaction of IDPs not only with increasing concentration, but also with increasing size of the crowding agents, at variance with the predictions from scaled-particle theory, the prevalent paradigm in the field. However, the observed behavior can be explained quantitatively if the polymeric nature of both the IDPs and the crowding molecules is taken into account explicitly. Our results suggest that excluded volume interactions between overlapping biopolymers and the resulting criticality of the system can be essential contributions to the physics governing the crowded cellular milieu. A surprisingly large number of eukaryotic proteins either contain substantial unstructured regions or are entirely unfolded under physiological conditions (1, 2). These "intrinsically disordered proteins" (IDPs) are involved in many crucial cellular processes, such as transcription, translation, and signal transduction; their functional and conformational properties are thus of great interest for a wide range of biological questions. Important advances in understanding the structures of IDPs have been made over the past decade, especially with spectroscopic techniques, e.g., NMR (3, 4), single-molecule fluorescence (5-7), and with atomistic and coarse-grained molecular simulations (8-10). In contrast with the stable folded structures we are familiar with from 50 y of structural biology, IDPs comprise highly heterogeneous and dynamic ensembles of conformations, which either lack stable tertiary structure altogether or fold only on binding their cellular targets (4). Important components of the cellular environment that affect IDPs include not only specific cellular ligands, but also pH and the concentration of salts (11,12). An additional contribution that has been difficult to investigate experimentally comes from the large number of different solutes present in a cell that do not interact with an IDP specifically, but result in an environment that is densely filled with macromolecules and metabolites (12)(13)(14). Given their lack of persistent structure, the conformations of IDPs are expected to be particularly sensitive to the effects of such molecular crowding. Indeed, first experiments indicate that some IDPs gain structure upon crowding (15), whereas others do not (16-18), but may change their dimensions (19)(20)(21). The question of how the conformational distributions of IDPs respond to crowded environments is of particular current interest because IDPs have a vital role in cellular compartments and regions with very high local concentrations of proteins and RNA, such as RNA granules and nuclear pore complexes (22)(23)(24)(25). Howeve...

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Protein-binding dynamics imaged in a living cell

Cited by 108 publications

References 39 publications

Protein–protein interactions in a crowded environment

Protein–protein interactions in a crowded environment

Effects of macromolecular crowding agents on protein folding in vitro and in silico

Single-molecule spectroscopy reveals polymer effects of disordered proteins in crowded environments

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