The macromolecular crowding effect – from in vitro into the cell

Gnutt, David; Ebbinghaus, Simon

doi:10.1515/hsz-2015-0161

Cited by 101 publications

(105 citation statements)

References 48 publications

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“…As such, the correlations in Fig. 6 not only add physical−chemical detail to the effects of intracellular crowding (4,12,27,53) but also provide a tool for rational protein surface design. Applications can include optimization of target proteins for in-cell NMR detection (54), surface optimization of protein therapeutics (55), and mutational examination of the yet poorly understood relation between protein motion, spatial localization, and function (7,56,57).…”

Section: Discussionmentioning

confidence: 99%

Physicochemical code for quinary protein interactions inEscherichia coli

Choi

Lang

et al. 2017

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

116

235

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How proteins sense and navigate the cellular interior to find their functional partners remains poorly understood. An intriguing aspect of this search is that it relies on diffusive encounters with the crowded cellular background, made up of protein surfaces that are largely nonconserved. The question is then if/how this protein search is amenable to selection and biological control. To shed light on this issue, we examined the motions of three evolutionary divergent proteins in the Escherichia coli cytoplasm by in-cell NMR. The results show that the diffusive in-cell motions, after all, follow simplistic physical−chemical rules: The proteins reveal a common dependence on (i) net charge density, (ii) surface hydrophobicity, and (iii) the electric dipole moment. The bacterial protein is here biased to move relatively freely in the bacterial interior, whereas the human counterparts more easily stick. Even so, the in-cell motions respond predictably to surface mutation, allowing us to tune and intermix the protein's behavior at will. The findings show how evolution can swiftly optimize the diffuse background of protein encounter complexes by just single-point mutations, and provide a rational framework for adjusting the cytoplasmic motions of individual proteins, e.g., for rescuing poor in-cell NMR signals and for optimizing protein therapeutics.in-cell NMR | protein surface properties | intracellular diffusion D espite considerable progress in mapping out how proteins interact functionally through structure and evolved interfaces (1-3), there is yet little known about how proteins interact nonspecifically upon random diffusive encounters (4-10). Although these nonspecific "quinary" (11) interactions are typically weak and short-lived, they are still expected to affect function because of their sheer numbers: Under crowded cellular conditions, they compete with specific binding (6-8), control diffusion (12), and skew structural stability (5,(13)(14)(15)(16)(17)(18)(19). The question is then to what extent this dynamic background of nonspecific interactions is biologically controlled and optimized. Part of the answer is hinted by the tendency of soluble proteins, nucleic acids, and membranes to carry a repulsive net-negative charge (20,21 , and PO 4 2− (22). However, proteins expose also positive, polar, and hydrophobic moieties that operate against the net-negative charge repulsion by engaging in attractive interactions upon diffusive encounters. The strength and duration of these attractive interactions depend on the proteins' detailed surface composition, relative orientations, and ability to adapt complementary shapes. Following Elcock's estimate for the Escherichia coli cytoplasm, each protein experiences at all times approximately five putative interaction partners in its immediate cellular environment (8). Sometimes, mutual fits enable strong functional binding (1, 2), but, most often, the proteins just separate after a brief tête-à-tête (3), in search of higher-affinity partners. A key detail is that the eff...

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Section: Discussionmentioning

confidence: 99%

Physicochemical code for quinary protein interactions inEscherichia coli

Choi

Lang

et al. 2017

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

116

235

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“…Although recognized long ago 66, 79, 93, 94 , the importance of weak, nonspecific interactions as modulators of biochemical processes in vivo has only recently begun to be widely appreciated 5, 11, 65, 95 . Although emphasis has been placed upon attractive interactions and the formation of weakly associated complexes, it is important to keep in mind that some of the most influential weak, nonspecific interactions that have been identified as significant modulators of biochemical equilibria and rates are repulsive steric and electrostatic interactions between macromolecules that do not form complexes.…”

Section: Concluding Remarks and Future Perspectivesmentioning

confidence: 99%

Macromolecular Crowding In Vitro , In Vivo , and In Between

Rivas

Minton

2016

Trends in Biochemical Sciences

405

370

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Biochemical processes take place in heterogeneous and highly volume occupied or crowded environments that can considerably influence the reactivity and distribution of participating macromolecules. Here we summarize the thermodynamic consequences of excluded volume and longer-ranged nonspecific intermolecular interactions for macromolecular reactions in volume-occupied media. In addition, we summarize and compare the information content of studies of crowding in vitro and in vivo. We emphasize the importance of characterizing the behavior not only of labeled tracer macromolecules, but also the composition and behavior of unlabeled macromolecules in the immediate vicinity of the tracer. Finally, we propose strategies for extending quantitative analyses of crowding in simple model systems to increasingly complex media up to and including intact cells.

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“…A protein in this crowded environment will endure forces due to excluded volume and nonspecific chemical interactions with the other macromolecules (2)(3)(4). Its thermodynamic activity will furthermore be affected by the solvent properties.…”

Section: Introductionmentioning

confidence: 99%

Design and Properties of Genetically Encoded Probes for Sensing Macromolecular Crowding

Zhang

Åberg

Eerden

et al. 2017

Biophysical Journal

100

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Cells are highly crowded with proteins and polynucleotides. Any reaction that depends on the available volume can be affected by macromolecular crowding, but the effects of crowding in cells are complex and difficult to track. Here, we present a set of Fö rster resonance energy transfer (FRET)-based crowding-sensitive probes and investigate the role of the linker design. We investigate the sensors in vitro and in vivo and by molecular dynamics simulations. We find that in vitro all the probes can be compressed by crowding, with a magnitude that increases with the probe size, the crowder concentration, and the crowder size. We capture the role of the linker in a heuristic scaling model, and we find that compression is a function of size of the probe and volume fraction of the crowder. The FRET changes observed in Escherichia coli are more complicated, where FRETincreases and scaling behavior are observed solely with probes that contain the helices in the linker. The probe with the highest sensitivity to crowding in vivo yields the same macromolecular volume fractions as previously obtained from cell dry weight. The collection of new probes provides more detailed readouts on the macromolecular crowding than a single sensor.

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The macromolecular crowding effect – from in vitro into the cell

Cited by 101 publications

References 48 publications

Physicochemical code for quinary protein interactions inEscherichia coli

Physicochemical code for quinary protein interactions inEscherichia coli

Macromolecular Crowding In Vitro , In Vivo , and In Between

Design and Properties of Genetically Encoded Probes for Sensing Macromolecular Crowding

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