<sup>15</sup>N NMR Spin Relaxation Dispersion Study of the Molecular Crowding Effects on Protein Folding under Native Conditions

Ai, Xuan; Zhou, Zheng; Bai, Yawen; Choy, Wing‐Yiu

doi:10.1021/ja057832n

Cited by 84 publications

(80 citation statements)

References 13 publications

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“…In contrast to the significant stabilization effects seen for confinement, experimental studies on macromolecular crowding from different laboratories have consistently found a modest effect on protein stability [43][44][45][46][47][48]. The theoretical prediction of modest stabilizing effect by crowding, presented earlier, thus appears to have experimental support.…”

Section: Effect Of Macromolecular Crowding On Folding Stabilitymentioning

confidence: 77%

“…[46] studied the effect of dextran 30 on the heat and cold denaturation of the molten globule form of apomyoglobin; a stabilization effect of ~0.5k B T was observed at 270 g/l of the crowding agent. Based on NMR relaxation dispersion data obtained under native conditions, Ai et al [47] found that the folding stability of a redesigned four helix-bundle protein is increased bỹ 04k B T in the presence of 85 g/l PEG 20000. Ignatova et al [48] even reported preliminary data indicating a small destabilizing effect by Ficoll 70 on cellular retinoic acid-binding protein I.…”

Section: Effect Of Macromolecular Crowding On Folding Stabilitymentioning

confidence: 99%

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Protein folding in confined and crowded environments

Zhou

2008

Archives of Biochemistry and Biophysics

148

137

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Confinement and crowding are two major factors that can potentially impact protein folding in cellular environments. Theories based on considerations of excluded volumes predict disparate effects on protein folding stability for confinement and crowding: confinement can stabilize proteins by over 10k B T but crowding has a very modest effect on stability. On the other hand, confinement and crowding are both predicted to favor conformations of the unfolded state which are compact, and consequently may increase the folding rate. These predictions are largely borne out by experimental studies of protein folding under confined and crowded conditions in the test tube. Protein folding in cellular environments is further complicated by interactions with surrounding surfaces and other factors. Concerted theoretical modeling and test-tube and in vivo experiments promise to elucidate the complexity of protein folding in cellular environments.The complexity of the cellular environments in which proteins function is increasingly been appreciated [1]. Potential impacts of two related, but distinct factors, confinement and macromolecular crowding, on protein folding are receiving extensive attention [2][3][4][5]. Confinement arises from encapsulation of proteins in compartments with dimensions on the scales of 10's to 100's Å, which are only moderately larger than the sizes of the proteins themselves. Examples of such compartments include the Anfinsen cage of chaperonins and the exit tunnel of ribosomes. Crowding refers to the exclusion of volumes to proteins of interest by the presence of other macromolecules. The aims of this paper are to discuss potential impacts of confinement and crowding on protein folding and to review the progress made by recent studies on protein folding in confined and crowded conditions and in cellular environments.

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Section: Effect Of Macromolecular Crowding On Folding Stabilitymentioning

confidence: 77%

Section: Effect Of Macromolecular Crowding On Folding Stabilitymentioning

confidence: 99%

Protein folding in confined and crowded environments

Zhou

2008

Archives of Biochemistry and Biophysics

148

137

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“…Earlier experimental work has focused on the effects of crowding on kinetic refolding of complex proteins and on protein nonnative states at extreme conditions, whereas theoretical approaches have instead involved simple lattice models or small peptide systems (1,(9)(10)(11)(12)). …”

Section: Discussionmentioning

confidence: 99%

“…Experimental and theoretical work has demonstrated large effects of crowding on the thermodynamics and kinetics of many biological processes, including protein binding, folding, and aggregation (1,(9)(10)(11)(12).…”

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confidence: 99%

Molecular crowding enhances native structure and stability of α/β protein flavodoxin

Stagg

Zhang²,

Cheung³

et al. 2007

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

256

276

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To investigate the consequences of macromolecular crowding on the behavior of a globular protein, we performed a combined experimental and computational study on the 148-residue singledomain ␣/␤ protein, Desulfovibrio desulfuricans apoflavodoxin. In vitro thermal unfolding experiments, as well as assessment of native and denatured structures, were probed by using far-UV CD in the presence of various amounts of Ficoll 70, an inert spherical crowding agent. Ficoll 70 has a concentration-dependent effect on the thermal stability of apoflavodoxin (⌬T m of 20°C at 400 mg/ml; pH 7). As judged by CD, addition of Ficoll 70 causes an increase in the amount of secondary structure in the native-state ensemble (pH 7, 20°C) but only minor effects on the denatured state. Theoretical calculations, based on an off-lattice model and hardsphere particles, are in good agreement with the in vitro data. The simulations demonstrate that, in the presence of 25% volume occupancy of spheres, native flavodoxin is thermally stabilized, and the free energy landscape shifts to favor more compact structures in both native and denatured states. The difference contact map reveals that the native-state compaction originates in stronger interactions between the helices and the central ␤-sheet, as well as by less fraying in the terminal helices. This study demonstrates that macromolecular crowding has structural effects on the folded ensemble of polypeptides.energy landscape theory ͉ excluded volume effect ͉ molecular simulations ͉ protein folding

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“…Slower folding is consistent with the viscosity increase. Slower unfolding in Ficoll is consistent with both viscosity and an entropic pressure for protein compaction (25,26); however, limiting the explanation to viscosity and compaction effects is probably too simple. In contrast, BSA had only small effects, whereas lysozyme slowed folding fivefold but had no effect on unfolding.…”

Section: Folding and Unfolding Rates Confirm Preferential Interactionmentioning

confidence: 92%

In-cell thermodynamics and a new role for protein surfaces

Smith

Zhou

Gorensek

et al. 2016

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

147

223

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There is abundant, physiologically relevant knowledge about protein cores; they are hydrophobic, exquisitely well packed, and nearly all hydrogen bonds are satisfied. An equivalent understanding of protein surfaces has remained elusive because proteins are almost exclusively studied in vitro in simple aqueous solutions. Here, we establish the essential physiological roles played by protein surfaces by measuring the equilibrium thermodynamics and kinetics of protein folding in the complex environment of living Escherichia coli cells, and under physiologically relevant in vitro conditions. Fluorine NMR data on the 7-kDa globular N-terminal SH3 domain of Drosophila signal transduction protein drk (SH3) show that charge-charge interactions are fundamental to protein stability and folding kinetics in cells. Our results contradict predictions from accepted theories of macromolecular crowding and show that cosolutes commonly used to mimic the cellular interior do not yield physiologically relevant information. As such, we provide the foundation for a complete picture of protein chemistry in cells.protein NMR | protein thermodynamics | protein folding | in-cell NMR C lassic theories about the effects of complex environments consider only hard-core repulsions (volume exclusion) and so predict entropy-driven protein stabilization (1-3). Here, we use the 7-kDa globular N-terminal SH3 domain of Drosophila signal transduction protein drk (SH3) as a model to test this idea in living cells. SH3 exists in a dynamic equilibrium between the folded state and the unfolded ensemble (4). This two-state behavior (5) is ideal for NMR-based studies of folding. Fluorine labeling (6) of its sole tryptophan leads to only two 19 F resonances (7): one from the folded state, the other from the unfolded ensemble (Fig. 1A). The area under each resonance is proportional to its population, ρ f and ρ u, respectively. These populations are used to quantify protein stability via the modified standard state free energy of unfolding,where R is the gas constant and T is the absolute temperature. Furthermore, the width at half height of each resonance is proportional to the transverse relaxation rate, which is an approximate measure of intermolecular interactions (8-10). Thus, this simple system yields both quantitative thermodynamic knowledge and information about interactions involving the folded state and the unfolded ensemble.To assess the enthalpic (ΔH°′ U ) and entropic (ΔS°′ U ) components, we measured the temperature dependence of ΔG°′ U .

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¹⁵N NMR Spin Relaxation Dispersion Study of the Molecular Crowding Effects on Protein Folding under Native Conditions

Cited by 84 publications

References 13 publications

Protein folding in confined and crowded environments

Protein folding in confined and crowded environments

Molecular crowding enhances native structure and stability of α/β protein flavodoxin

In-cell thermodynamics and a new role for protein surfaces

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15N NMR Spin Relaxation Dispersion Study of the Molecular Crowding Effects on Protein Folding under Native Conditions

Cited by 84 publications

References 13 publications

Protein folding in confined and crowded environments

Protein folding in confined and crowded environments

Molecular crowding enhances native structure and stability of α/β protein flavodoxin

In-cell thermodynamics and a new role for protein surfaces

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¹⁵N NMR Spin Relaxation Dispersion Study of the Molecular Crowding Effects on Protein Folding under Native Conditions