Efficacy of macromolecular crowding in forcing proteins to fold

Qu, Youxing; Bolen, D. Wayne

doi:10.1016/s0301-4622(02)00148-5

Cited by 92 publications

(109 citation statements)

References 19 publications

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“…This picture is entirely consistent with the NMR study of Loria and coworker (72), which showed that TMAO can significantly rigidify the protein backbone, and with the simulations of Thirumalai and coworkers (44), which demonstrated that TMAO can act as a nano-crowder, thus increasing protein stability via the excluded volume effect. Despite this consistency, we note that a study by Qu and Bolen (73) indicated that the efficacy of the macromolecular crowding in forcing proteins to fold may be modest and may also depend on the crowding agents. Thus, it would be very useful if new experiments can be designed to directly quantify the crowding efficacy of TMAO.…”

Section: Discussionmentioning

confidence: 73%

Microscopic insights into the protein-stabilizing effect of trimethylamine N-oxide (TMAO)

Pazos

Gai

2014

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

228

237

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Although it is widely known that trimethylamine N-oxide (TMAO), an osmolyte used by nature, stabilizes the folded state of proteins, the underlying mechanism of action is not entirely understood. To gain further insight into this important biological phenomenon, we use the C≡N stretching vibration of an unnatural amino acid, p-cyano-phenylalanine, to directly probe how TMAO affects the hydration and conformational dynamics of a model peptide and a small protein. By assessing how the lineshape and spectral diffusion properties of this vibration change with cosolvent conditions, we are able to show that TMAO achieves its protein-stabilizing ability through the combination of (at least) two mechanisms: (i) It decreases the hydrogen bonding ability of water and hence the stability of the unfolded state, and (ii) it acts as a molecular crowder, as suggested by a recent computational study, that can increase the stability of the folded state via the excluded volume effect.N ature employs a variety of small organic molecules to cope with osmotic stress. Trimethylamine N-oxide (TMAO) is one such naturally occurring osmolyte that protects intracellular components against disruptive stress conditions (1). In particular, previous studies have shown that TMAO is able to enhance protein stability and to counteract the denaturing effect of urea (2, 3). TMAO (Fig. 1, Inset) adopts a skewed tetrahedral structure with a charged oxygen capable of accepting hydrogen bonds (H bonds) and three hydrophobic (methyl) groups. This amphiphilic structural arrangement makes TMAO a rather special cosolvent, because it can form H bonds with water, self-associate in a manner similar to surfactants, and show preferential interactions with or exclusion (4-12) from certain protein functional groups (13-23). Indeed, these molecular properties of TMAO have been used, either individually or in combination, to rationalize its biological activities. For example, a prevailing view about TMAO is that its osmotic and protective role is caused by the molecule's tendency to be preferentially depleted from protein surfaces, as suggested by physicochemical measurements (24-28). This thermodynamic picture, as described by Bolen and coworkers (29), implies that the protein is preferentially hydrated. Although this notion is consistent with TMAO's being a protecting osmolyte, to the best of our knowledge no experimental studies have been carried out to directly examine the effect of TMAO on protein hydration dynamics, an aspect that is also important to protein function. Herein, we use twodimensional infrared (2D IR) spectroscopy and a site-specific IR probe to explore this critical issue and gain insight toward achieving a microscopic understanding of the molecular mechanism of the protecting action of TMAO.2D IR spectroscopy is capable of assessing the frequencyfrequency correlation function (FFCF) of a given IR probe, which reports on the underlying dynamics of events that lead to fluctuations of its vibrational frequency (30). For an IR probe that is able ...

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Section: Discussionmentioning

confidence: 73%

Microscopic insights into the protein-stabilizing effect of trimethylamine N-oxide (TMAO)

Pazos

Gai

2014

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

228

237

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“…• to the incident beam [19] was monitored at six different excitation-emission wavelengths between 320 and 520 nm using an Aminco Bowman UV-vis spectrometer and no change in scattering intensity was observed (data not shown). It was concluded that no appreciable aggregation of the native state of WT* occurred in concentrations up to 300 g/L glucose.…”

Section: Test For Native-state Aggregationmentioning

confidence: 99%

“…In vitro experiments with carbohydrate-based macromolecules and osmolytes suggest that most proteins increase in stability with cosolute concentration in a linear, additive fashion [16,17], with typical increases in stability of around 1-2 kcal/mol in 200-300 g/L of cosolute [16,[18][19][20][21][22]. These stability changes approximate those made by excluded volume theory, which estimates the change 2 in stability of a protein on the basis of the difference between the compactness of the native and denatured states [23].…”

Section: Introductionmentioning

confidence: 99%

Destabilised mutants of ubiquitin gain equal stability in crowded solutions

Roberts¹,

Jackson²

2007

Biophysical Chemistry

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This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT AbstractThis paper investigates the thermodynamic and kinetic response of WT* ubiquitin (F45W) and three mutants to high concentrations of glucose, sucrose and dextran under physiological temperature and pH. WT* ubiquitin was stabilised by the same amount when comparing each cosolute on a weight to volume ratio, with cosolute effects largely independent of denaturant concentration. The energy difference between the mutants and WT* ubiquitin also remained the same in high concentrations of cosolute. An apparent decrease in transition-state surface burial in the presence of the cosolutes was attributed to increased compaction of the denatured state, and not to the Hammond effect. Together, these results suggest higher thermodynamic stabilities and folding rates for proteins in vivo compared to in vitro, in addition to more compact denatured states. Because the effects of mutation are the same in dilute solution and crowded conditions used to mimic the cellular environment, there is validity in using measurements of mutant stabilities made in dilute solutions to inform on how the mutations may affect stability in vivo.

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“…For example, the effects of crowding agents on the refolding of reduced denatured lysozyme (1,9), oligomerization of GroEL subunits (13), self-assembly of the cell division protein FtsZ (14) and the capsid protein of HIV (15), and amyloid formation of the human apolipoprotein C-II (16) have been reported. A few studies have focused on the ability of crowding agents to induce conformational changes in unfolded states of proteins (17,18). For example, it was shown that unfolded cytochrome c adopts a molten globule state in the presence of crowding agents at low pH (19).…”

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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.

258

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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Efficacy of macromolecular crowding in forcing proteins to fold

Cited by 92 publications

References 19 publications

Microscopic insights into the protein-stabilizing effect of trimethylamine N-oxide (TMAO)

Microscopic insights into the protein-stabilizing effect of trimethylamine N-oxide (TMAO)

Destabilised mutants of ubiquitin gain equal stability in crowded solutions

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

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