The Effect of D2O on the Thermal Stability of Proteins. Thermodynamic Parameters for the Transfer of Model Compounds from H2O to D2O1,2

Kresheck, Gordon C.; Schneider, Henry; Scheraga, Harold A.

doi:10.1021/j100893a054

Cited by 213 publications

(122 citation statements)

References 4 publications

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“…We investigated this possibility by means of kinetic solvent isotope effects caused by replacing H 2 O by D 2 O in the samples. The stronger hydrogen bonding in D 2 O is thought to increase the hydrophobic effect and stabilize proteins (53)(54)(55)(56)(57). If water dominated the entropy change during the chaperone-mediated folding reaction, the kinetic solvent isotope effect in the chaperone should be significantly different from that of the autonomous folding reaction.…”

Section: Resultsmentioning

confidence: 99%

Single-molecule spectroscopy of protein folding in a chaperonin cage

Hofmann

Hillger

Pfeil

et al. 2010

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

113

124

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Molecular chaperones are known to be essential for avoiding protein aggregation in vivo, but it is still unclear how they affect protein folding mechanisms. We use single-molecule Förster resonance energy transfer to follow the folding of a protein inside the GroEL/GroES chaperonin cavity over a time range from milliseconds to hours. Our results show that confinement in the chaperonin decelerates the folding of the C-terminal domain in the substrate protein rhodanese, but leaves the folding rate of the N-terminal domain unaffected. Microfluidic mixing experiments indicate that strong interactions of the substrate with the cavity walls impede the folding process, but the folding hierarchy is preserved. Our results imply that no universal chaperonin mechanism exists. Rather, a competition between intra-and intermolecular interactions determines the folding rates and mechanisms of a substrate inside the GroEL/GroES cage.I n the recent past, a large number of components have been identified that control and modulate protein folding in vivo. This machinery includes molecular chaperones (1-3), sophisticated quality control systems, and complex mechanisms for protein translocation and degradation (3, 4), reflecting the importance of regulating the delicate balance of protein folding, misfolding, and aggregation in the cell. Such cellular factors exert conformational constraints on protein molecules that are expected to have a strong effect on the corresponding free-energy surfaces for folding (5). However, while the combination of cellular, biochemical, and structural data has led to some plausible qualitative models for the processes involved, mechanistic investigations comparable to those of autonomous protein folding in vitro (5-8) have been complicated by the complexity of the systems and the conformational heterogeneity involved (9). Even the autonomous folding of chaperone substrate proteins has been difficult to investigate because of their strong aggregation tendency (10). Contributions from confinement and crowding have been addressed in numerous studies using molecular simulations and theory (11)(12)(13)(14)(15)(16)(17)(18)(19)(20), but many of these concepts have eluded experimental examination.Here, we take a step towards closing this gap by investigating the GroEL/GroES chaperonin (1-3, 9) with single-molecule fluorescence spectroscopy (21-24), a method that is starting to provide previously inaccessible information on chaperonemediated protein folding (25)(26)(27)(28)(29)(30). GroEL/GroES is a remarkable molecular machine that binds nonnative proteins and allows them to fold within a cavity formed by the heptameric rings of GroEL and GroES. However, the cavity is only slightly larger than the folded structure of typical proteins known to interact with the chaperonin. The large volume of unconfined unfolded protein chains compared to the size of the cavity raises the question of whether and how such strong confinement affects the folding reaction (12-16, 18, 31, 32). By labeling the classic substrate prote...

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Section: Resultsmentioning

confidence: 99%

Single-molecule spectroscopy of protein folding in a chaperonin cage

Hofmann

Hillger

Pfeil

et al. 2010

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

113

124

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“…For the solvation of large biomolecules (94,95), small molecules (96)(97)(98), and ions (99) proton transfer by a factor of ~3 (121,122). Experimentally proton diffusion has been observed to 14 be slowed by a factor of ~1.5 upon deuteration (123).…”

Section: Solvation Propertiesmentioning

confidence: 99%

Nuclear Quantum Effects in Water and Aqueous Systems: Experiment, Theory, and Current Challenges

et al. 2016

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Nuclear quantum effects influence the structure and dynamics of hydrogen bonded systems, such as water, which impacts their observed properties with widely varying magnitudes. This review highlights the recent significant developments in the experiment, theory and simulation of nuclear quantum effects in water. Novel experimental techniques, such as deep inelastic neutron scattering, now provide a detailed view of the role of nuclear quantum effects in water's 2 properties. These have been combined with theoretical developments such as the introduction of the competing quantum effects principle that allows the subtle interplay of water's quantum effects and their manifestation in experimental observables to be explained. We discuss how this principle has recently been used to explain the apparent dichotomy in water's isotope effects, which can range from very large to almost nonexistent depending on the property and conditions. We then review the latest major developments in simulation algorithms and theory that have enabled the efficient inclusion of nuclear quantum effects in molecular simulations, permitting their combination with on-the-fly evaluation of the potential energy surface using electronic structure theory. Finally, we identify current challenges and future opportunities in the area.3

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“…The increase in ionic strength may act to shield repulsive electrostatic interactions among the large number of positively charged residues, whereas D 2 O may enhance the hydrophobic effect. 38,39 Neither increased ionic strength nor D 2 O appeared to cause significant compaction of the monomeric protein, however, suggesting that the expanded nature of the protein is quite robust. A predominance of expanded conformations may be functionally important for kN, which must form interactions with multiple components of the transcriptional complex to facilitate antitermination.…”

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confidence: 98%

Fractal dimension of an intrinsically disordered protein: Small‐angle X‐ray scattering and computational study of the bacteriophage λ N protein

2011

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Small-angle X-ray scattering (SAXS) was used to characterize the bacteriophage k N protein, a 107 residue intrinsically disordered protein (IDP) that functions as a transcriptional antitermination factor. The SAXS data were used to estimate both the average radius of gyration and the fractal dimension, a measure of the protein's internal scaling properties, under a variety of solution conditions. In the absence of denaturants, the radius of gyration was 38 6 3.5 Å and the fractal dimension was 1.76 6 0.05, slightly larger than the value predicted for a well-solvated polymer with excluded volume (1.7). Neither the radius of gyration nor the fractal dimension changed significantly on the addition of urea, further indicating that the protein is extensively unfolded and well solvated in the absence of denaturant. The addition of NaCl or D 2 O was found to promote aggregation, but did not appear to affect the properties of the monomeric form. The experimental SAXS profiles were also compared with those predicted by a computational model for a random-coil polypeptide, with an adjustable solvation energy term. The experimental data were well fit to the model with the solvation energy close to zero. These results indicate that the k N protein is among the more expanded members of the broad class of IDPs, most likely because of its high content of charged residues and a large net charge (115 at neutral pH). The expanded nature of the conformational ensemble may play a role in facilitating the interactions of the protein with other components of the dynamic transcriptional complex.

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The Effect of D₂O on the Thermal Stability of Proteins. Thermodynamic Parameters for the Transfer of Model Compounds from H₂O to D₂O^1,2

Cited by 213 publications

References 4 publications

Single-molecule spectroscopy of protein folding in a chaperonin cage

Single-molecule spectroscopy of protein folding in a chaperonin cage

Nuclear Quantum Effects in Water and Aqueous Systems: Experiment, Theory, and Current Challenges

Fractal dimension of an intrinsically disordered protein: Small‐angle X‐ray scattering and computational study of the bacteriophage λ N protein

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