Diffusional limits to the speed of protein folding: fact or friction?

Hagen, Stephen J.; Qiu, Linlin; Pabit, Suzette A.

doi:10.1088/0953-8984/17/18/008

Cited by 50 publications

(61 citation statements)

References 46 publications

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“…5B). In a plot of ic versus solvent viscosity and for a purely chain-entropy-controlled process the extrapolated, limiting time constant at zero solvent viscosity ( ic,0 ) crosses the origin ( ic,0 ϭ 0) (13)(14)(15). For D phys , however, we found ic,0 ϭ 260 Ϯ 90 ns.…”

Section: The Denatured State Of Bbl At Solvent Conditions That Favor mentioning

confidence: 68%

Direct observation of ultrafast folding and denatured state dynamics in single protein molecules

Neuweiler

Johnson

Fersht

2009

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

116

133

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Single-molecule fluorescence resonance energy transfer (smFRET) experiments are extremely useful in studying protein folding but are generally limited to time scales of greater than Ϸ100 s and distances greater than Ϸ2 nm. We used single-molecule fluorescence quenching by photoinduced electron transfer, detecting short-range events, in combination with fluorescence correlation spectroscopy (PET-FCS) to investigate folding dynamics of the small binding domain BBL with nanosecond time resolution. The kinetics of folding appeared as a 10-s decay in the autocorrelation function, resulting from stochastic fluctuations between denatured and native conformations of individual molecules. The observed rate constants were probe independent and in excellent agreement with values derived from conventional temperaturejump (T-jump) measurements. A submicrosecond relaxation was detected in PET-FCS data that reported on the kinetics of intrachain contact formation within the thermally denatured state. We engineered a mutant of BBL that was denatured under the reaction conditions that favored folding of the parent wild type (''D phys''). D phys had the same kinetic signature as the thermally denatured state and revealed segmental diffusion with a time constant of intrachain contact formation of 500 ns. This time constant was more than 10 times faster than folding and in the range estimated to be the ''speed limit'' of folding. D phys exhibited significant deviations from a random coil. The solvent viscosity and temperature dependence of intrachain diffusion showed that chain motions were slaved by the presence of intramolecular interactions. PET-FCS in combination with protein engineering is a powerful approach to study the early events and mechanism of ultrafast protein folding.fluorescence correlation spectroscopy ͉ intrachain diffusion ͉ photoinduced electron transfer ͉ protein folding ͉ speed limit S ingle-molecule spectroscopy detects molecular heterogeneities that are hidden in the ensemble average of conventional spectroscopy and provides complementary insights into the conformationally heterogeneous process of protein folding (1, 2). Commonly applied single-molecule fluorescence resonance energy transfer (smFRET) methods have limitations in revealing conformational changes of less than Ϸ2 nm or measuring very fast processes occurring in under Ϸ1 ms because of inherently low fluorescence photon statistics and interference from photophysical effects. The investigation of small and ultrafast folding protein domains, which often represent the building blocks of larger assemblies, is of particular importance to our fundamental understanding of folding. Importantly, their time scales of conformational motions can overlap with those of atomistic computer simulations of protein folding pathways (3).Current limitations of smFRET methods can be overcome using new developments in fluorescence correlation spectroscopy (FCS). FCS measures the kinetics of stochastic fluorescence fluctuations arising from individual fluorescent molecul...

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Section: The Denatured State Of Bbl At Solvent Conditions That Favor mentioning

confidence: 68%

Direct observation of ultrafast folding and denatured state dynamics in single protein molecules

Neuweiler

Johnson

Fersht

2009

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

116

133

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“…An example is shown in Fig. 3a, where k f Ϫ1 is plotted as a function of viscosity at 293 K (14,15). The data up to 5 cP can be fit equally well with a linear fit and a power law, Eq.…”

Section: Fractional Viscosity Dependence Of Protein Motionsmentioning

confidence: 75%

“…The temperature dependence of folding from the collapsed state of cytochrome c at constant viscosity also supports the arguments given here. Measured from 290 to 303 K, k f (T) has an apparent activation enthalpy of Ϸ30 kJ͞mol at constant viscosity (15,21). Most or all of the apparent activation enthalpy is due to the effect of the solvent on the protein.…”

Section: Fractional Viscosity Dependence Of Protein Motionsmentioning

confidence: 99%

“…The observation that U and N are nearly equally populated provides more evidence that folded proteins are not in a unique state, as is often stated, but can assume a very large number of conformational substates. Hagen et al (15) have made the observation that there seems to be a ''speed limit'' near 10 6 s -1 . This also implies some minimum number of steps n f for protein folding.…”

Section: Fractional Viscosity Dependence Of Protein Motionsmentioning

confidence: 99%

“…Although proteins may possess some internal viscosity (14,15), the slaving model does not require internal protein dissipation to explain the data. Thus, internal viscosity must be a negligibly small effect in existing experiments.…”

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Protein folding is slaved to solvent motions

Frauenfelder

Fenimore

Chen

et al. 2006

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

268

281

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Proteins, the workhorses of living systems, are constructed from chains of amino acids, which are synthesized in the cell based on the instructions of the genetic code and then folded into working proteins. The time for folding varies from microseconds to hours. What controls the folding rate is hotly debated. We postulate here that folding has the same temperature dependence as the ␣-fluctuations in the bulk solvent but is much slower. We call this behavior slaving. Slaving has been observed in folded proteins: Large-scale protein motions follow the solvent fluctuations with rate coefficient k␣ but can be slower by a large factor. Slowing occurs because large-scale motions proceed in many small steps, each determined by k␣. If conformational motions of folded proteins are slaved, so a fortiori must be the motions during folding. The unfolded protein makes a Brownian walk in the conformational space to the folded structure, with each step controlled by k␣. Because the number of conformational substates in the unfolded protein is extremely large, the folding rate coefficient, kf, is much smaller than k␣. The slaving model implies that the activation enthalpy of folding is dominated by the solvent, whereas the number of steps nf ‫؍‬ k␣͞kf is controlled by the number of accessible substates in the unfolded protein and the solvent. Proteins, however, undergo not only ␣-but also ␤-fluctuations. These additional fluctuations are local protein motions that are essentially independent of the bulk solvent fluctuations and may be relevant at late stages of folding.folding energy landscape ͉ fractional viscosity dependence ͉ internal viscosity ͉ Maxwell relation ͉ protein-solvent interaction P roteins in cells fold and unfold continuously. Consequently, an understanding of folding rates is key. The distribution and strength of contacts in the native state is one ingredient that influences the rate of folding (1). A second ingredient is the effect of the solvent, because protein motions are intimately linked to the motions of the environment. Our slaving model quantifies the linkage. The model is based on three concepts: Proteins assume a large number of different conformations or substates (2), their organization is described by a hierarchic energy landscape (3), and large-scale protein fluctuations follow the ␣-relaxation (Debye or dielectric relaxation) in the bulk solvent (4-6). Here we propose that these concepts also are valid for folding and that they lead to the model pictured in Fig. 1. Fig. 1a is a cartoon of folding and a 1D cross-section through the high-dimensional energy landscape. Fig. 1b is a 2D cross-section.

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Single‐Molecule FRET of Protein‐Folding Dynamics

Nettels

Schuler

2011

Single‐Molecule Biophysics

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Diffusional limits to the speed of protein folding: fact or friction?

Cited by 50 publications

References 46 publications

Direct observation of ultrafast folding and denatured state dynamics in single protein molecules

Direct observation of ultrafast folding and denatured state dynamics in single protein molecules

Protein folding is slaved to solvent motions

Single‐Molecule FRET of Protein‐Folding Dynamics

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