The transition state transit time of WW domain folding is controlled by energy landscape roughness

Liu, Feng; Nakaema, M. K. K.; Gruebele, Martin

doi:10.1063/1.3262489

Cited by 68 publications

(170 citation statements)

References 57 publications

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“…This difference has two counterbalancing effects on folding. More hydrophobicity means more nonnative contacts reducing the prefactor k 0 in the rate constant expression k ¼ k 0 e −ΔG † ∕RT (as observed in vitro) (24,26). At the same time, more hydrophobicity stabilizes the native state and decreases the activation free energy ΔG † .…”

Section: Discussionmentioning

confidence: 99%

Temperature dependence of protein folding kinetics in living cells

Guo¹,

Gruebele

2012

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

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We measure the stability and folding rate of a mutant of the enzyme phosphoglycerate kinase (PGK) inside bone tissue cells as a function of temperature from 38 to 48°C. To facilitate measurement in individual living cells, we developed a rapid laser temperature stepping method capable of measuring complete thermal melts and kinetic traces in about two min. We find that this method yields improved thermal melts compared to heating a sample chamber or microscope stage. By comparing results for six cells with in vitro data, we show that the protein is stabilized by about 6 kJ∕mole in the cytoplasm, but the temperature dependence of folding kinetics is similar to in vitro. The main difference is a slightly steeper temperature dependence of the folding rate in some cells that can be rationalized in terms of temperature-dependent crowding, local viscosity, or hydrophobicity. The observed rate coefficients can be fitted within measurement uncertainty by an effective two-state model, even though PGK folds by a multistate mechanism. We validate the effective two-state model with a three-state free energy landscape of PGK to illustrate that the effective fitting parameters can represent a more complex underlying free energy landscape. A s the ability of molecular dynamics and physics-based force fields to simulate protein folding kinetics improves (1-3), folding kinetics in complex environments such as crowding agents (4-6) or living cells (7) is receiving increased attention. Energy landscape theory predicts that the free energies of different protein states are similar, and that the free energy barriers connecting these states are small (8); hence, one might expect that even small perturbations in vivo (on the order of RT ≈ 2.5 kJ∕mole) can have significant effects on folding and function: the relative population of two pathways is exponentially sensitive to their free energy difference (Boltzmann factor). In this manner, different microenvironments in the cell could exert localized "postpost-translational" control over protein structure, folding, and function (9).Due to their dynamic nature and small folding equilibrium constant, proteins spontaneously unfold and refold many times in vivo between translational synthesis and degradation. We recently developed Fast Relaxation Imaging (FReI) to study posttranslational unfolding and refolding kinetics inside cells (10). The technique applies small temperature upward or downward jumps to living cells and monitors a target protein that has been FRET-labeled to distinguish native and unfolded states by fluorescence microscopy. As our prototype protein, we chose a FRETlabeled phosphoglycerate kinase construct (FRET-PGK) for comparison between in cell and in vitro. This multistate folder should be particularly sensitive to cellular modulation of its folding rate and mechanism. We found that unfolding was highly reversible in the cytoplasm up to 43°C (10). The local viscosity experienced by the polypeptide chain is about twice as large as in aqueous solvent (7). Significant kine...

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Section: Discussionmentioning

confidence: 99%

Temperature dependence of protein folding kinetics in living cells

Guo¹,

Gruebele

2012

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

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124

156

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“…Any theoretical treatment of internal polypeptide motion must account for this behavior. In particular, this may have important consequences for the theoretical treatment of onedimensional diffusion along the (folding) reaction coordinate, e.g., for estimating reaction rates using Kramers' theory (12) or simulating diffusion in idealized potentials (8,13). Currently, such simulations include the roughness of the multidimensional energy surface by assuming an effective friction coefficient; an investigation of the extent to which subdiffusional behavior (in 3D real space) does affect the validity of this approach is beyond the scope of the current paper.…”

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 93%

“…It is noteworthy that energy landscape roughness has been implicated directly in causing nonexponential folding kinetics (5, 6). Similarly, the experimentally observed increase of the transition state transit time at higher temperatures was explained by stronger internal friction due to increased energy landscape roughness (13,14).Some progress has been made in experimentally observing motion over the energy landscape on short timescales. In particular, intrapeptide contact formation has been investigated extensively using fluorescence and triplet quenching (15-19).…”

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confidence: 99%

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Measurement of energy landscape roughness of folded and unfolded proteins

Milanesi

Waltho

Hunter

et al. 2012

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

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The dynamics of protein conformational changes, from protein folding to smaller changes, such as those involved in ligand binding, are governed by the properties of the conformational energy landscape. Different techniques have been used to follow the motion of a protein over this landscape and thus quantify its properties. However, these techniques often are limited to short timescales and low-energy conformations. Here, we describe a general approach that overcomes these limitations. Starting from a nonnative conformation held by an aromatic disulfide bond, we use time-resolved spectroscopy to observe nonequilibrium backbone dynamics over nine orders of magnitude in time, from picoseconds to milliseconds, after photolysis of the disulfide bond. We find that the reencounter probability of residues that initially are in close contact decreases with time following an unusual power law that persists over the full time range and is independent of the primary sequence. Model simulations show that this power law arises from subdiffusional motion, indicating a wide distribution of trapping times in local minima of the energy landscape, and enable us to quantify the roughness of the energy landscape (4-5 k B T). Surprisingly, even under denaturing conditions, the energy landscape remains highly rugged with deep traps (>20 k B T) that result from multiple nonnative interactions and are sufficient for trapping on the millisecond timescale. Finally, we suggest that the subdiffusional motion of the protein backbone found here may promote rapid folding of proteins with low contact order by enhancing contact formation between nearby residues. photochemical trigger | subdiffusion M ajor advances have been made in recent years in understanding dynamic aspects of protein conformational changes, particularly protein folding; however, many issues remain to be solved (1). Among these are the properties of the unfolded protein ensemble and the role of residual structure of denatured proteins in promoting folding (2), the heterogeneity of microscopic folding pathways (3), and the existence of multiple distinct, but only transiently populated, intermediates (4). Particularly for fast-folding proteins, the idea of downhill folding, i.e., the absence of a significant barrier, has been suggested as an alternative mechanism (5, 6), but it is not clear to what extent fast-folding proteins make use of this mechanism. On the other hand, technical progress has made it possible to observe multiple folding and unfolding events in millisecond all-atom molecular dynamics simulations. Such simulations have shown that some proteins always follow the same folding pathway, whereas others have several different pathways (7). Moreover, individual folding events occur with submicrosecond transit times through a distinct transition state but are separated by long waiting times, which yield the experimentally observed folding times (8).The idea of motion on a rugged energy landscape (9-11) has been used widely to describe conformational changes in protei...

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“…A counter argument is suggested by the results of Liu at al. [64] that suggested the source of roughness in unfolding-folding of protein FiP35 is hydrophobic forces.…”

Section: Discussionmentioning

confidence: 99%

Correlation Force Spectroscopy for Single Molecule Measurements

Radiom

2015

Springer Theses

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This thesis addresses development of a new force spectroscopy tool, correlation force spectroscopy (CFS), for the measurement of the mechanical properties of very small volumes of material (molecular to µm 3 ) at kHz-MHz time-scales. CFS is based on atomic force microscopy (AFM) and the principles of CFS resemble those of dual-trap optical tweezers. CFS consists of was validated by comparison between experimental data and finite element modeling of the deterministic vibrations of the cantilevers using the known viscosity and density of fluids. Work in this thesis shows that the data can also be accurately fitted using a simple harmonic oscillator model, which can be used for rapid rheometric measurements, after calibration. The mechanical properties of biomolecules such as dextran and single stranded DNA (ssDNA) are also described. Working with this prominent scientist and researcher, and learning from him, were not only highlights of an academic life, but advent of a new lifestyle. This is due to his special character and charismatic behavior. His insights, ideas, perseverance and encouragements were great source of success in this project.

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The transition state transit time of WW domain folding is controlled by energy landscape roughness

Cited by 68 publications

References 57 publications

Temperature dependence of protein folding kinetics in living cells

Temperature dependence of protein folding kinetics in living cells

Measurement of energy landscape roughness of folded and unfolded proteins

Correlation Force Spectroscopy for Single Molecule Measurements

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