Single-Molecule Fluorescence Studies of Fast Protein Folding

Wang, Z.; Campos, Luis Alberto; Muñoz, Víctor

doi:10.1016/bs.mie.2016.09.011

Cited by 11 publications

(7 citation statements)

References 146 publications

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“…For the topic discussed in this article, it is also important to demonstrate the highly ordered hydrophobic core observed in titin [ 43 ]. This molecule, used in the SM-FRET method [ 21 ] as a frame for the analysis of the protein unfolding process, has a single-step unfolding form. Therefore, easy identification of the unfolding of the frame for other proteins enables a detailed analysis of the unfolding process of other proteins with more complex kinetics.…”

Section: Discussionmentioning

confidence: 99%

“…New perspectives are emerging with the appearance of techniques based on the “single molecule” experiment, including nuclear magnetic resonance, relaxation dispersion NMR spectroscopy [ 18 ] (in particular, photo-protection strategy [ 19 ]), as well as single molecule fluorescence spectroscopy sub-millisecond conformational dynamics [ 20 ]. Single molecule fluorescence resonance energy transfer (SM-FRET) utilizing fluorescent labeling and immobilization of proteins opens the possibility of quantitative (temporal) analysis of the folding process for fast-folding proteins [ 21 ].…”

Section: Introductionmentioning

confidence: 99%

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Downhill, Ultrafast and Fast Folding Proteins Revised

Banach

Stąpor

Konieczny

et al. 2020

IJMS

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Research on the protein folding problem differentiates the protein folding process with respect to the duration of this process. The current structure encoded in sequence dogma seems to be clearly justified, especially in the case of proteins referred to as fast-folding, ultra-fast-folding or downhill. In the present work, an attempt to determine the characteristics of this group of proteins using fuzzy oil drop model is undertaken. According to the fuzzy oil drop model, a protein is a specific micelle composed of bi-polar molecules such as amino acids. Protein folding is regarded as a spherical micelle formation process. The presence of covalent peptide bonds between amino acids eliminates the possibility of free mutual arrangement of neighbors. An example would be the construction of co-micelles composed of more than one type of bipolar molecules. In the case of fast folding proteins, the amino acid sequence represents the optimal bipolarity system to generate a spherical micelle. In order to achieve the native form, it is enough to have an external force field provided by the water environment which directs the folding process towards the generation of a centric hydrophobic core. The influence of the external field can be expressed using the 3D Gaussian function which is a mathematical model of the folding process orientation towards the concentration of hydrophobic residues in the center with polar residues exposed on the surface. The set of proteins under study reveals a hydrophobicity distribution compatible with a 3D Gaussian distribution, taken as representing an idealized micelle-like distribution. The structure of the present hydrophobic core is also discussed in relation to the distribution of hydrophobic residues in a partially unfolded form.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Downhill, Ultrafast and Fast Folding Proteins Revised

Banach

Stąpor

Konieczny

et al. 2020

IJMS

View full text Add to dashboard Cite

show abstract

“…It has been shown that the shape of transition paths and their distribution can provide sufficient information to dissect the rate expression ,, and hence to infer the underlying mechanisms . There currently are two options to access transition paths by experiment: single-molecule Förster resonance energy transfer (SM-FRET) and force spectroscopy (FS) . Recent FS experiments and simulations have provided strong evidence, , and direct observation, of multiple mechanical unfolding paths in otherwise “two-state” folding proteins.…”

Section: Introductionmentioning

confidence: 99%

Protein Folding Dynamics as Diffusion on a Free Energy Surface: Rate Equation Terms, Transition Paths, and Analysis of Single-Molecule Photon Trajectories

Mothi

Muñoz

2021

J. Phys. Chem. B

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The rates of protein (un)folding are often described as diffusion on the projection of a hyperdimensional energy landscape onto a few (ideally one) order parameters. Testing such an approximation by experiment requires resolving the reactive transition paths of individual molecules, which is now becoming feasible with advanced single-molecule spectroscopic techniques. This has also sparked the interest of theorists in better understanding reactive transition paths. Here we focus on these issues aiming to establish (i) practical guidelines for the mechanistic interpretation of transition path times (TPT) and (ii) methods to extract the free energy surface and protein dynamics from the maximum likelihood analysis of photon trajectories (MLA-PT). We represent the (un)folding rates as diffusion on a 1D free energy surface with the FRET efficiency as a reaction coordinate proxy. We then perform diffusive kinetic simulations on surfaces with two minima and a barrier, but with different shapes (curvatures, barrier height, and symmetry), coupled to stochastic simulations of photon emissions that reproduce current SM-FRET experiments. From the analysis of transition paths, we find that the TPT is inversely proportional to the barrier height (difference in free energy between minimum and barrier top) for any given surface shape, and that dividing the TPT into climb and descent segments provides key information about the barrier's symmetry. We also find that the original MLA-PT procedure used to determine the TPT from experiments underestimates its value, particularly for the cases with smaller barriers (e.g., fast folders), and we suggest a simple strategy to correct for this bias. Importantly, we also demonstrate that photon trajectories contain enough information to extract the 1D free energy surface's shape and dynamics (if TPT is >4−5-fold longer than the interphoton time) using the MLA-PT directly implemented with a diffusive free energy surface model. When dealing with real (unknown) experimental data, the comparison between the likelihoods of the free energy surface and discrete kinetic three-state models can be used to evaluate the statistical significance of the estimated free energy surface.

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“…Molecular understanding of the biological machinery and its mechanisms requires detailed knowledge of conformational changes and biomolecular dynamics . Single-molecule Förster resonance energy transfer (FRET) is a method that enables the investigation of such processes via measurements of distances and distance dynamics in biological macromolecules. − Its high sensitivity on the nanometer length scale can be used to probe the mechanisms of conformational changes, dynamics, and interactions on time scales from nanoseconds to hours . A particular strength of the technique is the identification of individual conformational subpopulations, whose time-dependent behavior often allows structural and kinetic heterogeneity to be resolved.…”

Section: Introductionmentioning

confidence: 99%

Combining Rapid Microfluidic Mixing and Three-Color Single-Molecule FRET for Probing the Kinetics of Protein Conformational Changes

et al. 2021

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Single-molecule Forster resonance energy transfer (FRET) is well suited for studying the kinetics of protein conformational changes, owing to its high sensitivity and ability to resolve individual subpopulations in heterogeneous systems. However, the most common approach employing two fluorophores can only monitor one distance at a time, and the use of three fluorophores for simultaneously monitoring multiple distances has largely been limited to equilibrium fluctuations. Here we show that three-color single-molecule FRET can be combined with rapid microfluidic mixing to investigate conformational changes in a protein from milliseconds to minutes. In combination with manual mixing, we extended the kinetics to 1 h, corresponding to a total range of 5 orders of magnitude in time. We studied the monomer-to-protomer conversion of the pore-forming toxin cytolysin A (ClyA), one of the largest protein conformational transitions known. Site-specific labeling of ClyA with three fluorophores enabled us to follow the kinetics of three intramolecular distances at the same time and revealed a previously undetected intermediate. The combination of three-color single-molecule FRET with rapid microfluidic mixing thus provides an approach for probing the mechanisms of complex biomolecular processes with high time resolution.

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Single-Molecule Fluorescence Studies of Fast Protein Folding

Cited by 11 publications

References 146 publications

Downhill, Ultrafast and Fast Folding Proteins Revised

Downhill, Ultrafast and Fast Folding Proteins Revised

Protein Folding Dynamics as Diffusion on a Free Energy Surface: Rate Equation Terms, Transition Paths, and Analysis of Single-Molecule Photon Trajectories

Combining Rapid Microfluidic Mixing and Three-Color Single-Molecule FRET for Probing the Kinetics of Protein Conformational Changes

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