Single‐Molecule Fluorescence Studies of Protein Folding and Conformational Dynamics

Michalet, Xavier; Weiss, Shimon; Jaeger, Marcus

doi:10.1002/chin.200631292

Cited by 95 publications

(127 citation statements)

References 69 publications

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“…Here, we focus on one new and exciting area in this category, the application of fluorescence at the single-molecule level [41][42][43][44] . This technique brings to life a dream that biochemists have had for many years -watching a single protein molecule functioning in real time.…”

Section: Experimental Low-resolution and Local-site Methodsmentioning

confidence: 99%

Dynamic personalities of proteins

Henzler-Wildman

Kern

2007

Nature

2,095

2,222

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Life is marked by change over time, and biologists explore this phenomenon by watching, for example, Caenorhabditis elegans developing from embryos into adults, mice running in a cage, and nerve cells firing. In search of how and why, biology arrived at the molecular level. Understanding protein function on an atomic level has been revolutionized by high-resolution X-ray crystallography, resulting in a surge in studies of structure-function relationships. The detail in these colourful structures flooding the covers of modern journals can be deceptive, suggesting that one unique structure, the 'folded state' , is the final answer. Ironically, the dynamic nature of biology seems to have been forgotten at this microscopic level.Physicists, however, will object to a static picture: they see proteins as soft materials that sample a large ensemble of conformations around the average structure as a result of thermal energy. A complete description of proteins requires a multidimensional energy landscape that defines the relative probabilities of the conformational states (thermodynamics) and the energy barriers between them (kinetics). In biology, this concept has recently gained traction, leading to an extension of the structure-function paradigm to include dynamics. To understand proteins in action, the fourth dimension, time, must be added to the snapshots of proteins frozen in crystal structures. A major obstacle is that it is not possible to watch experimentally individual atoms moving within a protein. Instead, sophisticated biophysical methods are needed to measure the physical properties from which the dynamics can be inferred.In this review, we discuss how protein function is rooted in the energy landscape. The basic concepts and the biophysical methods are illustrated by several examples. To avoid past semantic confusion about the term protein dynamics, we define it as any time-dependent change in atomic coordinates. Protein dynamics thus includes both equilibrium fluctuations and non-equilibrium effects. The fluctuations observed at equilibrium seem to govern biological function in processes both near and far from equilibrium; therefore, we focus on these motions. Non-equilibrium effects are also called dynamical effects 1 (the source of confusion 2 ), and they have a minimal effect on the overall rates of biological processes 3,4 . Biological motors that convert chemical energy to mechanical energy 5,6 are not discussed here. The energy landscapeAlthough the idea of an energy landscape might be most familiar in the context of protein folding (for example, the folding funnel hypothesis) [7][8][9] , this concept had already been applied to folded proteins more than 30 years ago by Frauenfelder and co-workers . Subsequent studies on myoglobin led to the idea that substates are in thermal equilibrium and that both solvent 13-15 and ligands influence the landscape (Fig. 1a). At the glass transition temperature 10,12 , an increase in anharmonic dynamics occurs in proteins, and this is interpreted as the protein no...

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Section: Experimental Low-resolution and Local-site Methodsmentioning

confidence: 99%

Dynamic personalities of proteins

Henzler-Wildman

Kern

2007

Nature

2,095

2,222

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“…To better understand the complexities of ␣-synuclein folding, we turned to single-molecule experiments, which avoid loss of information due to ensemble averaging (35)(36)(37)(38)(39)(40) and thus benefit studies of protein folding landscapes (41)(42)(43) by permitting multiple structural distributions and coexisting populations to be resolved and examined in a more straightforward and modelindependent manner. Here, we use single-molecule fluorescence resonance energy transfer (smFRET) as a long-range distance ruler (44) to provide unequivocal evidence for the structural interplay between the broken and extended ␣-helix structures observed for the protein, induced by binding to SDS and phospholipid SUVs.…”

mentioning

confidence: 99%

Interplay of α-synuclein binding and conformational switching probed by single-molecule fluorescence

Ferreon

Gambin²,

Lemke³

et al. 2009

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

378

449

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We studied the coupled binding and folding of ␣-synuclein, an intrinsically disordered protein linked with Parkinson's disease. Using single-molecule fluorescence resonance energy transfer and correlation methods, we directly probed protein membrane association, structural distributions, and dynamics. Results revealed an intricate energy landscape on which binding of ␣-synuclein to amphiphilic small molecules or membrane-like partners modulates conformational transitions between a natively unfolded state and multiple ␣-helical structures. ␣-Synuclein conformation is not continuously tunable, but instead partitions into 2 main classes of folding landscape structural minima. The switch between a broken and an extended helical structure can be triggered by changing the concentration of binding partners or by varying the curvature of the binding surfaces presented by micelles or bilayers composed of the lipid-mimetic SDS. Single-molecule experiments with lipid vesicles of various composition showed that a low fraction of negatively charged lipids, similar to that found in biological membranes, was sufficient to drive ␣-synuclein binding and folding, resulting here in the induction of an extended helical structure. Overall, our results imply that the 2 folded structures are preencoded by the ␣-synuclein amino acid sequence, and are tunable by small-molecule supramolecular states and differing membrane properties, suggesting novel control elements for biological and amyloid regulation of ␣-synuclein.A lpha-synuclein, a highly acidic 140-residue protein expressed at high levels in the human brain and enriched in presynaptic nerve termini, is a member of the growing class of intrinsically disordered proteins that adopt ordered structure upon interaction with cellular partners (1-5). This natively unfolded protein plays crucial roles in the pathogenesis of several neurodegenerative disorders including Parkinson's disease and Alzheimer's disease (6-9). Although several physiological functions have been proposed for the protein, including roles in the regulation of distinct pools of presynaptic vesicles (10, 11), maintenance of SNARE protein complexes (12), modulation of neural plasticity (13), control of dopamine neurotransmission (14), and ER-Golgi trafficking (15), its precise biological role remains unclear. Nevertheless, membrane interaction is generally believed to be a key modulator of ␣-synuclein function (16,17).Sequence analysis predicts ␣-synuclein interaction with lipid membranes through amphipathic ␣-helices encoded by 7 imperfect 11-residue repeats, approximately 4 of which are located in the highly basic N-terminal region of the protein, and 3 in the highly acidic and hydrophobic NAC region (non-A␤ component of Alzheimer's disease amyloid) (13, 18). Not surprisingly, the protein undergoes structural transitions upon binding to either brain-derived or synthetic acidic phospholipid vesicles, adopting ␣-helical conformations in the membrane-bound form (17)(18)(19). Similarly, ␣-synuclein assumes helical structu...

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“…Here, we take a step towards closing this gap by investigating the GroEL/GroES chaperonin (1-3, 9) with single-molecule fluorescence spectroscopy (21)(22)(23)(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.…”

mentioning

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.

109

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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Single‐Molecule Fluorescence Studies of Protein Folding and Conformational Dynamics

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Cited by 95 publications

References 69 publications

Dynamic personalities of proteins

Dynamic personalities of proteins

Interplay of α-synuclein binding and conformational switching probed by single-molecule fluorescence

Single-molecule spectroscopy of protein folding in a chaperonin cage

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