Single-molecule biophysics: at the interface of biology, physics and chemistry

Deniz, Ashok A.; Mukhopadhyay, Samrat; Lemke, Edward A.

doi:10.1098/rsif.2007.1021

Cited by 266 publications

(209 citation statements)

References 327 publications

(403 reference statements)

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“…A host of single molecule manipulation techniques [1][2][3][4][5], employing atomic force microscopes (AFMs) [6], microneedles [7], optical [8,9] and magnetic [10] tweezers, have provided quite detailed studies of shapes and forces in long polymers. The positional accuracy of an AFM tip [5,11] can be as good as a few nm, while forces of order of 1 pN can be measured, with measurements carried out in nearly biological conditions [12,13].…”

Section: Introductionmentioning

confidence: 99%

Polymer-mediated entropic forces between scale-free objects

2012

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The number of configurations of a polymer is reduced in the presence of a barrier or an obstacle. The resulting loss of entropy adds a repulsive component to other forces generated by interaction potentials. When the obstructions are scale invariant shapes (such as cones, wedges, lines or planes) the only relevant length scales are the polymer size R0 and characteristic separations, severely constraining the functional form of entropic forces. Specifically, we consider a polymer (single strand or star) attached to the tip of a cone, at a separation h from a surface (or another cone). At close proximity, such that h ≪ R0, separation is the only remaining relevant scale and the entropic force must take the form F = A kBT /h. The amplitude A is universal, and can be related to exponents η governing the anomalous scaling of polymer correlations in the presence of obstacles. We use analytical, numerical and ǫ-expansion techniques to compute the exponent η for a polymer attached to the tip of the cone (with or without an additional plate or cone) for ideal and selfavoiding polymers. The entropic force is of the order of 0.1pN at 0.1µm for a single polymer, and can be increased for a star polymer.

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Section: Introductionmentioning

confidence: 99%

Polymer-mediated entropic forces between scale-free objects

2012

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“…1 A), resulting in a mixture of Donor-Acceptor-and Acceptor-Donor-labeled proteins at the two positions. On the basis of the NMR structure of SDS spherical micelle-bound ␣-synuclein (26), these labeling positions flank the helical regions of the protein, and are ideal for FRET detection of global structural transitions involving long-range distance changes (20-70 Å) (36), like that between the broken and extended helical protein states (20-22, 26, 28). The smFRET data reported here are in good agreement with our previous ensemble data on wild-type ␣-synuclein (22), showing that dye-labeling does not introduce significant perturbations of protein properties.…”

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confidence: 99%

“…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.

381

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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“…In the recent past, fluorescence microscopy of single molecules has become a common mode of experimentation to study molecular interactions and conformational changes, where the unsynchronized behavior of many molecules renders the conventional ensemble measurements insufficient (1,2). Among the different single-molecule (SM) microscopy techniques the Förster resonance energy transfer (FRET) approach is one of the most fruitful (3)(4)(5).…”

Section: Introductionmentioning

confidence: 99%

DNA-Endonuclease Complex Dynamics by Simultaneous FRET and Fluorophore Intensity in Evanescent Field

Tutkus

Marčiulionis

Sasnauskas

et al. 2017

Biophysical Journal

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The single-molecule Fö rster resonance energy transfer (FRET) is a powerful tool to study interactions and conformational changes of biological molecules in the distance range from a few to 10 nm. In this study, we demonstrate a method to augment this range with longer distances. The method is based on the intensity changes of a tethered fluorophore, diffusing in the exponentially decaying evanescent excitation field. In combination with FRET it allowed us to reveal and characterize the dynamics of what had been inaccessible conformations of the DNA-protein complex. Our model system, restriction enzyme Ecl18kI, interacts with a FRET pair-labeled DNA fragment to form two different DNA loop conformations. The DNA-protein interaction geometry is such that the efficient FRET is expected for one of these conformations-''antiparallel'' loop. In the alternative ''parallel'' loop, the expected distance between the dyes is outside the range accessible by FRET. Therefore, ''antiparallel'' looping is observed in a single-molecule time trajectory as discrete transitions to a state of high FRET efficiency. At the same time, transitions to a high-intensity state of the directly excited acceptor fluorophore on a DNA tether are due to a change of its average position in the evanescent field of excitation and can be associated with a loop of either ''parallel'' or ''antiparallel'' configuration. Simultaneous analysis of FRET and acceptor intensity trajectories then allows us to discriminate different DNA loop conformations and access the average lifetimes of different states.

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Single-molecule biophysics: at the interface of biology, physics and chemistry

Cited by 266 publications

References 327 publications

Polymer-mediated entropic forces between scale-free objects

Polymer-mediated entropic forces between scale-free objects

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

DNA-Endonuclease Complex Dynamics by Simultaneous FRET and Fluorophore Intensity in Evanescent Field

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