Single‐Molecule Fluorescence Spectroscopy of Protein Folding

Abstract

Single-molecule spectroscopy is an important new approach for studying the intrinsically heterogeneous process of protein folding. This Review illustrates how different single-molecule fluorescence techniques have improved our understanding of mechanistic aspects in protein folding, exemplified by a series of recent experiments on a small protein.

Shot-Noise Limited Single-Molecule FRET Histograms: Comparison between Theory and Experiments

“…This is therefore also the probability of observing a PR value equal to x = A/S for bursts of S photons (11) Limiting ourselves to rational values x, this can be rewritten (12) where we define χ N (t) as the characteristic function of natural numbers (13) The total probability to observe the PR value x requires summing eq 12 over all burst sizes S, weighted by their probability, given by the burst size distribution (14) where B is the total number of bursts in the BSD, and S min (respectively, S max ) the smallest (respectively, largest) burst size taken into consideration. Introducing the characteristic function of rational numbers in [0, 1] (15) one obtains (16) Using eq 10, we end up with our final prediction of the probability distribution of PR values (in the absence of background) (17) To obtain the predicted PRH, a natural way could be to compute the number of predicted occurrences of each PR value x by multiplying eq 17 by the total number of bursts B and histogram these values with the same bin numbers as the PRH.…”

“…2,3 These approaches have been used to study (i) the conformation and conformational dynamics of nucleic acids, ribozymes, 4 and proteins, 5 (ii) the interaction of proteins with nucleic acids or other proteins, 6,7 (iii) the dimerization of membrane receptors in live cells, 8 (iv) the folding and unfolding of proteins, 5,9 and many other questions described in recent reviews. 2,3,5,[10][11][12][13] Although FRET in principle provides a way to obtain distances between dyes, and therefore to obtain structural information on molecular species, in practice extracting accurate distances from smFRET measurements can be delicate due to the difficulty of measuring critical experimental parameters such as the dyes' rotational freedom of motion (and hence the orientational factor κ 2 ), detection efficiencies, dye quantum yields, donor fluorescence leakage in the acceptor channel, or direct excitation of the acceptor, to cite only a few. 14,15 Luckily, even without a complete knowledge of all these parameters, smFRET measurements have demonstrated their utility to detect structural changes and quantify conformational subpopulations, as well as, in certain conditions, giving access to the time scales of various fluctuations taking place in biomolecules.…”

Resonance Energy Transfer

“…This derives from a quantitative assessment of the inter-chromophore separations, based on comparisons between the corresponding RET efficiencies. [35][36][37][38] Such a technique is popularly known as a 'spectroscopic ruler'. The elucidations of molecular structure by such means usually lack information on the relative orientations of the groups involved, and as an expedient the calculations usually ignore the kappa parameter (4).…”

“…the variation in proximity of one chromophore with respect to another, a number of valuable RET applications arise; including the detection of conformational changes and folding in proteins, 36,[44][45][46] and the inspection of intracellular protein-protein [47][48][49][50] and protein-DNA 51,52 interactions (see for example Figs 5 and 6). These and other such processes can be registered by selectively exciting one chromophore using laser light, and monitoring either the decrease in fluorescence from that chromophore, or the rise in the generally longer-wavelength fluorescence from the other chromophore as it adopts the role of acceptor.…”

Non-Markovian properties and multiscale hidden Markovian network buried in single molecule time series

“…Single-molecule (SM) spectroscopy provides a means to uncover the complex mechanism of molecular machinery at single molecule level that one cannot address in bulk measurements. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16] For example, it was found in an in vitro reconstituted system that the recognition (i.e., dissociation and association) kinetics between epidermal growth factor receptor (EGFR) on the plasma membrane and its adaptor protein Ash/Grb2 (Grb2) show non-exponential kinetics, and association kinetics depends nonlinearly on the Grb2 concentration, suggesting the existence of molecular memory in the signaling process. 16 Such innovative experimental developments of synthetic approaches using intact plasma membrane fractions a) Email: tamiki@es.hokudai.ac.jp have offered new challenges in theoretical modeling of the underlying mechanism of molecular machinery.…”