Characterizing the unfolded states of proteins using single-molecule FRET spectroscopy and molecular simulations

Merchant, Kusai A.; Best, Robert B.; Louis, John M.; Gopich, Irina V.; Eaton, William A.

doi:10.1073/pnas.0607097104

Cited by 336 publications

(480 citation statements)

References 46 publications

(59 reference statements)

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“…where P (R ee ) is the end-to-end distance, R ee , probability distribution function of the protein, FRET overestimates radius of gyration in high [GuHCl]: We mimicked the FRET experiments 21,22 to estimate R F RET g from E Burst . The Gaussian polymer chain end-toend probability distribution is given by…”

Section: Resultsmentioning

confidence: 99%

“…The difference in the R g predictions of FRET 21,22 and SAXS 24,25 experiments for Protein L during the burst-phase of folding is statistically significant. The reasons for the disagreement between these experiments for Protein L are not completely clear.…”

Section: Introductionmentioning

confidence: 85%

“…In this manuscript, we study the burst-phase folding of Protein L to understand the origin of discrepancy between the FRET 21,22 and SAXS 24,25 experiments using the native-centric self-organised polymer model with side chains (SOP-SC) 41,42 and molecular dynamics simulations. The effect of [GuHCl] on Protein L conformations is taken into account using the molecular transfer model (MTM) 11,43 .…”

Section: Introductionmentioning

confidence: 99%

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Folding of Protein L with Implications for Collapse in the Denatured State Ensemble

Maity

Reddy

2016

J. Am. Chem. Soc.

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A fundamental question in protein folding is whether the coil to globule collapse transition occurs during the initial stages of folding (burst-phase) or simultaneously with the protein folding transition. Single molecule fluorescence resonance energy transfer (FRET) and small angle X-ray scattering (SAXS) experiments disagree on whether Protein L collapse transition occurs during the burst-phase of folding. We study Protein L folding using a coarse-grained model and molecular dynamics simulations. The collapse transition in Protein L is found to be concomitant with the folding transition. In the burst-phase of folding, we find that FRET experiments overestimate radius of gyration, R g , of the protein due to the application of Gaussian polymer chain end-to-end distribution to extract R g from the FRET efficiency. FRET experiments estimate ≈ 6Å decrease in R g when the actual decrease is ≈ 3Å on Guanidinium Chloride denaturant dilution from 7.5M to 1M, and thereby suggesting pronounced compaction in the protein dimensions in the burst-phase.The ≈ 3Å decrease is close to the statistical uncertainties of the R g data measured from SAXS experiments, which suggest no compaction, leading to a disagreement with the FRET experiments.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 85%

Section: Introductionmentioning

confidence: 99%

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Folding of Protein L with Implications for Collapse in the Denatured State Ensemble

Maity

Reddy

2016

J. Am. Chem. Soc.

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“…A sensitive tool for monitoring the assembly of macromolecules and their kinetics is fluorescence microscopy, by virtue of imaging the dynamics of fluorescent dyes attached to the molecules of interest. Recently, fluorescent imaging techniques have been developed for individual molecules, allowing the monitoring of molecular interactions at the single molecule level (Blanchard et al 2004;Coban et al 2006;Deniz et al 1999;Dietrich et al 2002;Friedman et al 2006;Gell et al 2006;Ha 2001Ha , 2004Merchant et al 2007;Murphy et al 2004;Myong et al 2006;Rueda et al 2004;Yasuda et al 2003). These new techniques provide unique information about the natural heterogeneity (structural and dynamics) of individual macromolecules, and allow the analysis of interactions between the molecules, the dynamics of complex formation, determination of the stoichiometry of the complex and calculation of the spatial distance between specific molecules in a complex.…”

Section: Introductionmentioning

confidence: 99%

“…In many cases, the information about inter-dye distance is extracted from FRET efficiency histograms which are often approximated by a Gaussian function (Agrawal et al 2008;Coban et al 2006;Deniz et al 1999;Dietrich et al 2002;Ha 2001;Iqbal et al 2008a; Kapanidis et al 2004;Koopmans et al 2007; Kuzmenkina et al 2006;Lee et al 2005;Merchant et al 2007;Murphy et al 2004;Nir et al 2006;Sabanayagam et al 2005;Schuler et al 2005;Sugawa et al 2007;Yildiz and Selvin 2005;Yim et al 2005;Zhang et al 2007). The apparent width of thus extracted histograms is usually in the range of 0.07-0.2 (on a scale of 0-1) and often ascribed to variations of the interdye distance (Coban et al 2006;Dietrich et al 2002;Merchant et al 2007;Schuler et al 2005;Sugawa et al 2007). Here we describe an approach to discern the contributions of various factors to the apparent width of FRET distribution, showing that photon shot noise and modulations of orientation factor due to helix flexibilities are the main sources for the experimentally observed width of the FRET distribution.…”

Section: Introductionmentioning

confidence: 99%

Probing complexes with single fluorophores: factors contributing to dispersion of FRET in DNA/RNA duplexes

2008

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Single molecule fluorescent microscopy is a method for the analysis of the dynamics of biological macromolecules by detecting the fluorescence signal produced by fluorophores associated with the macromolecule. Two fluorophores located in a close proximity may result in Förster resonance energy transfer (FRET), which can be detected at the single molecule level and the efficiency of energy transfer calculated. In most cases, the experimentally observed distribution of FRET efficiency exhibits a significant width corresponding to 0.07-0.2 (on a scale of 0-1).Here, we present a general approach describing the analysis of experimental data for a DNA/RNA duplex. We have found that for a 15 bp duplex with Cy3 and Cy5 fluorophores attached to the opposite ends of the helix, the width of the energy transfer distribution is mainly determined by the photon shot noise and the orientation factor, whereas the variation of inter-dye distances plays a minor role.

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Single‐Molecule Spectroscopy of Unfolded Proteins

Schuler

2010

Instrumental Analysis of Intrinsically Disordered Proteins

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13Single -molecule spectroscopy has developed into a versatile method to probe distances, distance distributions, and dynamics of unfolded proteins. Singlemolecule F ö rster resonance energy transfer has been used most extensively to study long -range intramolecular distance distributions and dynamics of unfolded proteins from timescales of nanoseconds to seconds . The methods developed in this context will also be helpful for studying the behavior of intrinsically disordered proteins. INTRODUCTIONThe folding of a protein is the fi rst part of its function: it has to fi nd its native three -dimensional structure -encoded in the amino acid sequence -to be able to act as an enzyme, a signal transducer, a membrane channel, or a stabilizing element of a cell, to name but a few of the many roles that proteins can take. Contrary to widespread belief, folding is not a unique, singular event in the life of a protein. Many proteins are marginally stable and will fold and unfold many times during their functional life. As described in the other chapters of ABSTRACT this book, in many cases proteins even fold only in the presence of stabilizing ligands or binding partners, illustrating the close coupling of folding and function. Correspondingly, the unfolded or denatured state is of central relevance to understanding the protein -folding reaction, especially in the context of intrinsically disordered proteins (IDPs) (24, 102) . SINGLE -MOLECULE SPECTROSCOPYIn the past decade, single -molecule methods have increasingly been employed to study protein folding. The two main methods used are force -probe techniques and F ö rster resonance energy transfer (FRET). Experiments using atomic force microscopy and laser tweezers have provided a lot of previously inaccessible information on the mechanical stability and folding of proteins, and the behavior of unfolded polypeptides under force, and the reader is referred to a number of recent reviews on this topic (7,11,27,28,111) . Here, we focus on the investigation of protein folding and especially unfolded proteins using single -molecule FRET ( Fig. 13.1 ). First demonstrated about 10 years ago (35) , single -molecule FRET has since become an important approach to study intramolecular distances and dynamics in biomolecules (106) , including protein folding (37, 62, 76, 86 -88) . FRETThe fi rst quantitative test of F ö rster ' s theory (29, 103) and the crucial experiment that put FRET on the map of biochemistry was published by Stryer and Haugland in 1967 (97) . They attached dansyl and naphthyl groups to the 372 INSTRUMENTAL ANALYSIS OF INTRINSICALLY DISORDERED PROTEINS

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Characterizing the unfolded states of proteins using single-molecule FRET spectroscopy and molecular simulations

Cited by 336 publications

References 46 publications

Folding of Protein L with Implications for Collapse in the Denatured State Ensemble

Folding of Protein L with Implications for Collapse in the Denatured State Ensemble

Probing complexes with single fluorophores: factors contributing to dispersion of FRET in DNA/RNA duplexes

Single‐Molecule Spectroscopy of Unfolded Proteins

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