Abstract
To follow the folding process of the protein, single-molecule burst experiments on CTPR31C-Alexa molecules were performed. Protein molecules were set to diffuse through the focus of the confocal microscope. Bursts of emitting photons from donor and acceptor fluorophores were registered by two avalanche photo diodes (APDs). Signal detection was performed using photon counting instrumentation (PicoHarp, PicoQuant). A running-average of 15 photons was first used to smooth the photon trajectory. Photons arriving at any of the detectors with a time difference smaller than 50 µs were considered as belonging to the same fluorescent burst. Bursts with more than 35 photons per burst were analyzed.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Schultz PG et al (2000) Single-molecule protein folding: Diffusion fluorescence resonance energy transfer studies of the denaturation of chymotrypsin inhibitor 2. Proc Natl Acad Sci USA 97(10):5179–5184
Gopich IV, Szabo A (2010) FRET efficiency distributions of multistate single molecules. J Phys Chem B 114(46):15221–15226
Fersht A (1999) Structure and mechanism in protein science: a guide to enzyme catalysis and protein folding. W.H. Freeman, New York. xxi, p 631
Kalinin S et al (2010) On the origin of broadening of single-molecule FRET efficiency distributions beyond shot noise limits. J Phys Chem B 114(18):6197–6206
Hagen SJ et al (1996) Diffusion-limited contact formation in unfolded cytochrome c: estimating the maximum rate of protein folding. Proc Natl Acad Sci USA 93(21):11615–11617
Pirchi M et al (2011) Single-molecule fluorescence spectroscopy maps the folding landscape of a large protein. Nat Commun 2:493
Javadi Y, Main ER (2009) Exploring the folding energy landscape of a series of designed consensus tetratricopeptide repeat proteins. Proc Natl Acad Sci USA 106(41):17383–17388
Wahl M et al (2003) Fast calculation of fluorescence correlation data with asynchronous time-correlated single-photon counting. Opt Express 11(26):3583–3591
Torres T, Levitus M (2007) Measuring conformational dynamics: a new FCS-FRET approach. J Phys Chem B 111(25):7392–7400
Krichevsky O, Bonnet G (2002) Fluorescence correlation spectroscopy: the technique and its applications. Rep Prog Phys 65:251–297
Price ES, DeVore MS, Johnson CK (2010) Detecting intramolecular dynamics and multiple Forster resonance energy transfer states by fluorescence correlation spectroscopy. J Phys Chem B 114(17):5895–5902
Lakowicz JR (1999) Principles of fluorescence spectroscopy. 2nd edn, Kluwer Academic/Plenum., New York. xxiii, p 698
Haas E, Katchalski-Katzir E, Steinberg IZ (1978) Effect of the orientation of donor and acceptor on the probability of energy transfer involving electronic transitions of mixed polarization. Biochemistry 17(23):5064–5070
Gould H, Tobochnik J (1996) An introduction to computer simulation methods. 2nd edn, Addison Wesley Publishing Company, Boston
Kohn JE et al (2004) Random-coil behavior and the dimensions of chemically unfolded proteins. Proc Natl Acad Sci USA 101(34):12491–12496
Sherman E, Haran G (2006) Coil-globule transition in the denatured state of a small protein. Proc Natl Acad Sci USA 103(31):11539–11543
Main ER et al (2005) Local and long-range stability in tandemly arrayed tetratricopeptide repeats. Proc Natl Acad Sci USA 102(16):5721–5726
Cortajarena AL, Mochrie SG, Regan L (2008) Mapping the energy landscape of repeat proteins using NMR-detected hydrogen exchange. J Mol Biol 379(3):617–626
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
Copyright information
© 2016 Springer International Publishing Switzerland
About this chapter
Cite this chapter
Cohen, S. (2016). Results. In: Single-Molecule Fluorescence Spectroscopy of the Folding of a Repeat Protein. Springer Theses. Springer, Cham. https://doi.org/10.1007/978-3-319-09558-5_3
Download citation
DOI: https://doi.org/10.1007/978-3-319-09558-5_3
Published:
Publisher Name: Springer, Cham
Print ISBN: 978-3-319-09557-8
Online ISBN: 978-3-319-09558-5
eBook Packages: Chemistry and Materials ScienceChemistry and Material Science (R0)