Experimental tools of increasing sophistication have been employed in recent years to study protein folding and misfolding. Folding is considered a complex process, and one way to address it is by studying small proteins, which seemingly possess a simple energy landscape with essentially only two stable states, either folded or unfolded. The B1-IgG binding domain of protein L (PL) is considered a model two-state folder, based on measurements using a wide range of experimental techniques. We applied single-molecule fluorescence resonance energy transfer (FRET) spectroscopy in conjunction with a hidden Markov model analysis to fully characterize the energy landscape of PL and to extract the kinetic properties of individual molecules of the protein. Surprisingly, our studies revealed the existence of a third state, hidden under the two-state behavior of PL due to its small population, ∼7%. We propose that this minority intermediate involves partial unfolding of the two C-terminal β strands of PL. Our work demonstrates that single-molecule FRET spectroscopy can be a powerful tool for a comprehensive description of the folding dynamics of proteins, capable of detecting and characterizing relatively rare metastable states that are difficult to observe in ensemble studies.
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Two states or not two states: Single-molecule folding studies of protein L
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28 March 2018
Research Article|
October 31 2017
Two states or not two states: Single-molecule folding studies of protein L

Haim Yuval Aviram;
Haim Yuval Aviram
1
Department of Chemical Physics, Weizmann Institute of Science
, Rehovot 7610001, Israel
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Menahem Pirchi;
Menahem Pirchi
1
Department of Chemical Physics, Weizmann Institute of Science
, Rehovot 7610001, Israel
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Yoav Barak;
Yoav Barak
2
Chemical Research Support, Weizmann Institute of Science
, Rehovot 7610001, Israel
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Inbal Riven;
Inbal Riven
1
Department of Chemical Physics, Weizmann Institute of Science
, Rehovot 7610001, Israel
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Gilad Haran
Gilad Haran
a)
1
Department of Chemical Physics, Weizmann Institute of Science
, Rehovot 7610001, Israel
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a)
Author to whom correspondence should be addressed: [email protected]
J. Chem. Phys. 148, 123303 (2018)
Article history
Received:
July 25 2017
Accepted:
October 04 2017
Connected Content
A companion article has been published:
Important model protein found to fold in unexpected way by single-molecule FRET spectroscopy
Citation
Haim Yuval Aviram, Menahem Pirchi, Yoav Barak, Inbal Riven, Gilad Haran; Two states or not two states: Single-molecule folding studies of protein L. J. Chem. Phys. 28 March 2018; 148 (12): 123303. https://doi.org/10.1063/1.4997584
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