Understanding pathways of protein folding and unfolding, and mapping partially folded intermediate states, are of utmost importance for our ability to decipher the physiology of misfolding diseases. The generation of maps of folding pathways on the energy landscapes of proteins is the long range goal of our research. Here we propose to address this goal by applying powerful single-molecule fluorescence methodology to study the folding of selected protein systems. We will use fluorescence resonance energy transfer (FRET) to follow conformational dynamics of protein molecules trapped within surface-tethered lipid vesicles, a unique method developed in our lab. Vesicles will be prepared under conditions where molecules have equal folding and unfolding rates, so that single- molecule trajectories will show frequent transitions between conformational states, including partially-folded intermediates. Analysis of trajectories will yield a wealth of information on folding pathways and rates of interconversion between folding intermediates. In addition, studies on freely-diffusing molecules will probe global structural changes of the proteins during folding. We will first study the 62 amino-acid protein L, known to be a two-state folder from bulk experiments. We will verify this observation on the single molecule level, as well as obtain structural information on the denatured state of the protein. We will also study a larger protein, adenylate kinase, which, as we already showed, possesses a very heterogeneous folding energy lansdcape. Intermediates observed during folding of this protein will be carefully mapped. A low-resolution structure of these intermdiates will be obtained by measuring several pairs of labeling sites. In a strong collaboration with Lynne Regan from Yale we will study single-molecule folding of the tetratricopeptide repeat proteins. This family of modular proteins, constructed of 34 amino-acid repeat units, has been suggested to obey a new folding paradigm, based on the classical Ising model, which predicts a large population of partially folded states near the transition midpoint. Single-molecule measurements will probe this intriguing assertion. This study will have impact on our understanding of misfolding diseases, like Alzheimer's and type II diabetes. Our experiments should help us identifying intermediate structures that act as 'weak points' during folding, leading to protein aggregates which are responsible for disease. ? ? ?

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Macromolecular Structure and Function B Study Section (MSFB)
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Wehrle, Janna P
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Weizmann Institute of Science
Rehovot, Israel
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Stone, Lindsey B; Mennies, Rebekah J; Waller, Jennifer M et al. (2018) Help me Feel Better! Ecological Momentary Assessment of Anxious Youths' Emotion Regulation with Parents and Peers. J Abnorm Child Psychol :
De Rosa, Lucia; Cortajarena, Aitziber L; Romanelli, Alessandra et al. (2012) Site-specific protein double labeling by expressed protein ligation: applications to repeat proteins. Org Biomol Chem 10:273-80
Haran, Gilad (2012) How, when and why proteins collapse: the relation to folding. Curr Opin Struct Biol 22:14-20
Yoo, Tae Yeon; Meisburger, Steve P; Hinshaw, James et al. (2012) Small-angle X-ray scattering and single-molecule FRET spectroscopy produce highly divergent views of the low-denaturant unfolded state. J Mol Biol 418:226-36
Sherman, Eilon; Haran, Gilad (2011) Fluorescence correlation spectroscopy of fast chain dynamics within denatured protein L. Chemphyschem 12:696-703
England, Jeremy L; Haran, Gilad (2011) Role of solvation effects in protein denaturation: from thermodynamics to single molecules and back. Annu Rev Phys Chem 62:257-77
Cortajarena, Aitziber L; Mochrie, Simon G J; Regan, Lynne (2011) Modulating repeat protein stability: the effect of individual helix stability on the collective behavior of the ensemble. Protein Sci 20:1042-7
Pirchi, Menahem; Ziv, Guy; Riven, Inbal et al. (2011) Single-molecule fluorescence spectroscopy maps the folding landscape of a large protein. Nat Commun 2:493
Cortajarena, Aitziber L; Regan, Lynne (2011) Calorimetric study of a series of designed repeat proteins: modular structure and modular folding. Protein Sci 20:336-40
Frank, Gabriel A; Goomanovsky, Mila; Davidi, Amit et al. (2010) Out-of-equilibrium conformational cycling of GroEL under saturating ATP concentrations. Proc Natl Acad Sci U S A 107:6270-4

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