title: Single-molecule fluorescence spectroscopy maps the folding landscape of a large protein.
abstract: Proteins attain their function only after folding into a highly organized three-dimensional structure. Much remains to be learned about the mechanisms of folding of large multidomain proteins, which may populate metastable intermediate states on their energy landscapes. Here we introduce a novel method, based on high-throughput single-molecule fluorescence experiments, which is specifically geared towards tracing the dynamics of folding in the presence of a plethora of intermediates. We employ this method to characterize the folding reaction of a three-domain protein, adenylate kinase. Using thousands of single-molecule trajectories and hidden Markov modelling, we identify six metastable states on adenylate kinase's folding landscape. Remarkably, the connectivity of the intermediates depends on denaturant concentration; at low concentration, multiple intersecting folding pathways co-exist. We anticipate that the methodology introduced here will find broad applicability in the study of folding of large proteins, and will provide a more realistic scenario of their conformational dynamics.
authors: Chemical Physics Department, Weizmann Institute of Science, Rehovot, Israel.
authors: Menahem
authors: M
authors: Pirchi
authors: Guy
authors: G
authors: Ziv
authors: Inbal
authors: I
authors: Riven
authors: Sharona Sedghani
authors: SS
authors: Cohen
authors: Nir
authors: N
authors: Zohar
authors: Yoav
authors: Y
authors: Barak
authors: Gilad
authors: G
authors: Haran
mesh_terms: D000465:Algorithms; D008390:Markov Chains; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D011506:Proteins