2003/02/28 by Elizabeth Rhoades, Eugene Gussakovsky, Gilad Haran · 347 citations
Biochemistry, Genetics and Molecular Biology · Chemistry · Physics and Astronomy · #Biochemistry #Biology #Biophysics #Chemical physics #Chemistry #Crystallography #Downhill folding #Energy landscape #Fluorescence #Fluorophore #Folding (DSP implementation) #Förster resonance energy transfer #Lipid Membrane Structure and Behavior #Molecule #Phi value analysis #Physics #Protein Structure and Dynamics #Protein folding #Single-molecule experiment #Spectroscopy and Quantum Chemical Studies
paper · doi:10.1073/pnas.2628068100
published in Proceedings of the National Academy of Sciences 100(6), 3197-3202 (National Academy of Sciences)
openalex publication_date 2003/02/28 · openalex created_date 2025/10/10 · openalex updated_date 2026/07/22
Recent theoretical work suggests that protein folding involves an ensemble of pathways on a rugged energy landscape. We provide direct evidence for heterogeneous folding pathways from single-molecule studies, facilitated by a recently developed immobilization technique. Individual fluorophore-labeled molecules of the protein adenylate kinase were trapped within surface-tethered lipid vesicles, thereby allowing spatial restriction without inducing any spurious interactions with the environment, which often occur when using direct surface-linking techniques. The conformational fluctuations of these protein molecules, prepared at the thermodynamic midtransition point, were studied by using fluorescence resonance energy transfer between two specifically attached labels. Folding and unfolding transitions appeared in experimental time traces as correlated steps in donor and acceptor fluorescence intensity. The size of the steps, in fluorescence resonance energy transfer efficiency units, shows a very broad distribution. This distribution peaks at a relatively low value, indicating a preference for small-step motion on the energy landscape. The time scale of the transitions is also distributed, and although many transitions are too fast to be time-resolved here, the slowest ones may take >1 sec to complete. These extremely slow changes during the folding of single molecules highlight the possible importance of correlated, non-Markovian conformational dynamics.