title: What causes hyperfluorescence: folding intermediates or conformationally flexible native states?
abstract: Hyperfluorescent intensity maxima during protein unfolding titrations are often taken as a sign for a thermodynamic folding intermediate. Here we explore another possibility: that hyperfluorescence could be the signature of a "pretransition" conformationally loosened native state. To model such native states, we study mutants of a fluorescent ubiquitin variant, placing cavities at various distances from the tryptophan fluorophore. We examine the correlation between protein flexibility and enhanced fluorescence intensity by using circular dichroism, fluorescence intensity unfolding titrations, fluorescence anisotropy measurements, and molecular dynamics. Based on experiment and simulation, we propose a simple model for hyperfluorescence in terms of static and dynamic conformational properties of the native state during unfolding. Apomyoglobin denaturant unfolding and phosphoglycerate kinase cold denaturation are discussed as examples. Our results do not preclude the existence of thermodynamic intermediates but do raise caution that by itself, hyperfluorescence during unfolding titrations is not conclusive proof of thermodynamic folding intermediates.
authors.affiliation: Department of Chemistry, Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Illinois 61801, USA.
authors.forename: John
authors.initials: J
authors.lastname: Ervin
authors.forename: Edgar
authors.initials: E
authors.lastname: Larios
authors.forename: Szabolcs
authors.initials: S
authors.lastname: Osváth
authors.forename: Klaus
authors.initials: K
authors.lastname: Schulten
authors.forename: Martin
authors.initials: M
authors.lastname: Gruebele
mesh_terms: D016880:Anisotropy; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D014644:Genetic Variation; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D011487:Protein Conformation; D017510:Protein Folding; D012984:Software; D013696:Temperature; D013816:Thermodynamics; D013997:Time Factors; D025801:Ubiquitin / Q000737:chemistry*
chemical_list: D025801:Ubiquitin