Test for cooperativity in the early kinetic intermediate in lysozyme folding.
During the folding of many proteins, collapsed globular states are formed prior to the native structure. The role of these states for the folding process has been widely discussed. Comparison with properties of synthetic homo and heteropolymers had suggested that the initial collapse represented a shift of the ensemble of unfolded conformations to more compact states without major energy barriers. We investigated the folding/unfolding transition of a collapsed state, which transiently populates early in lysozyme folding. This state forms within the dead-time of stopped-flow mixing and it has been shown to be significantly more compact and globular than the denaturant-induced unfolded state. We used the GdmCl-dependence of the dead-time signal change to characterize the unfolding transition of the burst phase intermediate. Fluorescence and far-UV CD give identical unfolding curves, arguing for a cooperative two-state folding/unfolding transition between unfolded and collapsed lysozyme. These results show that collapse leads to a distinct state in the folding process, which is separated from the ensemble of unfolded molecules by a significant energy barrier. NMR, fluorescence and small angle X-ray scattering data further show that some local interactions in unfolded lysozyme exist at denaturant concentrations above the coil-collapse transition. These interactions might play a crucial role in the kinetic partitioning between fast and slow folding pathways.
Bachmann
Annett
A
Biozentrum der Universität Basel, Abteilung Biophysikalische Chemie, Basel, Switzerland.
Segel
Daniel
D
Kiefhaber
Thomas
T
D000818:Animals; D002645:Chickens; D004305:Dose-Response Relationship, Drug; D019791:Guanidine / Q000494:pharmacology; D007700:Kinetics; D009113:Muramidase / Q000737:chemistry*; D011487:Protein Conformation; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D020673:Protein Renaturation; D013057:Spectrum Analysis; D014961:X-Ray Diffraction
D009113:Muramidase; D019791:Guanidine