title: Isothermal calorimetry as a tool to investigate slow conformational changes in proteins and peptides.
abstract: A new calorimetric method has been developed to follow the time course of slow conformational changes during the refolding of denatured proteins. The method is based on the ability of isothermal titration calorimeters (ITC) to detect small amounts of heat continuously over a minute to an hour time range without being disturbed by baseline drift. We benchmarked the method on the basis of the slow kinetic phases resulting from prolyl cis/trans isomerization of oligopeptides. Using this method, the simultaneous investigation of the kinetics and thermodynamics of slow phases in the refolding of GdmCl-denatured RNase A by single jump techniques was performed. Time traces of heat production in the presence of a peptidyl prolyl cis/trans isomerase support the classical model of rate-limiting prolyl trans to cis isomerizations in the folding reactions of RNase A. However, we also observed that, unlike prolyl cis/trans isomerizations in oligopeptides, those found in RNase A refolding are highly exothermic. It appears that coupling between slow prolyl trans to cis isomerization and relocation of remote backbone segments increases the number of contacting sites during formation of the native protein. The results demonstrate that calorimetrically monitored folding kinetics will be of relevance in the detection of otherwise silent folding events.
authors.affiliation: Max-Planck-Forschungsstelle für Enzymologie der Proteinfaltung, Weinbergweg 22, D-06120 Halle (Saale), Germany. joergfanghaenel@web.de
authors.forename: Jörg
authors.initials: J
authors.lastname: Fanghänel
authors.forename: Stephan
authors.initials: S
authors.lastname: Wawra
authors.forename: Christian
authors.initials: C
authors.lastname: Lücke
authors.forename: Dirk
authors.initials: D
authors.lastname: Wildemann
authors.forename: Gunter
authors.initials: G
authors.lastname: Fischer
mesh_terms: D002151:Calorimetry / Q000379:methods*; D007700:Kinetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D010455:Peptides / Q000737:chemistry*; D011487:Protein Conformation; D011489:Protein Denaturation; D011506:Proteins / Q000737:chemistry*; D013816:Thermodynamics
chemical_list: D010455:Peptides; D011506:Proteins