title: Effect of multiple prolyl isomerization reactions on the stability and folding kinetics of the notch ankyrin domain: experiment and theory.
abstract: Studies on the folding kinetics of the Notch ankyrin domain have demonstrated that the major refolding phase is slow, the minor refolding phase is limited by the isomerization of prolyl peptide bonds, and that unfolding is multiexponential. Here, we explore the relationship between prolyl isomerization and folding heterogeneity using a combination of experiment and simulation. Proline residues were replaced with alanine, both singly and in various combinations. These destabilizing substitutions combine to eliminate the minor refolding phase, although unfolding heterogeneity persists even when all seven proline residues are replaced. To test whether prolyl isomerization influences the major refolding phase, we modeled folding and prolyl isomerization as a system of sequential reactions. Simulations that use rate constants of the major folding phase of the Notch ankyrin domain to represent intrinsic folding indicate that even with seven prolyl isomerization reactions, only two significant phases should be observed, and that the fast observed phase provides a good approximation of the intrinsic folding in the absence of prolyl isomerization. These results indicate that the major refolding phase of the Notch ankyrin domain reflects an intrinsically slow folding transition, rather than coupling of fast folding events with slow prolyl isomerization steps. This is consistent with the observation that the single observed refolding phase of a construct in which all proline residues are replaced remains slow. Finally, the simulation fails to produce a second unfolding phase at high urea concentrations, indicating that prolyl isomerization does not play a role in the three-state mechanism that leads to this heterogeneity.
authors.affiliation: T. C. Jenkins Department of Biophysics, Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218, USA.
authors.forename: Christina Marchetti
authors.initials: CM
authors.lastname: Bradley
authors.forename: Doug
authors.initials: D
authors.lastname: Barrick
mesh_terms: D000409:Alanine / Q000737:chemistry; D019943:Amino Acid Substitution; D017089:Ankyrin Repeat; D003198:Computer Simulation; D007536:Isomerism; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D008956:Models, Chemical; D011392:Proline / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D051880:Receptors, Notch
chemical_list: D008565:Membrane Proteins; D051880:Receptors, Notch; D011392:Proline; D000409:Alanine