title: An on-pathway intermediate in the folding of a PDZ domain.
abstract: The folding pathways of some proteins include the population of partially structured species en route to the native state. Identification and characterization of these folding intermediates are particularly difficult as they are often only transiently populated and play different mechanistic roles, being either on-pathway productive species or off-pathway kinetic traps. To define the role of folding intermediates, a quantitative analysis of the folding and unfolding rate constants over a wide range of denaturant concentration is often required. Such a task is further complicated by the reversible nature of the folding reaction, which implies the observed kinetics to be governed by a complex combination of different microscopic rate constants. Here, we tackled this problem by measuring directly the folding rate constant under highly denaturing conditions, namely by inducing the folding of a PDZ domain through a quasi-irreversible binding reaction with a specific peptide. In analogy with previous works based on hydrogen exchange experiments, we present evidence that the folding pathway of the PDZ domain involves the formation of an obligatory on-pathway intermediate. The results presented exemplify a novel type of kinetic test to detect an on-pathway folding intermediate.
authors.affiliation: Istituto di Biologia e Patologia Molecolari del Consiglio Nazionale delle Ricerche, Dipartimento di Scienze Biochimiche A. Rossi Fanelli, Università di Roma La Sapienza, Piazzale A. Moro 5, 00185 Rome, Italy.
authors.forename: Ylva
authors.initials: Y
authors.lastname: Ivarsson
authors.forename: Carlo
authors.initials: C
authors.lastname: Travaglini-Allocatelli
authors.forename: Per
authors.initials: P
authors.lastname: Jemth
authors.forename: Francesco
authors.initials: F
authors.lastname: Malatesta
authors.forename: Maurizio
authors.initials: M
authors.lastname: Brunori
authors.forename: Stefano
authors.initials: S
authors.lastname: Gianni
mesh_terms: D020816:Amino Acid Motifs; D001671:Biochemistry / Q000379:methods*; D004305:Dose-Response Relationship, Drug; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D008956:Models, Chemical; D010455:Peptides / Q000737:chemistry; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013696:Temperature; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry; D014508:Urea / Q000494:pharmacology
chemical_list: D010455:Peptides; D014364:Tryptophan; D014508:Urea