An On-pathway Intermediate in the Folding of a PDZ Domain

Maurizio Brunori,Stefano Gianni,Per Jemth,Ylva Ivarsson,Francesco Malatesta,Carlo Travaglini-Allocatelli

doi:10.1074/jbc.m611026200

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.

Highlights

An early folding intermediate was proposed for murine PDZ2 from PTP-BL (PDZ2, the second PDZ domain from protein-tyrosine phosphatase Bas-like) based on a rollover effect occurring in the chevron plot under certain folding conditions [12, 13]
This bindingdriven experiment provides an unequivocal determination of the folding rate constant only when and if (i) the binding step is faster than the folding reaction, and (ii) the binding partner interacts only with the fully native state, as depicted in Scheme I, kF
In the case of PDZ2, we have shown previously that the observed rate constant for binding of the specific peptide EQVSAV is in the submillisecond time domain, i.e. faster than the folding rate constants at any ligand concentration [13, 16]

Summary

EXPERIMENTAL PROCEDURES

The V44A site-directed mutant was produced using a QuikChange site-directed mutagenesis kit (Stratagene). PDZ2 was purified as described previously [13]. All experiments were performed in the presence of 50 mM phosphate buffer, pH 7, at 25 °C. An engineered variant of PDZ2 with Tyr-43 replaced with Trp (pWT43 PDZ2) and a dansylated peptide, D-EQVSAV, were used, as described previously [16]. The fluorescence of the introduced Trp-43 is decreased upon peptide binding by Forster resonance transfer between the Trp-43 and the dansyl group. Kinetic experiments were carried out on an Applied Photophysics Pi-star stopped-flow instrument (Leatherhead, United Kingdom); peptide induced binding experiments were carried out by mixing unfolded PDZ2 (i.e. in the presence of 6 – 8 M urea) with different concentrations of peptide. The excitation wavelength was 280 nm, and the fluorescence emission was measured using a 330 Ϯ 20 nm band-pass glass filter

RESULTS

DISCUSSION

Brunori and Stefano Gianni