Kinetic folding mechanism of PDZ2 from PTP-BL.
PDZ domains represent a large family of protein-interaction modules associated with a variety of unrelated proteins with different functions. We report a complete characterization of the kinetic folding mechanism of a fluorescent variant of PDZ2 from PTP-BL, investigated under a variety of different experimental conditions. For this purpose, we engineered a fluorescent variant of this protein Y43W (called pseudo-wild-type, pWT43). The results suggest the presence of a high-energy intermediate in the folding of PDZ2, as revealed by a pronounced non-linear dependence of the unfolding rate constant on denaturant concentration. Such an intermediate may or may not be detectable depending on the experimental conditions, giving rise to apparent two-state folding under stabilizing conditions (e.g. in the presence of sodium sulfate). Interestingly, even under these conditions, three-state folding can be restored by selectively destabilizing the native-like rate-limiting barrier by one specific mutation (V44A). Finally, we show that data taken on pWT43 under different experimental conditions (e.g. different pH values from 2.1 to 8.0 or in the presence of a stabilizing salt) and also data on a site-directed conservative mutant can be rationalized in terms of a simple reaction scheme involving a single set of intermediates and transition states.
Gianni
Stefano
S
Istituto Pasteur-Fondazione Cenci Bolognetti e Istituto di Biologia e Patologia Molecolari del CNR, Dipartimento di Scienze Biochimiche A. Rossi Fanelli, Università di Roma La Sapienza, Piazzale A. Moro 5, 00185 Rome, Italy.
Calosci
Nicoletta
N
Aelen
Jan M A
JM
Vuister
Geerten W
GW
Brunori
Maurizio
M
Travaglini-Allocatelli
Carlo
C
D019943:Amino Acid Substitution; D002942:Circular Dichroism; D003001:Cloning, Molecular; D005456:Fluorescent Dyes / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D054595:Protein Tyrosine Phosphatase, Non-Receptor Type 13; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013431:Sulfates / Q000737:chemistry; D013816:Thermodynamics; D014508:Urea / Q000737:chemistry
D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D013431:Sulfates; C012036:sodium sulfate; D014508:Urea; D054595:Protein Tyrosine Phosphatase, Non-Receptor Type 13; D017027:Protein Tyrosine Phosphatases