Effect of Modulating Unfolded State Structure on the Folding Kinetics of the Villin Headpiece Subdomain

• Published in: Proceedings of the National Academy of Sciences - PNAS 2005-11, Vol.102 (46), p.16662-16667
• Main Authors: Scott H. Brewer, Dung M. Vu, Yuefeng Tang, Li, Ying, Franzen, Stefan, Raleigh, Daniel P., Dyer, R. Brian
• Format: Article
• Language: English
• Subjects: Absorption spectra; Amides; Amplitude; Biological Sciences; Fourier transforms; Infrared radiation; Kinetics; Laser beams; Microfilament Proteins - chemistry; Mutation; Nuclear Magnetic Resonance, Biomolecular; Protein Conformation; Protein Folding; Proteins; Solvents; Spectroscopy, Fourier Transform Infrared; Spectrum analysis; Temperature; Temperature dependence; Thermodynamic equilibrium; Thermodynamics
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0505432102

Equilibrium Fourier transform infrared (FTIR) and temperature-jump (T-jump) IR spectroscopic techniques were used to study the thermodynamics and kinetics of the unfolding and folding of the villin headpiece helical subdomain (HP36), a small three-helix protein. A double phenylalanine mutant (HP36 F47L, F51L) that destabilizes the hydrophobic core of this protein also was studied. The double mutant is less stable than wild type (WT) and has been shown to contain less residual secondary structure and tertiary contacts in its unfolded state. The relaxation kinetics after a T-jump perturbation were studied for both HP36 and HP36 F47L, F51L. Both proteins exhibited biphasic relaxation kinetics in response to a T-jump. The folding times for the WT (3.23 µs at 60.2°C) and double phenylalanine mutant (3.01 µs at 49.9°C) at the approximate midpoints of their thermal unfolding transitions were found to be similar. The folding time for the WT was determined to be 3.34 µts at 49.9µC, similar to the folding time of the double phenylalanine mutant at that temperature. The double phenylalanine mutant, however, unfolds faster with an unfolding time of 3.01 µs compared with 6.97 µs for the WT at 49.9°C.