Arbely E.,Center for Protein Engineering |
Arbely E.,Cambridge Chemical Laboratories |
Rutherford T.J.,Center for Protein Engineering |
Rutherford T.J.,Cambridge Chemical Laboratories |
And 8 more authors.
Journal of Molecular Biology | Year: 2010
The protein BBL undergoes structural transitions and acid denaturation between pH1.2 and 8.0. Using NMR spectroscopy, we measured the pKa values of all the carboxylic residues in this pH range. We employed 13C direct-detection two-dimensional IPAP (in-phase antiphase) CACO NMR spectroscopy to monitor the ionization state of different carboxylic groups and demonstrated its advantages over other NMR techniques in measuring pKa values of carboxylic residues. The two residues Glu161 and Asp162 had significantly lowered pKa values, showing that these residues are involved in a network of stabilizing electrostatic interactions, as is His166. The other carboxylates had unperturbed values. The pH dependence of the free energy of denaturation was described quantitatively by the ionizations of those three residues of perturbed pKa, and, using thermodynamic cycles, we could calculate their pKas in the native and denatured states as well as the equilibrium constants for denaturation of the different protonation states. We also measured 13Cα chemical shifts of individual residues as a function of pH. These shifts sense structural transitions rather than ionizations, and they titrated with pH consistent with the change in equilibrium constant for denaturation. Kinetic measurements of the folding of BBL E161Q indicated that, at pH7, the stabilizing interactions with Glu161 are formed mainly in the transition state. We also found that local interactions still exist in the acid-denatured state of BBL, which attenuate somewhat the flexibility of the acid-denatured state. © 2010 Elsevier Ltd.
Dodson C.A.,Center for Protein Engineering |
Dodson C.A.,Institute of Cancer Research |
Ferguson N.,Center for Protein Engineering |
Ferguson N.,University College Dublin |
And 4 more authors.
Protein Engineering, Design and Selection | Year: 2010
The SAP domain from the Saccharomyces cerevisiae THO1 protein contains a hydrophobic core and just two -helices. It could provide a system for studying protein folding that bridges the gap between studies on isolated helices and those on larger protein domains. We have engineered the SAP domain for protein folding studies by inserting a tryptophan residue into the hydrophobic core (L31W) and solved its structure. The helical regions had a backbone root mean-squared deviation of 0.9 from those of wild type. The mutation L31W destabilised wild type by 0.8 ± 0.1 kcal mol -1. The mutant folded in a reversible, apparent two-state manner with a microscopic folding rate constant of around 3700 s -1 and is suitable for extended studies of folding. © The Author 2010.
Arbely E.,Medical Research Council Center for Protein Engineering |
Neuweiler H.,Medical Research Council Center for Protein Engineering |
Sharpe T.D.,Cambridge Chemical Laboratories |
Johnson C.M.,Medical Research Council Center for Protein Engineering |
And 2 more authors.
Protein Science | Year: 2010
Peripheral subunit binding domains (PSBDs) are integral parts of large multienzyme complexes involved in carbohydrate metabolism. PSBDs facilitate shuttling of prosthetic groups between different catalytic subunits. Their protein surface is characterized by a high density of positive charges required for binding to subunits within the complex. Here, we investigated folding thermodynamics and kinetics of the human PSBD (HSBD) using circular dichroism and tryptophan fluorescence experiments. HSBD was only marginally stable under physiological solvent conditions but folded within microseconds via a barrier-limited apparent two-state transition, analogous to its bacterial homologues. The high positive surface-charge density of HSBD leads to repulsive Coulomb forces that modulate protein stability and folding kinetics, and appear to even induce native-state movement. The electrostatic strain was alleviated at high solution-ionic-strength by Debye-Hückel screening. Differences in ionic-strength dependent characteristics among PSBD homologues could be explained by differences in their surface charge distributions. The findings highlight the trade-off between protein function and stability during protein evolution. Published by Wiley-Blackwell. © 2010 The Protein Society.