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Lau WL , DeGrado WF , Roder H
The Effects of pK(a) Tuning on the Thermodynamics and Kinetics of Folding: Design of a Solvent-Shielded Carboxylate Pair at the a-Position of a Coiled-Coil
Biophysical Journal. 2010 Oct;99(7) :2299-2308
PMID: ISI:000282850600036    PMCID: PMC3042555   
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Abstract
The tuning of the pK(a) of ionizable residues plays a critical role in various protein functions, such as ligand-binding, catalysis, and allostery. Proteins harness the free energy of folding to position ionizable groups in highly specific environments that strongly affect their pK(a) values. To investigate the interplay among protein folding kinetics, thermodynamics, and pK(a) modulation, we introduced a pair of Asp residues at neighboring interior positions of a coiled-coil. A single Asp residue was replaced for an Asn side chain at the a-position of the coiled-coil from GCN4, which was also crosslinked at the C-terminus via a flexible disulfide bond. The thermodynamic and kinetic stability of the system was measured by circular dichroism and stopped-flow fluorescence as a function of pH and concentration of guanidine HCl. Both sets of data are consistent with a two-state equilibrium between fully folded and unfolded forms. Distinct pK(a) values of 6.3 and 5.35 are assigned to the first and second protonation of the Asp pair; together they represent an energetic difference of 5 kcal/mol relative to the protonation of two Asp residues with unperturbed pK(a) values. Analysis of the rate data as a function of pH and denaturant concentration allowed calculation of the kinetic constants for the conformational transitions of the peptide with the Asp residues in the doubly protonated, singly protonated, and unprotonated forms. The doubly and singly protonated forms fold rapidly, and a phi-value analysis shows that their contribution to folding occurs subsequent to the transition state ensemble for folding. By contrast, the doubly charged state shows a reduced rate of folding and a phi-value near 0.5 indicative of a repulsive interaction, and possibly also heterogeneity in the transition state ensemble.
Notes
Lau, Wai Leung DeGrado, William F. Roder, Heinrich National Institutes of Health [GM056250, GM54616]; National Cancer Institute [CA06927] The work was supported by grants from the National Institutes of Health (No. GM056250 to H.R. and No. GM54616 to W.F.D.), a grant from the National Cancer Institute (No. CA06927), and an Appropriation by the Commonwealth of Pennsylvania to the Fox Chase Cancer Center. 68 Cell press; 600 technology square, 5th floor, cambridge, ma 02139 usa 662yq