Factors determining the pKa values of the ionizable groups in proteins: their intrinsic pKas and the effects of hydrogen bonding on buried carboxyl groups

摘要

A goal of the modern protein chemist is the design of novel proteins with specificactivities or functions. One hurdle to overcome is the ability to accurately predict thepKas of ionizable groups upon their burial in the interior of a protein, where they aretypically perturbed from their intrinsic pKas. Most discussion of intrinsic pKas is basedon model compound data collected prior to the 1960's. We present here a new set ofintrinsic pKas based on model peptides, which we think are more applicable than themodel compound values. We observe some differences with the model compoundvalues, and discuss these by critically examining the compounds originally used for thedataset. One interaction affecting the pKas of ionizable groups in proteins that is notwell understood is the effect of hydrogen bonds. The side chain carboxyl of Asp33 inRNase Sa is buried, forms 3 intramolecular hydrogen bonds, and has a pKa of 2.4 in thefolded protein. One of these hydrogen bonds is to the side chain hydroxyl of Thr56. Wemutated Thr56 to alanine and valine and observed that the mutations relieves theperturbation on the carboxyl group and elevates its pKa by 1.5 and 2 units, respectively.The side chain carboxyl of Asp76 in RNase T1 is completely buried, forms 3intramolecular hydrogen bonds to other side chain groups, and has a pKa of 0.5 in the folded protein. Mutating any of the hydrogen bonding groups to the carboxyl affects itspKa differently, depending on the group mutated. Mutating all of the hydrogen bondinggroups, creating a triple mutant of RNase T1, reverses the perturbation on the pKa andelevates it to about 6.4, very near the observed pKa of other carboxyl groups buried inhydrophobic environments. We compared these experimental results with predictedresults from theoretical models based on the Solvent Accessibility Corrected Tanford-Kirkwood Equation and the finite difference solution to the linearized Poisson-Boltzmann Equation. The comparisons revealed that these models, most often used bytheoreticians, are flawed when typically applied, and some possible improvements areproposed.