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

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Title: Factors determining the pKa values of the ionizable groups in proteins: their intrinsic pKas and the effects of hydrogen bonding on buried carboxyl groups
Author: Thurlkill, Richard Lee
Abstract: A goal of the modern protein chemist is the design of novel proteins with specific activities or functions . One hurdle to overcome is the ability to accurately predict the pKas of ionizable groups upon their burial in the interior of a protein , where they are typically perturbed from their intrinsic pKas . Most discussion of intrinsic pKas is based on model compound data collected prior to the 1960's . We present here a new set of intrinsic pKas based on model peptides , which we think are more applicable than the model compound values . We observe some differences with the model compound values , and discuss these by critically examining the compounds originally used for the dataset . One interaction affecting the pKas of ionizable groups in proteins that is not well understood is the effect of hydrogen bonds . The side chain carboxyl of Asp33 in RNase Sa is buried , forms 3 intramolecular hydrogen bonds , and has a pKa of 2 .4 in the folded protein . One of these hydrogen bonds is to the side chain hydroxyl of Thr56 . We mutated Thr56 to alanine and valine and observed that the mutations relieves the perturbation 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 3 intramolecular 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 its pKa differently , depending on the group mutated . Mutating all of the hydrogen bonding groups , creating a triple mutant of RNase T1 , reverses the perturbation on the pKa and elevates it to about 6 .4 , very near the observed pKa of other carboxyl groups buried in hydrophobic environments . We compared these experimental results with predicted results 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 by theoreticians , are flawed when typically applied , and some possible improvements are proposed .
URI: http : / /hdl .handle .net /1969 .1 /4832
Date: 2007-04-25

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Factors determining the pKa values of the ionizable groups in proteins: their intrinsic pKas and the effects of hydrogen bonding on buried carboxyl groups. Available electronically from http : / /hdl .handle .net /1969 .1 /4832 .

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