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Description:
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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 . |