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.
| Identifer | oai:union.ndltd.org:tamu.edu/oai:repository.tamu.edu:1969.1/4832 |
| Date | 25 April 2007 |
| Creators | Thurlkill, Richard Lee |
| Contributors | Pace, C. Nick |
| Publisher | Texas A&M University |
| Source Sets | Texas A and M University |
| Language | en_US |
| Detected Language | English |
| Type | Book, Thesis, Electronic Dissertation, text |
| Format | 1381089 bytes, electronic, application/pdf, born digital |
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