Although validation studies show that theoretical models for predictin
g the pK(a)s of ionizable groups in proteins are increasingly accurate
, a number of important questions remain: (1) What factors limit the a
ccuracy of current models? (2) How can conformational flexibility of p
roteins best be accounted for? (3) Will use of solution structures in
the calculations, rather than crystal structures, improve the accuracy
of the computed pK(a)s? and (4) Why does accurate prediction of prote
in pK(a)s seem to require that a high dielectric constant be assigned
to the protein interior? This paper addresses these and related issues
. Among the conclusions are the following: (1) computed pK(a)s average
d over NMR structure sets are more accurate than those based upon sing
le crystal structures; (2) use of atomic parameters optimized to repro
duce hydration energies of small molecules improves agreement with exp
eriment when a low protein dielectric constant is assumed; (3) despite
use of NMR structures and optimized atomic parameters, pK(a)s compute
d with a protein dielectric constant of 20 are more accurate than thos
e computed with a low protein dielectric constant; (4) the pK(a) shift
s in ribonuclease A that result from phosphate binding are reproduced
reasonably well by calculations; (5) the substantial pK(a) shifts obse
rved in turkey ovomucoid third domain result largely from interactions
among ionized groups; and (6) both experimental data and calculations
indicate that proteins tend to lower the pK(a)s of Asp side chains bu
t have little overall effect upon the pK(a)s of other ionizable groups
.