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Biophys J, June 2002, p. 3289-3304, Vol. 82, No. 6
*Department of Biophysics, The Johns Hopkins University,
Baltimore, Maryland 21218 and Department of
Chemistry and Biochemistry, University of Arkansas, Fayetteville,
Arkansas 72701 USA
Lys-66 and Glu-66, buried in the hydrophobic
interior of staphylococcal nuclease by mutagenesis, titrate with
pKa values of 5.7 and 8.8, respectively (Dwyer et al.,
2000, Biophys. J. 79:1610-1620; García-Moreno
E. et al., 1997, Biophys. Chem. 64:211-224). Continuum calculations with static structures reproduced the pKa
values when the protein interior was treated with a dielectric constant (
in) of 10. This high apparent polarizability can be
rationalized in the case of Glu-66 in terms of internal water
molecules, visible in crystallographic structures, hydrogen bonded to
Glu-66. The water molecules are absent in structures with Lys-66; the
high polarizability cannot be reconciled with the hydrophobic
environment surrounding Lys-66. Equilibrium thermodynamic experiments
showed that the Lys-66 mutant remained folded and native-like after
ionization of the buried lysine. The high polarizability must therefore
reflect water penetration, minor local structural rearrangement, or
both. When in pKa calculations with continuum methods, the
internal water molecules were treated explicitly, and allowed to relax in the field of the buried charged group, the pKa values of
buried residues were reproduced with in in the range
4-5. The calculations show that internal waters can modulate
pKa values of buried residues effectively, and they support
the hypothesis that the buried Lys-66 is in contact with internal
waters even though these are not seen crystallographically. When only
the one or two innermost water molecules were treated explicitly,
in of 5-7 reproduced the pKa values. These
values of in > 4 imply that some conformational reorganization occurs concomitant with the ionization of the buried groups.
Biophys J, June 2002, p. 3289-3304, Vol. 82, No. 6
© 2002 by the Biophysical Society 0006-3495/02/06/3289/16 $2.00
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