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To determine the energetic and structural consequences of placing a charged
group within the core of a protein, two "buried charge" mutants, Met 102----Lys
(M102K) and Leu 133----Asp (L133D) were constructed in phage T4 lysozyme. Both
proteins fold at neutral pH, although they are substantially less stable than
wild type. The activity of M102K is about 35% that of wild type, while that of
L133D is about 4%. M102K could be crystallized, and its structure was determined
at high resolution. The crystal structure (at pH 6.8) of the mutant is very
similar to that of wild type except for the alpha-helix that includes residues
108-113. In wild-type lysozyme, one side of this helix is exposed to solvent and
the other contacts Met 102. In the M102K structure this alpha-helix becomes much
more mobile, possibly allowing partial access of Lys 102 to solvent. The
stability of M102K, determined by monitoring the unfolding of the protein with
CD, is pH-dependent, consistent with the charged form of the substituted amino
acid being more destabilizing than the uncharged form. The pKa of Lys 102 was
estimated to be 6.5 both by differential titration and also by NMR analysis of
isotopically labeled protein with 13C incorporated at the C epsilon position of
all lysines. As the pH is lowered below pH 6.5, the overall three-dimensional
structure of M102K at room temperature appears to be maintained to pH 3 or so,
although there is evidence for some structural adjustment possibly allowing
solvent accessibility to the protonated form of Lys 102.
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