  The relative solvent accessibility (RSA) of a residue in a protein measures
the extent of burial or exposure of that residue in the 3D structure. RSA is
frequently used to describe a protein's biophysical or evolutionary properties.
To calculate RSA, a residue's solvent accessibility (ASA) needs to be
normalized by a suitable reference value for the given amino acid; several
normalization scales have previously been proposed. However, these scales do
not provide tight upper bounds on ASA values frequently observed in empirical
crystal structures. Instead, they underestimate the largest allowed ASA values,
by up to 20%. As a result, many empirical crystal structures contain residues
that seem to have RSA values in excess of one. Here, we derive a new
normalization scale that does provide a tight upper bound on observed ASA
values. We pursue two complementary strategies, one based on extensive analysis
of empirical structures and one based on systematic enumeration of
biophysically allowed tripeptides. Both approaches yield congruent results that
consistently exceed published values. We conclude that previously published ASA
normalization values were too small, primarily because the conformations that
maximize ASA had not been correctly identified. As an application of our
results, we show that empirically derived hydrophobicity scales are sensitive
to accurate RSA calculation, and we derive new hydrophobicity scales that show
increased correlation with experimentally measured scales.
