Cellulases are increasingly being used for industrial
purposes, particularly in washing powders, yet little is
known of the factors governing the stability of proteins
in detergent solutions. We present a comparative analysis
of the behavior of the cellulase Cel45 from Humicola
insolens in the presence of the denaturant guanidinium
chloride and the anionic detergent C12-LAS. Although Cel45
unfolds in GdmCl according to a simple two-state model
under equilibrium conditions, it accumulates a transient
intermediate during refolding. The four disulfide bonds
do not contribute detectably to the stability of the native
state. Cel45 is unfolded by very low concentrations of
C12-LAS (1–4 mM). An analysis of 16 mutants of Cel45
shows a very weak correlation between unfolding rates in
denaturant and detergent; mutants that have the same unfolding
rate in GdmCl (within a factor of 1.5) vary 1,000-fold
in their unfolding rates in C12-LAS. The data support a
simple model for unfolding by detergent, in which the introduction
of positive charges or removal of negative charges greatly
increases detergent sensitivity, while interactions with
the hydrophobic detergent tail contribute to a smaller
extent. This implies that different detergent-mediated
unfolding pathways exist, whose accessibilities depend
on individual residues. Double-mutant cycles reveal that
mutations in two proximal residues lead to repulsion and
a destabilization greater than the sum of the individual
mutations as measured by GdmCl denaturation, but they also
reduce the affinity for LAS and therefore actually stabilize
the protein relative to wild-type. Ligands that interact
strongly with the denatured state may therefore alter the
unfolding process.