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In an attempt to view the onset of urea denaturation in ribonuclease we have
collected X-ray diffraction data on ribonuclease S crystals soaked in 0, 1.5, 2,
3, and 5 molar urea. At concentrations above 2 M urea, crystals were stabilized
by glutaraldehyde crosslinking. We have also collected data on ribonuclease S
crystals at low pH in an attempt to study the onset of pH denaturation. The
resolution of the datasets range from 1.9 to 3.0 A. Analysis of the structures
reveals an increase in disorder with increasing urea concentration. In the 5 M
urea structure, this increase in disorder is apparent all over the structure but
is larger in loop and helical regions than in the beta strands. The low pH
structure shows a very similar pattern of increased disorder. In addition there
is a major change in the position of the main chain (> 1 A) in the 65-72 turn
region. This region has previously been shown to be involved in one of the
initial steps of unfolding in the reduction of ribonuclease A. Crystallographic
analyses in the presence of denaturant, when combined with controlled
crosslinking, can thus provide detailed structural information that is related
to the initial steps of unfolding in solution. Proteins 1999;36:282-294.
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