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Title
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Solvent structure in crystals of trypsin determined by X-ray and neutron diffraction.
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Authors
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J.S.Finer-Moore,
A.A.Kossiakoff,
J.H.Hurley,
T.Earnest,
R.M.Stroud.
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Ref.
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Proteins, 1992,
12,
203-222.
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PubMed id
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Abstract
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The solvent structure in orthorhombic crystals of bovine trypsin has been
independently determined by X-ray diffraction to 1.35 A resolution and by
neutron diffraction to 2.1 A resolution. A consensus model of the water molecule
positions was obtained using oxygen positions identified in the electron density
map determined by X-ray diffraction, which were verified by comparison to
D2O-H2O difference neutron scattering density. Six of 184 water molecules in the
X-ray structure, all with B-factors greater than 50 A2, were found to be
spurious after comparison with neutron results. Roughly two-thirds of the water
of hydration expected from thermodynamic data for proteins was localized by
neutron diffraction; approximately one-half of the water of hydration was
located by X-ray diffraction. Polar regions of the protein are well hydrated,
and significant D2O-H2O difference density is seen for a small number of water
molecules in a second shell of hydration. Hydrogen bond lengths and angles
calculated from unconstrained refinement of water positions are distributed
about values typically seen in small molecule structures. Solvent models found
in seven other bovine trypsin and trypsinogen and rat trypsin structures
determined by X-ray diffraction were compared. Internal water molecules are well
conserved in all trypsin structures including anionic rat trypsin, which is 65%
homologous to bovine trypsin. Of the 22 conserved waters in trypsin, 19 were
also found in trypsinogen, suggesting that they are located in regions of the
apoprotein that are structurally conserved in the transition to the mature
protein. Seven waters were displaced upon activation of trypsinogen. Water
structure at crystal contacts is not generally conserved in different crystal
forms. Three groups of integral structural water molecules are highly conserved
in all solvent structures, including a spline of water molecules inserted
between two beta-strands, which may resemble an intermediate in the formation of
beta sheets during the folding of a protein.
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