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The three-dimensional structure of barley serine proteinase inhibitor, CI-2, has
been determined using nuclear magnetic resonance spectroscopy. The present
structure determination is a refinement of the structure previously determined
by us, using in the present case stereo-specific assignments, and a virtually
complete set of assignments of the two-dimensional nuclear Overhauser spectrum.
The structure determination is based on the identification of more than 1300
nuclear Overhauser effects, of which 961 were used in the structure calculation
as distance restraints, and on 94 dihedral angle restraints, of which 31 are for
chi 1 angles in defined chiral centers. These have been used to calculate a
series of 20 three-dimensional structures using a combination of distance
geometry, simulated annealing and restrained molecular dynamics. Each of the 20
structures was in agreement within less than 0.5 A of each of the distance
restraints and with all dihedral angle restraints. When compared to the
geometric average structure of the 20 refined structures the root-mean-square
differences for the backbone atoms were 0.8 (+/- 0.2) A and for all atoms were
1.6 (+/- 0.2) A. By comparison, the values obtained for the structures
determined previously were 1.4 (+/- 0.2) A and 2.1 (+/- 0.1) A, respectively.
The structures were also compared to the structure determined in the crystalline
state by X-ray diffraction showing root-mean-square differences of 1.6 (+/- 0.2)
A and 2.8 (+/- 0.2) A for the backbone and all atoms, respectively. Common
features of the solution structure and the two crystal structures are the
four-stranded beta-structure, composed of a pair of parallel strands, and three
pairs of antiparallel beta-strands flanked on one side by a 12-residue
alpha-helix and on the other side by a loop containing the serine proteinase
binding site. The new analysis of the structure has revealed an additional pair
of antiparallel beta-strands, consisting of residues 65 to 67 and 81 to 83, that
was not seen in either of the crystal structures or the previous solution
structure. Identification of this was based on nuclear magnetic resonance
evidence for the hydrogen bond (67HN to 81CO) not reported previously. Also the
presence of a bifurcated hydrogen bond involving Phe69 CO and HN atoms of Ala77
and Gln78 was observed in solution but not in crystals. Minor differences
between the two structures were observed in the phi-angles of residues Met59 and
Glu60 in the inhibitory site.
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