Crystal and molecular structure of the serine proteinase inhibitor CI-2 from barley seeds

Abstract

Chymotrypsin inhibitor 2 (CI-2), a serine proteinase inhibitor from barley seeds, has been crystallized and its three-dimensional structure determined at 2.0-A resolution by the molecular replacement method. The structure has been refined by restrained-parameter least-squares methods to a crystallographic R factor (= sigma parallel Fo magnitude of-Fo parallel/sigma magnitude of Fo) o of 0.198. CI-2 is a member of the potato inhibitor 1 family. It lacks the characteristic stabilizing disulfide bonds of most other members of serine proteinase inhibitor families. The body of CI-2 shows few conformational changes between the free inhibitor and the previously reported structure of CI-2 in complex with subtilisin Novo [McPhalen, C.A., Svendsen, I., Jonassen, I., & James, M.N.G. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 7242-7246]. However, the reactive site loop has some significant conformational differences between the free inhibitor and its complexed form. The residues in this segment of polypeptide exhibit relatively large thermal motion parameters and some disorder in the uncomplexed form of the inhibitor. The reactive site bond is between Met-59I and Glu-60I in the consecutive sequential numbering of CI-2 (Met-60-Glu-61 according to the alignment of Svendsen et al. [Svendsen, I., Hejgaard, J., & Chavan, J.K. (1984) Carlsberg Res. Commun. 49, 493-502]). The network of hydrogen bonds and electrostatic interactions stabilizing the conformation of the reactive site loop is much less extensive in the free than in the complexed inhibitor.

Single-molecule protein folding: Diffusion fluorescence resonance energy transfer studies of the denaturation of chymotrypsin inhibitor 2

“…Because the single-molecule E values exclude non-FRET contributions (see below), they are used in the discussion below. A value for the mean distance between the two dyes of 31 Å is calculated for the folded form, consistent with a number of about 32 Å estimated from the known crystal structure of the protein (25), and allowing a reasonable additional 7 Å because of the size of the dye͞tethers, assuming a right-angled geometry on average. For the unfolded form, the mean calculated distance (from mean E, using the same assumptions for 2 and R 0 ) is 48 Å.…”

Characterization of the transition state of protein unfolding by use of molecular dynamics: chymotrypsin inhibitor 2.

“…Fersht and coworkers (6, 7) have determined (DF values spanning all regions ofC12, where (F = AAGtu/AAGF1u (F, folded; U, unfolded; t, transition state) (27). In particular, we focus on the 4sF values for 10 hydrophobic-core mutants (6).…”

“…The structure of the first 19 residues is not resolved by either x-ray crystallography (10) or two-dimensional NMR studies (11)(12)(13)(14)(15). The structure of a pseudo-wild-type form of C12 in which these residues have been omitted has recently been solved to 1.7-A resolution ( Fig.…”

Structure of the transition state for the folding/unfolding of the barley chymotrypsin inhibitor 2 and its implications for mechanisms of protein folding.

“…It is small and monomeric. Its three-dimensional structure has been solved in both the crystal (26,27) and the solution states (28)(29)(30)(31). Despite its small size, there is considerable secondary and tertiary structure present and a small, compact hydrophobic core.…”