Studies of beta-sheet rearrangements in antichymotrypsin / Christine M. Lukacs.

Format:

Book

Manuscript

Microformat

Thesis/Dissertation

Author/Creator:

Lukacs, Christine M.

Contributor:

Christianson, David W., advisor.

University of Pennsylvania.

Language:

English

Subjects (All):

Penn dissertations--Chemistry.

Chemistry--Penn dissertations.

Local Subjects:

Penn dissertations--Chemistry.

Chemistry--Penn dissertations.

Physical Description:

xv, 182 pages : illustrations ; 29 cm

Production:

1997.

Summary:

A hallmark of serpin function is the massive $\beta$-sheet rearrangement involving the insertion of the cleaved reactive loop into $\beta$-sheet A as strand s4A. Although the structures of intact and cleaved serpins are known, structural details of what happens during the transition have eluded researchers. Furthermore, the extent of strand insertion involved in the inhibition process is widely debated.

Substitutions in the serpin reactive loop can affect specificity and/or function. I have determined the structure of cleaved antichymotrypsin-P3P3$\sp\prime,$ which has conservative substitutions in the six residues directly surrounding the scissile bond, altering the specificity of the serpin. Although this structure is nearly identical to that of the native protein, it nevertheless represents the cleaved counterpart to the structure of intact ACT-P3P3$\sp\prime$ and highlights the differences between native and recombinant proteins.

Small hydrophobic residues in the hinge region (around P14) of s4A facilitate strand insertion, since they eventually pack in the hydrophobic core. The substitution of bulky, charged residues at the P14 and P12 positions is postulated to change a serpin from inhibitor to substrate by slowing the rate of strand insertion. I support this theory with the structures of four cleaved s4A arginine variants of antichymotrypsin, demonstrating that strand insertion is not prevented by such substitutions.

In cleaved A347R-ACT (P12), strand s4A is fully inserted with normal $\beta$-sheet geometry; the R347 side chain is buried in the hydrophobic protein core. In cleaved T345R-ACT (P14), strand s4A is mostly inserted, but R345 twists in order to remain solvent exposed. Both of these variants are chymotrypsin substrates. The structure of cleaved A349R-ACT (P10) reveals full strand insertion and the burial of R349 with a salt-linked acetate counterion. A349R-ACT has a stoichiometry of inhibition of $\sim$5. Finally, the structure of cleaved A350R-ACT (P9) shows accommodation of the arginine on the other side of the s4A. In this inhibitory variant, full strand insertion is observed, but the adjacent surface loop becomes disordered.

Notes:

Supervisor: David W. Christianson.

Thesis (Ph.D. in Chemistry) -- University of Pennsylvania, 1997.

Includes bibliographical references.

Local Notes:

University Microfilms order no.: 98-00894.

OCLC:

244969740