Serpin conformational change in ovalbumin. Enhanced reactive center loop insertion through hinge region mutations.
Ovalbumin is a noninhibitory member of the serpin superfamily that does not spontaneously undergo the loop-to-sheet conformational change upon cleavage of its reactive center that is characteristic of inhibitory serpins. We tested the hypothesis that ovalbumin could be turned into a proteinase inhibitor by increasing the rate of loop insertion through hinge region mutations alone. We found that none of the three variants examined showed any detectable proteinase inhibitory properties. However, replacement of the P14 arginine residue of ovalbumin by serine, either alone or in combination with changes of P12-P10 to alanine, resulted in a large increase in the rate of loop insertion into beta-sheet A following cleavage at the P1-P1' bond by porcine pancreatic elastase (PPE), as shown by the spontaneous formation of a loop-inserted form upon cleavage that has increased the thermal stability. From the magnitude of the increase in stability of the cleaved, loop-inserted forms of the P14 ovalbumin variants, as well as the accessibility of the P1-P1'-cleaved reactive center loop to further proteolysis at P8-P7, we concluded that the reactive center loop can only partially insert into beta-sheet A and therefore that ovalbumin is also defective in the ability of beta-sheet A to expand to fully accommodate the whole of the reactive center loop. This defect, through its effect on the extent and/or rate of loop insertion, is likely to be a principal reason for ovalbumin not being a proteinase inhibitor.
Huntington
J A
JA
Department of Biochemistry, University of Illinois at Chicago, 60612, USA.
Fan
B
B
Karlsson
K E
KE
Deinum
J
J
Lawrence
D A
DA
Gettins
P G
PG
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D002942:Circular Dichroism; D004795:Enzyme Stability; D007700:Kinetics; D013058:Mass Spectrometry; D016297:Mutagenesis, Site-Directed; D010047:Ovalbumin / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D015843:Serpins / Q000737:chemistry* / Q000378:metabolism; D013381:Subtilisins / Q000378:metabolism; D013552:Swine
D011994:Recombinant Proteins; D015843:Serpins; D010047:Ovalbumin; D013381:Subtilisins