Probing nanosecond motions of plasminogen activator inhibitor-1 by time-resolved fluorescence anisotropy.
Plasminogen activator inhibitor-1 (PAI-1) is a member of the serpin (serine protease inhibitor) superfamily. Like most serpins, the inhibitory function of PAI-1 relies on a flexible reactive centre loop (RCL) undertaking a striking conformational transition. We have investigated the conformational dynamics of the RCL of PAI-1 by time-resolved fluorescence anisotropy. A heterogeneous population model with three rotational correlation times has been employed to account for the "dip and rise" observed in fluorescence anisotropy decay curves. The RCL becomes almost fully solvent exposed and exhibits faster rotation when PAI-1 interacts with a RCL-mimicking octapeptide which blocks the loop insertion pathway, indicating that the RCL is well displaced from the protein surface; while the binding of Somatomedin B (SMB) domain of vitronectin, only induces small changes in the RCL. Comparison of the fluorescence lifetime and anisotropy decay of the wild-type PAI-1 with that of the stabilised mutant suggests that there would be no major structural differences between them. Our results indicate that in a native serpin, the P14 residue of the hinge region can flip in and out of the central beta-sheet A more readily than previously thought, which is likely an inherent property for serpins' protease inhibitory function.
Ko
Chin Wing
CW
Molecular Medicine, National Heart and Lung Institute, Imperial College London, London, UKSW7 2AZ.
Wei
Zhenquan
Z
Marsh
Richard J
RJ
Armoogum
Daven A
DA
Nicolaou
Nick
N
Bain
Angus J
AJ
Zhou
Aiwu
A
Ying
Liming
L
D005454:Fluorescence Polarization / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D008958:Models, Molecular; D036103:Nanotechnology / Q000379:methods*; D009282:Naphthalenesulfonates / Q000737:chemistry; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins; D013002:Somatomedins / Q000378:metabolism; D019096:Vitronectin / Q000378:metabolism
D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D017395:Plasminogen Activator Inhibitor 1; D011994:Recombinant Proteins; D013002:Somatomedins; D019096:Vitronectin; C016756:somatomedin B; C006421:1,5-I-AEDANS