Dynamic structural and functional relationships in recombinant plasminogen activator inhibitor-1 (rPAI-1).
The conformational characteristics of active, latent, and denatured recombinant plasminogen activator inhibitor-1 (rPAI-1) were compared using UV spectroscopy, spectrofluorimetry and circular dichroism (CD) techniques. The UV absorbance wavelength maxima in all preparations approximated 280 nm, while the extinction coefficients of active and latent rPAI-1 differed by up to 60%. When incubated at 37 degrees C, the A280 of latent rPAI-1 was quite stable while the A280 of active rPAI-1 spontaneously increased, eventually approximating that of latent rPAI-1. Alkali difference spectroscopy yielded markedly divergent titration patterns for active and latent rPAI-1, suggesting that the tyrosine residues present in active and latent rPAI-1 differ in terms of solvent exposure. At an excitation wavelength of 280 nm, active rPAI-1 exhibited the greatest relative fluorescence quantum yield. The relative fluorescence of latent and denatured rPAI-1 were less than that of active PAI-1, and the emission maxima of both species were slightly red-shifted in comparison to that of active rPAI-1, suggesting that at least one of the four tryptophan residues present in rPAI-1 is less exposed to the aqueous environment in the active form of the molecule. In contrast, the derived secondary structures based on CD of active and latent rPAI-1 were nearly identical, with both moieties exhibiting approx. 40% alpha-helix and 15% beta-sheet. Taken together, these spectroscopic data provide evidence supporting the hypothesis that active and latent PAI-1 differ in terms of their tertiary conformation and aromatic residue exposure, while their secondary structures appear generally comparable. Furthermore, denaturant-induced reactivation of latent rPAI-1 produces a partially active rPAI-1 with spectroscopic properties similar to that of latent rPAI-1, suggesting that denatured rPAI-1 more closely resembles the latent rPAI-1 conformation after refolding. The spontaneous spectroscopic changes observed in rPAI-1 may reflect conformational transitions that are critical to the regulation of endogenous PAI-1 activity.
Vaughan
D E
DE
Cardiology Division, West Roxbury VAMC, Boston, MA.
Declerck
P J
PJ
Reilly
T M
TM
Park
K
K
Collen
D
D
Fasman
G D
GD
D002942:Circular Dichroism; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry* / Q000302:isolation & purification; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014364:Tryptophan / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry
D017395:Plasminogen Activator Inhibitor 1; D011994:Recombinant Proteins; D014443:Tyrosine; D014364:Tryptophan