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Conformational distributions of protease-serpin complexes: a partially translocated complex.

Biochemistry (2006-09-07)
Lu Liu, Nicole Mushero, Lizbeth Hedstrom, Anne Gershenson

Serpins regulate serine proteases by forming metastable covalent complexes with their targets. The protease docks with the serpin and cleaves the serpin's reactive center loop (RCL) forming an acylenzyme intermediate. Cleavage triggers insertion of the RCL into beta sheet A, translocating the attached protease approximately 70 A and disrupting the protease active site, trapping the acylenzyme intermediate. Using single-pair Förster resonance energy transfer (spFRET), we have measured the conformational distributions of trypsin and alpha(1)-proteinase inhibitor (alpha(1)PI) covalent complexes. Bovine trypsin (BTryp) complexes display a single set of conformations consistent with the full translocation of BTryp (E(full)I*). However, the range of spFRET efficiencies is large, suggesting that the region around the trypsin label is mobile. Most complexes between alpha(1)PI variants and the more stable rat trypsin (RTryp) also show a single set of conformations, but the conformational distribution is narrower, indicating less disruption of RTryp. Surprisingly, RTryp complexes containing alpha(1)PI labeled at Cys232 with a cationic fluorophore display two equally populated conformations, E(full)I* and a conformation in which RTryp is only partially translocated (E(part)I*). Destabilizing the RTryp active site, by substituting Ala for Ile16, increases the E(full)I* population. Thus, interactions between anionic RTryp and cationic dyes likely exert a restraining force on alpha(1)PI, increasing the energy needed to translocate trypsin, and this force can be counteracted by active site destabilization. These results highlight the role of protease stability in determining the conformational distributions of protease-serpin covalent complexes and show that full translocation is not required for the formation of metastable complexes.

Product Number
Product Description

Atto 610-NHS ester, BioReagent, suitable for fluorescence, ≥90.0% (coupling to amines)
Atto 610, BioReagent, suitable for fluorescence, ≥85.0% (HPCE/HPLC)
Atto 610-Biotin, BioReagent, suitable for fluorescence, ≥85% (HPCE)
Atto 610 maleimide, BioReagent, suitable for fluorescence, ≥90% (coupling to thiols)

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