Enthalpy measurement using calorimetry shows a significant difference in potential energy between the active and latent conformations of PAI-1

Westley A. Sherman 1. , Grant E. Blouse 2. , Michel J. Perron 3. , Timothy Tran 4. , Joseph D. Shore 5.  and Ari Gafni 6.
  • 1. Biological Chemistry Department, University of Michigan, Ann Arbor, MI 48109-1066, USA
  • 2. Division of Biochemical Research, Henry Ford Health System, Detroit, MI 48202, USA
  • 3. Division of Biochemical Research, Henry Ford Health System, Detroit, MI 48202, USA
  • 4. Biophysics Research Division, University of Michigan, Ann Arbor, MI 48109-1055, USA
  • 5. Division of Biochemical Research, Henry Ford Health System, Detroit, MI 48202, USA
  • 6. Biological Chemistry Department, University of Michigan, Ann Arbor, MI 48109-1066, USA, Biophysics Research Division, University of Michigan, Ann Arbor, MI 48109-1055, USA and Institute of Gerontology, University of Michigan, Ann Arbor, MI 48109-2007, USA

Abstract

A central feature of the serpin inhibition mechanism is insertion of the reactive center loop into the central β-sheet (β-sheet A). This insertion also occurs when the reactive center loop is cleaved without protease inhibition. Using this effect, we have measured the enthalpy (ΔH) of loop cleavage and insertion for plasminogen activator inhibitor 1 (PAI-1) as -38 kcal/mol. Because loop insertion can be blocked by incorporating a peptide into the central β-sheet, it was possible to assign -7 kcal/mol to loop cleavage and -31 kcal/mol to loop insertion. These values are lower than values reported for the serpins α1-proteinase inhibitor and antithrombin of -53 to -63 kcal/mol, respectively, for loop insertion with negligible enthalpy for loop cleavage. A free energy difference of -9 kcal/mol has been reported between the active and spontaneously loop inserted ‘latent forms’ of PAI-1, which is significantly smaller in magnitude than the -31 kcal/mol of enthalpy we measured for loop insertion. Because the enthalpy should relate closely to those regions of PAI-1 that have moved to lower potential energy, a difference distance matrix is presented that identifies regions of PAI-1 that move during loop insertion.

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