Alpha2-Macroglobulin (alpha2M) is a major inhibitor of endoproteases found in the plasma vertebrates. Alpha2M is also involved in a wide variety of physiological processes such as binding and transport of hormones and ions, and in modulating the immune response through various mechanisms. Additionally, alpha2M has been implicated in the development of arterial thrombosis and thus may have a role in the development of atherosclerosis. Alpha2M is a high molecular weight glycoprotein (Mr = 725,000) composed of four identical subunits (Mr = 180,000) when reduced and denatured. The mechanism of protease inhibition is accompanied by a drastic change in the conformation of alpha2M resulting in an entrapment of the protease. Although several forms of alpha2M have been observed by electron microscopy, it has been difficult to relate these morphologic structures with the molecular mechanism of the conformational change. The large size of alpha2M and the ability to crystallize it has precluded any attempt to determine the structure by X-ray diffraction. High resolution low dose microscopy with computer imaging seems to be the most logical approach for resolving the three dimensional structure of alpha2M both in its native and protease-exposed forms. The determination of the three-dimensional structures of alpha2M will provide some insight for relating the molecular mechanism of the conformational change with the morphological structures. We propose to determine the three dimensional structure of alpha2M in its native form and after exposure to various proteases (trypsin, chymotrypsin, plasmin) by using a conical tilt series of images obtained by low dose electron microscopy of the protein fixed with a non-denaturing stain at a neutral pH like methylamine tungstate. We will develop a model from the reconstructed three-dimensional structures and carry out neutron scattering experiments to conform and/or refine the structures obtained from the three dimensional reconstructions. We will carry out timed experiments to determine whether the two forms observed after exposure to proteases are different projections of a same prototype or whether one is an intermediary which will transform itself into the other form. We will attempt to locate the protease binding site on alpha2M by using plasmin as a model protease which, because of its higher molecule weight, is likely to be better resolved by computer imaging.
