The spatial orientation of the essential amino acid residues arginine and aspartate within the alpha 1 beta 1 integrin recognition site of collagen IV has been resolved using fluorescence resonance energy transfer

The interaction of collagen IV with cells is mediated mainly by the integri n alpha 1 beta 1 The recognition site has been located to a segment of the triple-helical domain 100 nm away from the N terminus of the collagen molec ule. The three essential amino acid residues of the alpha 1 beta 1 binding site, arginine alpha 2(IV)461 and the two aspartate residues alpha 1(IV)461 , are all located on different chains. Since the spatial array of the three residues depends on the stagger of the chains within the triple helix, the stagger has been elucidated using fluorescence resonance energy transfer w ith phenylalanine alpha 1(IV)473 and tryptophan alpha 2(IV)479 as the fluor escent donor/acceptor pair. The distance R between phenylalanine and trypto phan was determined by analysis of the energy transfer efficiency, E, and t he orientation factor, kappa(2). In parallel, distance R and orientation fa ctor, kappa(2) were also calculated from the coordinates of the triple heli x. Comparison of the calculated and empirically determined values unequivoc ally showed the stagger to be alpha 1'alpha 1 alpha 2. This arrangement of the three a chains describes the conformation of the alpha 1 beta 1 integri n recognition site, that is the distinct orientation of the side-chains of the essential residues aspartate and arginine in respect to the helix axis. (C) 2000 Academic Press.