Resolving pathways of functional coupling within protein assemblies by site-specific structural perturbation.

Site-specific structural modification is a powerful tool for studying functional mechanisms in proteins where the structures may be manipulated by direct chemical modification, by selection of naturally-occurring mutants, or by site-directed mutagenesis. Here, we present a general strategy for such studies, which we term "mapping by structure-function perturbation." A series of functional perturbations (i.e., deviations of functional behavior from that of the native protein) are mapped against the structural locations of the modified sites, obtained over a range of locations. The modifications are treated as arbitrary perturbations of structure at specific locations, in contrast to the conventional approach of trying to interpret their local stereochemistry. The map yields information on structural locations of functional events and pathways of coupling within protein assemblies. We have applied this approach to the ligand-linked subunit assembly of human hemoglobin, using both chemically-modified heme sites (CN-met), and amino acid residues altered by mutation and chemical modification.