PEPTIDE WALKING IS A NOVEL METHOD FOR MAPPING FUNCTIONAL DOMAINS IN PROTEINS - ITS APPLICATION TO THE RAC1-DEPENDENT ACTIVATION OF NADPH OXIDASE

Activation of the superoxide generating NADPH oxidase of phagocytes in volves the assembly of a multimolecular complex and is dependent on th e participation of the small molecular weight GTP-binding protein Rac (1 or 2). This model system was used for mapping functional domains in the primary sequence of Rad, based on assessing the inhibitory effect of 90 individual overlapping pentadecapeptides, spanning the entire l ength of Rad, on NADPH oxidase activation in two types of cell-free as say. Five functional domains mere identified, each consisting of a clu ster of contiguous residues shared by members of five groups of overla pping inhibitory peptides. Four of the five domains are exposed on the molecular surface of Rad and were not identified previously by mutati onal analysis; the fifth corresponds to a polybasic motif near the car boxyl terminus, confirming earlier reports. Screening the entire linea r sequence of a protein with a battery of overlapping peptides for int erference with its ability to interact with upstream or downstream mol ecules should be of wide applicability as a reliable, fast, and econom ical method for mapping of functionally relevant domains.