Methods for dual, site-specific derivatization of bovine pancreatic trypsin inhibitor: Trypsin protection of lysine-15 and attachment of fatty acids or hydrophobic peptides at the N-terminus

To produce a series of model membrane proteins, bovine pancreatic trypsin i nhibitor (BPTI) has been modified by specifically attaching reporter groups to Lys-15 and fatty acids or hydrophobic peptides at the N-terminus. Lys-1 5 of BPTI was protected by trypsin bound to BPTI, then O-methylisourea (OMI U) was used to guanidinate all unprotected lysines. The N-terminal amine wa s then reacted with several saturated fatty acid anhydrides from 8 to 18 ca rbons in length, or with an SMCC crosslinker. Cysteine-containing hydrophob ic peptides, cleaved from resin in the presence of sodium dodecyl sulfate ( SDS), were then attached to the protein via the N-terminal cross-linker. Th e methods described yield a unique, chemically modified protein which can c arry site-specific modifications at two distinct residues. The resulting pr oteins are ideal for diffusional or partitioning studies on model and biolo gical membranes.