Figure 1.

Identification of enriched phage display peptides that inhibit ROCK1 autophosphorylation. (a) Structure of ROCK1 and ROCK2 contains an N-terminal kinase domain (92% identity), followed by a coiled-coiled region containing a Rho-binding domain (RBD), and a C-terminal cysteine-rich domain located within the pleckstrin homology (PH) motif domain. (b) The catalytic domain of ROCK1 (5–348) was fused to the maltose-binding protein (MBP) and expressed in baculovirus expression system, and (c) was incubated with the Ph.D-12 phage display library of 12 amino acids in length under high ATP levels (1 mM) to screen for peptides with strong binding affinity to ROCK N′ terminal catalytic domain. (d) Identification of Rock inhibitory peptides by the luciferase-based kinase assay. Several inhibitory peptides shared overlapping amino acid sequences with the strongest ROCK1 inhibitor, peptide7 (ERTYSPSTAVRS) in the N′ terminal (blue lettering) and the C′ terminal (red lettering) ends. (A color version of this figure is available in the online journal.)