14] Prenylated peptides in identification of specific binding proteins

Abstract

Publisher Summary This chapter discusses the methods used in the identification of specific binding proteins for prenylated peptides, which was initially identified and characterized using biochemical binding analysis. The chemical cross-linking approaches, which lead to the visualization of membrane proteins that bind prenylated peptides and proteins, are also described. Using prenylated peptides that correspond to C-terminal sequences of proteins containing the CaaX motif as probes, a high-affinity binding protein for the prenylated peptides in the microsomal membrane fraction of bovine brain is identified. The binding protein may serve as the initial component in the processing/trafficking pathway for newly prenylated proteins. The specificity of the binding activity detected in the membrane fraction can be assessed by competition analysis. Typical competitors include prenyl peptides (GG-γ peptide and Farn-ras peptide), unprenylated counterpart peptides, peptides modified by other lipids, and the prenyl diphosphates (GGPP and FPP) at up to 400 n M .