Simultaneous Site-Specific Dual Protein Labeling Using Protein Prenyltransferases

Area of Science:

• Biochemistry
• Chemical Biology
• Molecular Biology

Background:

• Site-specific protein labeling is crucial for applications like protein conjugation and immobilization.
• Enzymatic prenylation, catalyzed by prenyltransferases (PFTase, GGTase-I), is a key method for site-specific protein modification.
• Existing methods often utilize protein farnesyltransferase (PFTase) with isoprenoid analogues.

Purpose of the Study:

• To explore the utility of rat geranylgeranyltransferase type I (rGGTase-I) for expanding prenyltransferase-mediated protein labeling.
• To characterize the isoprenoid analogue specificity of rGGTase-I.
• To develop a simultaneous dual labeling method using distinct prenyltransferases and specific isoprenoid analogues.

Main Methods:

• Evaluated isoprenoid analogue specificity of rat GGTase-I (rGGTase-I) using eight different analogues.
• Developed a simultaneous dual labeling strategy by exploiting differential substrate specificities of rat PFTase (rPFTase) and rGGTase-I.
• Utilized model proteins, GFP-CVLL and RFP-CVIA, with specific C-terminal motifs for selective labeling.

Main Results:

• rGGTase-I efficiently recognized isoprenoid analogues with bulky moieties and longer backbone lengths.
• Demonstrated selective labeling of GFP-CVLL with a ketone-functionalized analogue by rGGTase-I and RFP-CVIA with an alkyne-containing analogue by rPFTase.
• Successfully created protein tail-to-tail dimers via copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) by switching to an azide-containing analogue.

Conclusions:

• The study successfully expanded the scope of prenyltransferase-mediated protein labeling by incorporating rGGTase-I.
• A novel simultaneous dual labeling method was established, enabling selective modification of two distinct proteins.
• The developed system offers flexibility and enhances the utility of prenyltransferases for diverse protein engineering applications.