title: Simultaneous Site-Specific Dual Protein Labeling Using Protein Prenyltransferases.
abstract: Site-specific protein labeling is an important technique in protein chemistry and is used for diverse applications ranging from creating protein conjugates to protein immobilization. Enzymatic reactions, including protein prenylation, have been widely exploited as methods to accomplish site-specific labeling. Enzymatic prenylation is catalyzed by prenyltransferases, including protein farnesyltransferase (PFTase) and geranylgeranyltransferase type I (GGTase-I), both of which recognize C-terminal CaaX motifs with different specificities and transfer prenyl groups from isoprenoid diphosphates to their respective target proteins. A number of isoprenoid analogues containing bioorthogonal functional groups have been used to label proteins of interest via PFTase-catalyzed reaction. In this study, we sought to expand the scope of prenyltransferase-mediated protein labeling by exploring the utility of rat GGTase-I (rGGTase-I). First, the isoprenoid specificity of rGGTase-I was evaluated by screening eight different analogues and it was found that those with bulky moieties and longer backbone length were recognized by rGGTase-I more efficiently. Taking advantage of the different substrate specificities of rat PFTase (rPFTase) and rGGTase-I, we then developed a simultaneous dual labeling method to selectively label two different proteins by using isoprenoid analogue and CaaX substrate pairs that were specific to only one of the prenyltransferases. Using two model proteins, green fluorescent protein with a C-terminal CVLL sequence (GFP-CVLL) and red fluorescent protein with a C-terminal CVIA sequence (RFP-CVIA), we demonstrated that when incubated together with both prenyltransferases and the selected isoprenoid analogues, GFP-CVLL was specifically modified with a ketone-functionalized analogue by rGGTase-I and RFP-CVIA was selectively labeled with an alkyne-containing analogue by rPFTase. By switching the ketone-containing analogue to an azide-containing analogue, it was possible to create protein tail-to-tail dimers in a one-pot procedure through the copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) reaction. Overall, with the flexibility of using different isoprenoid analogues, this system greatly extends the utility of protein labeling using prenyltransferases.
authors.affiliation: Department of Chemistry, University of Minnesota , Minneapolis, Minnesota 55455, United States.
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Chemistry, Syracuse University , Syracuse, New York 13244, United States.
authors.forename: Melanie J
authors.initials: MJ
authors.lastname: Blanden
authors.affiliation: Department of Chemistry, University of Minnesota , Minneapolis, Minnesota 55455, United States.
authors.forename: Ch
authors.initials: Ch
authors.lastname: Sudheer
authors.affiliation: Department of Chemistry, Syracuse University , Syracuse, New York 13244, United States.
authors.forename: Soumyashree A
authors.initials: SA
authors.lastname: Gangopadhyay
authors.affiliation: Department of Biology, Massachusetts Institute of Technology , Cambridge, Massachusetts 02139, United States.
authors.forename: Mohammad
authors.initials: M
authors.lastname: Rashidian
authors.affiliation: Department of Chemistry, Syracuse University , Syracuse, New York 13244, United States.
authors.forename: James L
authors.initials: JL
authors.lastname: Hougland
authors.affiliation: Department of Chemistry, University of Minnesota , Minneapolis, Minnesota 55455, United States.
authors.forename: Mark D
authors.initials: MD
authors.lastname: Distefano
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000378:metabolism*; D000818:Animals; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism*; D008164:Luminescent Proteins / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D017368:Protein Prenylation; D051381:Rats; D013194:Staining and Labeling; D013379:Substrate Specificity; D013729:Terpenes / Q000737:chemistry / Q000378:metabolism*; D000097573:Red Fluorescent Protein
chemical_list: D019883:Alkyl and Aryl Transferases; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D013729:Terpenes; D000097573:Red Fluorescent Protein; C083291:geranylgeranyltransferase type-I; C064911:p21(ras) farnesyl-protein transferase