C-terminal incorporation of fluorogenic and affinity labels using wild-type and mutagenized carboxypeptidase Y

Analytical Biochemistry
H R StennickeK Breddam

Abstract

The ability to carry out specific C-terminal modification or labeling of peptides and proteins has a broad range of applications. It is well established that this may be achieved by protease-catalyzed transacylation reactions and that carboxypeptidase Y (CPD-Y) is suitable for this due to its broad specificity and stability in the presence of denaturants. Furthermore, CPD-Y is characterized by a S'1 binding site that is open to solvent and, thus, capable of catalyzing a transpeptidation reaction with nucleophiles that extend beyond the perimeter of the active site. However, one major drawback with CPD-Y is that the yield of the reaction is highly dependent on the nature of the leaving group; e.g., with large apolar leaving groups the yield of the reaction does not exceed 15%. In the present publication it is demonstrated that mutants of CPD-Y, designed for low leaving group dependence, efficiently incorporate biocytin amide as well as a new fluorescent nucleophile, N'-Abz-Lysine amide (ablysin amide), into peptides and proteins.

Citations

Aug 5, 2011·ACS Chemical Biology·Guoqiang XuSamie R Jaffrey
Jan 7, 2009·Peptides·Bernd Peschke, Sonja Bak
May 21, 2004·Protein Science : a Publication of the Protein Society·María T GarzónJosé L Neira
Jun 17, 2020·Biochemical Society Transactions·Clara L Frazier, Amy M Weeks
Jan 15, 2009·Bioconjugate Chemistry·Xiaohong ZhangChuan-Fa Liu
May 8, 2007·Bioorganic & Medicinal Chemistry·Bernd PeschkeKjeld Madsen

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