Quantifying the Kinetics of Pilus-Specific Sortase-Catalyzed Crosslinking Using High-Performance Liquid Chromatography

Nicole A Cheung; Mabel Song; Christopher K Sue; Robert T Clubb

doi:10.1007/978-1-0716-3491-2_11

Quantifying the Kinetics of Pilus-Specific Sortase-Catalyzed Crosslinking Using High-Performance Liquid Chromatography

Methods Mol Biol. 2024:2727:135-143. doi: 10.1007/978-1-0716-3491-2_11.

Authors

Nicole A Cheung^{1

2}, Mabel Song³, Christopher K Sue^{3

1}, Robert T Clubb^{4

5

6}

Affiliations

¹ UCLA-DOE Institute for Genomics and Proteomics, University of California, Los Angeles, Los Angeles, CA, USA.
² Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA, USA.
³ Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, CA, USA.
⁴ Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, CA, USA. [email protected].
⁵ UCLA-DOE Institute for Genomics and Proteomics, University of California, Los Angeles, Los Angeles, CA, USA. [email protected].
⁶ Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA, USA. [email protected].

PMID: 37815714
DOI: 10.1007/978-1-0716-3491-2_11

Abstract

Gram-positive bacteria display pili whose protein components (pilins) are covalently crosslinked by pilus-specific sortase enzymes. These cysteine transpeptidase enzymes catalyze a transpeptidation reaction that joins the pilins together via lysine isopeptide bonds. The crosslinking reaction that builds the SpaA pilus in Corynebacterium diphtheriae is mediated by the SrtA sortase (^CdSrtA) and has been reconstituted in vitro. Here, we present a protocol that can be used to measure the kinetics of ^CdSrtA-catalyzed crosslinking using high-performance liquid chromatography (HPLC). In principle, this biochemical procedure can be used to measure the in vitro crosslinking activity of any pilus-specific sortase.

Keywords: High-performance liquid chromatography; Kinetics; Pili; Sortase.

Publication types

Research Support, N.I.H., Extramural

MeSH terms

Aminoacyltransferases* / chemistry
Bacterial Proteins / metabolism
Cadmium / metabolism
Catalysis
Chromatography, High Pressure Liquid
Fimbriae Proteins* / chemistry
Fimbriae, Bacterial / metabolism

Substances

Fimbriae Proteins
Bacterial Proteins
Cadmium
Aminoacyltransferases

Grants and funding

T32 GM145388/GM/NIGMS NIH HHS/United States