Abstract

Adenylation domains (A-domains) are responsible for the selective incorporation of carboxylic acid substrates in the biosynthesis of nonribosomal peptides and related natural products. The A-domain transfers an acyl substrate onto its cognate carrier protein (CP). The proper interactions between an A-domain and the cognate CP are important for functional substrate transfer. To stabilize the transient interactions sufficiently for structural analysis of A-domain-CP complex, vinylsulfonamide adenosine inhibitors have been traditionally used as molecular probes. Recently, we have developed an alternative strategy using a synthetic pantetheine-type probe that enables site-specific cross-linking between an A-domain and a CP. In this chapter, we describe the laboratory protocols for this cross-linking reaction.

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