Polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs) are large multidomain proteins present in microorganisms that produce bioactive compounds. Curacin A is such a bioactive compound with potent anti-proliferative activity. During its biosynthesis the growing substrate is bound covalently to an acyl carrier protein (ACP) that is able to access catalytic sites of neighboring domains for chain elongation and modification. While ACP domains usually occur as monomers, the curacin A cluster codes for a triplet ACP (ACPI-ACPII-ACPIII) within the CurA PKS module. We have determined the structure of the isolated holo-ACPI and show that the ACPs are independent of each other within this tridomain system. In addition, we have determined the structure of the 3-hydroxyl-3-methylglutaryl-loaded holo-ACPI, which is the substrate for the unique halogenase (Hal) domain embedded within the CurA module. We have identified the interaction surface of both proteins using mutagenesis and MALDI-based identification of product formation. Amino acids affecting product formation are located on helices II and III of ACPI and form a contiguous surface. Since the CurA Hal accepts substrate only when presented by one of the ACPs within the ACPI-ACPII-ACPIII tridomain, our data provide insight into the specificity of the chlorination reaction.

Der richtige Griff: Mit einem natürlich gespaltenen Intein wurde eine effiziente Dreiwege‐Ligationsmethode entwickelt, die ohne Rückfaltungsschritte auskommt. Auf diese Art gelingt die selektive Markierung einer zentralen Domäne in einem Dreidomänenprotein mit NMR‐aktiven Isotopen (siehe Bild), was die Untersuchung von Domänen‐Domänen‐Wechselwirkungen in Mehrdomänenproteinen ermöglicht.

A split intein saves nine: A naturally split intein is used to create an efficient three‐way ligation method that does not require any refolding steps. This method enables the selective labeling of a central domain within a three‐domain protein, with NMR active isotopes (see picture) allowing domain–domain interactions in multidomain proteins to be investigated.

Background: Protein trans-splicing as a molecular design tool has been demonstrated for soluble but not yet for membrane proteins. Results: Two separate polypeptides have been spliced in vivo, yielding correctly folded and functional proteorhodopsin. Conclusion: Trans-splicing of ␣-helical membrane proteins under native conditions is possible. Significance: Our findings are important for the folding, assembly, and engineering of membrane proteins.

Split intein enabled protein trans-splicing (PTS) is a powerful method for the ligation of two protein fragments, thereby paving the way for various protein modification or protein function control applications. PTS activity is strongly influenced by the amino acids directly flanking the splice junctions. However, to date no reliable prediction can be made whether or not a split intein is active in a particular foreign extein context. Here we describe SPLICEFINDER, a PCR-based method, allowing fast and easy screening for active split intein insertions in any target protein. Furthermore we demonstrate the applicability of SPLICEFINDER for segmental isotopic labeling as well as for the generation of multi-domain and enzymatically active proteins.

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