While it is widely accepted for proteins that structure implies function, some proteins contain intrinsically disordered regions (IDR) or regions that do not fold into a defined structure. Due to their undefined structure and unstable nature, proteins with large IDRs are difficult to study. NMR can be used for structural characterization of IDRs but severe spectral overlap limits its application to small segments of IDRs. To circumvent this issue, segmental isotopic labeling can be employed to reduce spectral overlap while also maintaining the innate environment of IDRs. Here, we describe our efforts to segmentally label an IDR within plant villin 4 using sortase-mediated ligation. A C-terminal 110aa modular fragment of plant villin 4 (V4HP-L110) was prepared that includes a sortase recognition motif (LPATG) between the 60aa headpiece domain (HP) and a 40aa IDR linker. We investigated V4HP-L110 as a substrate for sortase cleavage using heptamutant Sortase A (7-mut SrtA). Our results demonstrate that 7-mut SrtA cleaves V4HP-L110 at the recognition site. Furthermore, a model reaction showed that the cleaved HP can be re-ligated to a small synthetic polypeptide that contains the sortase site. These studies lay the foundation for using sortase in the construction of larger segmentally labeled IDRs.

Toward segmental isotopic labeling of large disordered polypeptides

255th National Meeting (2018)

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