A Conserved Histidine Residue Drives Extein Dependence in an Enhanced Atypically Split Intein.

TitleA Conserved Histidine Residue Drives Extein Dependence in an Enhanced Atypically Split Intein.
Publication TypeJournal Article
Year of Publication2022
AuthorsSekar, G, Stevens, AJ, Mostafavi, AZ, Sashi, P, Muir, TW, Cowburn, D
JournalJ Am Chem Soc
Volume144
Issue41
Pagination19196-19203
Date Published2022 Oct 19
ISSN1520-5126
KeywordsExteins, Histidine, Inteins, Protein Splicing, Proteins
Abstract

<p>Split intein-mediated protein trans-splicing (PTS) is widely applied in chemical biology and biotechnology to carry out traceless and specific protein ligation. However, the external residues immediately flanking the intein (exteins) can reduce the splicing rate, thereby limiting certain applications of PTS. Splicing by a recently developed intein with atypical split architecture ("Cat") exhibits a stark dependence on the sequence of its N-terminal extein residues. Here, we further developed Cat using error-prone polymerase chain reaction (PCR) and a cell-based selection assay to produce Cat*, which exhibits greatly enhanced PTS activity in the presence of unfavorable N-extein residues. We then applied solution nuclear magnetic resonance spectroscopy and molecular dynamics simulations to explore how the dynamics of a conserved B-block histidine residue (His) contribute to this extein dependence. The enhanced extein tolerance of Cat* reported here should expand the applicability of atypically split inteins, and the mechanism highlights common principles that contribute to extein dependence.</p>

DOI10.1021/jacs.2c08985
Alternate JournalJ Am Chem Soc
PubMed ID36194550
Grant ListR37 GM086868 / GM / NIGMS NIH HHS / United States
P30 CA013330 / CA / NCI NIH HHS / United States
S10 OD016432 / OD / NIH HHS / United States