Modulation of Intein Activity by Its Neighboring Extein Substrates

Overview

Journal Proc Natl Acad Sci U S A

Specialty Science

Date 2009 Jun 23

PMID 19541659

Citations 62

Authors

Gil Amitai

Brian P Callahan

Matt J Stanger

Georges Belfort

Marlene Belfort

Affiliations

Soon will be listed here.

Abstract

Inteins comprise a large family of phylogenetically widespread self-splicing protein catalysts that colonize diverse host proteins. The evolutionary and functional relationship between the intein and the split-host protein, the exteins, is largely unknown. To probe an association, we developed an in vivo and in vitro intein assay based on FRET. The FRET assay reports cleavage of the intein from its N-terminal extein. Applying this assay to randomized extein libraries, we show that the nature of the extein substrate bordering the intein can profoundly influence intein activity. Residues proximal to the intein-splicing junction in both N- and C-terminal exteins can accelerate the N-terminal cleavage rate by >4-fold or attenuate cleavage by 1,000-fold, both resulting in compromised self-splicing efficiency. The existence and the magnitude of extein effects require consideration for maximizing the utility of inteins in biotechnological applications, and they predict biases in intein integration sites in nature.

Citing Articles

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References

Klabunde T, Sharma S, Telenti A, Jacobs Jr W, Sacchettini J . Crystal structure of GyrA intein from Mycobacterium xenopi reveals structural basis of protein splicing. Nat Struct Biol. 1998; 5(1):31-6. DOI: 10.1038/nsb0198-31. View

Chong S, Williams K, Wotkowicz C, Xu M . Modulation of protein splicing of the Saccharomyces cerevisiae vacuolar membrane ATPase intein. J Biol Chem. 1998; 273(17):10567-77. DOI: 10.1074/jbc.273.17.10567. View

Hackeng T, Griffin J, Dawson P . Protein synthesis by native chemical ligation: expanded scope by using straightforward methodology. Proc Natl Acad Sci U S A. 1999; 96(18):10068-73. PMC: 17843. DOI: 10.1073/pnas.96.18.10068. View

Chong S, Montello G, Zhang A, Cantor E, Liao W, Xu M . Utilizing the C-terminal cleavage activity of a protein splicing element to purify recombinant proteins in a single chromatographic step. Nucleic Acids Res. 1998; 26(22):5109-15. PMC: 147948. DOI: 10.1093/nar/26.22.5109. View

Pietrokovski S . Intein spread and extinction in evolution. Trends Genet. 2001; 17(8):465-72. DOI: 10.1016/s0168-9525(01)02365-4. View

Nichols N, Benner J, Martin D, Evans Jr T . Zinc ion effects on individual Ssp DnaE intein splicing steps: regulating pathway progression. Biochemistry. 2003; 42(18):5301-11. DOI: 10.1021/bi020679e. View

Chong S, Mersha F, COMB D, Scott M, Landry D, Vence L . Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element. Gene. 1997; 192(2):271-81. DOI: 10.1016/s0378-1119(97)00105-4. View

Southworth M, Amaya K, Evans T, Xu M, Perler F . Purification of proteins fused to either the amino or carboxy terminus of the Mycobacterium xenopi gyrase A intein. Biotechniques. 1999; 27(1):110-4, 116, 118-20. DOI: 10.2144/99271st04. View

Perler F, Adam E . Protein splicing and its applications. Curr Opin Biotechnol. 2000; 11(4):377-83. DOI: 10.1016/s0958-1669(00)00113-0. View

10.

Moore S, Jencks W . Model reactions for CoA transferase involving thiol transfer. Anhydride formation from thiol esters and carboxylic acids. J Biol Chem. 1982; 257(18):10882-92. View

11.

Gangopadhyay J, Jiang S, van Berkel P, Paulus H . In vitro splicing of erythropoietin by the Mycobacterium tuberculosis RecA intein without substituting amino acids at the splice junctions. Biochim Biophys Acta. 2003; 1619(2):193-200. DOI: 10.1016/s0304-4165(02)00495-6. View

12.

Shi J, Muir T . Development of a tandem protein trans-splicing system based on native and engineered split inteins. J Am Chem Soc. 2005; 127(17):6198-206. DOI: 10.1021/ja042287w. View

13.

Nguyen A, Daugherty P . Evolutionary optimization of fluorescent proteins for intracellular FRET. Nat Biotechnol. 2005; 23(3):355-60. DOI: 10.1038/nbt1066. View

14.

Muralidharan V, Muir T . Protein ligation: an enabling technology for the biophysical analysis of proteins. Nat Methods. 2006; 3(6):429-38. DOI: 10.1038/nmeth886. View

15.

Pearl E, Bokor A, Butler M, Poulter R, Wilbanks S . Preceding hydrophobic and beta-branched amino acids attenuate splicing by the CnePRP8 intein. Biochim Biophys Acta. 2007; 1774(8):995-1001. DOI: 10.1016/j.bbapap.2007.05.015. View

16.

Flavell R, Kothari P, Bar-Dagan M, Synan M, Vallabhajosula S, Friedman J . Site-specific (18)F-labeling of the protein hormone leptin using a general two-step ligation procedure. J Am Chem Soc. 2008; 130(28):9106-12. DOI: 10.1021/ja801666z. View

17.

Iwai H, Zuger S, Jin J, Tam P . Highly efficient protein trans-splicing by a naturally split DnaE intein from Nostoc punctiforme. FEBS Lett. 2006; 580(7):1853-8. DOI: 10.1016/j.febslet.2006.02.045. View

18.

Nogami S, SATOW Y, Ohya Y, Anraku Y . Probing novel elements for protein splicing in the yeast Vma1 protozyme: a study of replacement mutagenesis and intragenic suppression. Genetics. 1997; 147(1):73-85. PMC: 1208124. DOI: 10.1093/genetics/147.1.73. View

19.

Martin D, Xu M, EVANS Jr T . Characterization of a naturally occurring trans-splicing intein from Synechocystis sp. PCC6803. Biochemistry. 2001; 40(5):1393-402. DOI: 10.1021/bi001786g. View

20.

Paulus H . Protein splicing and related forms of protein autoprocessing. Annu Rev Biochem. 2000; 69:447-96. DOI: 10.1146/annurev.biochem.69.1.447. View