N-methylcysteine-mediated total chemical synthesis of ubiquitin thioester.
Ubiquitin thioester is a key intermediate in the ubiquitylation of proteins and is formed enzymatically through the activation of alpha-COOH of ubiquitin in an ATP dependent manner using the E1 enzyme. The current methods used for the preparation of ubiquitin thioester rely on either the enzymatic machinery or on expressed protein ligation technology. In this article, we report a new chemical strategy, combining native chemical ligation and N-methylcysteine containing peptides, to chemically prepare ubiquitin thioester for the first time. The N-methylcysteine is utilized as an N-->S acyl transfer device, and in its protected form serves as a latent thioester functionality. This enabled us to trigger the formation of ubiquitin thioester subsequent to the assembly of the ubiquitin polypeptide via native chemical ligation. The synthetic ubiquitin thioester showed a similar behavior in peptide ubiquitylation to the one obtained via expression. This approach should allow for higher flexibility in the chemical manipulation of ubiquitin thioester in a wide variety of ubiquitylated peptides and proteins for structural and biochemical analysis and for the synthesis of ubiquitin chains.
Erlich
Lesly A
LA
Department of Chemistry, Ben Gurion University, Beer Sheva, Israel 84105.
Kumar
K S Ajish
KS
Haj-Yahya
Mahmood
M
Dawson
Philip E
PE
Brik
Ashraf
A
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000031:analogs & derivatives* / Q000737:chemistry; D050091:Dendrimers / Q000737:chemistry; D006020:Glycopeptides / Q000138:chemical synthesis / Q000737:chemistry; D008969:Molecular Sequence Data; D025801:Ubiquitin / Q000138:chemical synthesis* / Q000737:chemistry*; D014452:Ubiquitins / Q000138:chemical synthesis* / Q000737:chemistry*
D050091:Dendrimers; D006020:Glycopeptides; D025801:Ubiquitin; D014452:Ubiquitins; D003545:Cysteine
Org Lett. 2008 Aug 21;10(16):3531-3
18642828
J Am Chem Soc. 2009 Sep 30;131(38):13592-3
19728708
Org Lett. 2009 Feb 19;11(4):823-6
19161341
Org Lett. 2006 Sep 28;8(20):4465-8
16986926
Bioorg Med Chem. 2005 Aug 1;13(15):4622-6
15953729
Biochem Biophys Res Commun. 1978 Apr 28;81(4):1100-5
666810
Angew Chem Int Ed Engl. 2007;46(16):2814-8
17366504
Angew Chem Int Ed Engl. 2007;46(48):9248-52
18046687
Science. 1994 Nov 4;266(5186):776-9
7973629
Org Lett. 2005 Jun 23;7(13):2647-50
15957912
Chembiochem. 2005 Dec;6(12):2176-80
16270365
Angew Chem Int Ed Engl. 2009;48(43):8090-4
19780082
Angew Chem Int Ed Engl. 2006 Jun 12;45(24):3985-8
16639756
Proc Natl Acad Sci U S A. 1998 Jun 9;95(12):6705-10
9618476
Proc Natl Acad Sci U S A. 1999 Aug 31;96(18):10068-73
10468563
Chem Commun (Camb). 2009 Jan 28;(4):407-9
19137167
Chem Biol. 2002 Oct;9(10):1149-59
12401499
Org Lett. 2002 Nov 14;4(23):4041-4
12423081
EMBO Rep. 2008 Jun;9(6):536-42
18516089
Annu Rev Biochem. 2001;70:503-33
11395416
Angew Chem Int Ed Engl. 2008;47(36):6851-5
18651678
Nat Cell Biol. 2006 Aug;8(8):834-42
16862145
J Am Chem Soc. 2001 Jan 31;123(4):526-33
11456564
Angew Chem Int Ed Engl. 2005 Jun 20;44(25):3852-6
15834850