Method of generating linkage-specific di-and polyprotein probes
Deubiquitinating enzyme (DUB) probes are provided that resemble native diubiquitin (diUB) with a similar linkage size and that may contain a Michael acceptor for trapping the DUB active-site cysteine. For example, both K63- and K48-linked diubiquitin probes are generated using a facile chemical ligation method, utilizing the linker compound 3-(2-(bromomethyl)-1,3-dioxolan-2-yl)prop-2-en-1-amine. The diUb probes are capable of labelling DUBs from different families and may be employed to reveal intrinsic linkage specificities of DUBs.
1. A compound having a structure in accordance with Formula (I):
H 2 NCH 2 —R—C(Diox)CH 2 X (I)
wherein R is —CH 2 CH 2 — or —CH═CH—, Diox is —O(CH 2 ) n O— with n=2 or 3 and forms a 1,3-dioxolane ring or 1,3-dioxane ring which includes the carbon atom to which the oxygen atoms of —O(CH 2 ) n O— are both bonded, and X is Br, Cl or I.
2. The compound of claim 1 , wherein R is —CH═CH—, n=2, and X is Br.
3. The compound of claim 1 , wherein R is —CH 2 CH 2 —, n=2, and X is Br.
4. A method of making the compound of claim 1 , comprising hydrolyzing a precursor compound having a structure in accordance with Formula (II):
(Isoindol)CH 2 —R—C(Diox)CH 2 X (II)
wherein Isoindol is an isoindoline-1,3-dione moiety, R is —CH 2 CH 2 — or —CH═CH—, and Diox is —O(CH 2 ) n O— with n=2 or 3 and forms a 1,3-dioxolane ring or 1,3-dioxane ring which includes the carbon atom to which the oxygen atoms of —O(CH 2 ) n O— are both bonded, and X is Br, Cl or I.
5. The method of claim 4 , wherein the precursor compound has been prepared by reacting a compound having a structure in accordance with Formula (III):
(Isoindol)CH 2 —R—C(═O)CH 2 X (III)
with ethylene glycol or 1,3-propanediol.
6. A method of making a di- or polyprotein, comprising linking a first polypeptide molecule and a second polypeptide molecule using a compound having a structure in accordance with Formula (I):
H 2 NCH 2 —R—C(Diox)CH 2 X (I)
wherein R is —CH 2 CH 2 — or —CH═CH—, Diox is —O(CH 2 ) n O— with n=2 or 3 and forms a 1,3-dioxolane ring or 1,3-dioxane ring which includes the carbon atom to which the oxygen atoms of —O(CH 2 ) n O— are both bonded, and X is Br, Cl or I.
7. The method of claim 6 , wherein the first polypeptide molecule is Ub 1-75 .
8. The method of claim 6 , wherein the second polypeptide molecule contains a cysteine residue.
9. The method of claim 6 , wherein the second polypeptide molecule is a ubiquitin having a cysteine residue at position 6, 11, 27, 29, 33, 48 or 63.
10. A method of making a di- or polyprotein, comprising the steps of:
a) reacting PP1-C(═O)—S—R 1 , wherein PP1 is a first polypeptide and —C(═O)—S—R 1 is a thioester group, with a compound having a structure in accordance with Formula (I):
H 2 NCH 2 —R—C(Diox)CH 2 X (I)
wherein R is —CH 2 CH 2 — or —CH═CH—, Diox is —O(CH 2 ) n O— with n=2 or 3 and forms a 1,3-dioxolane ring or 1,3-dioxane ring which includes the carbon atom to which the oxygen atoms of —O(CH 2 ) n O— are both bonded, and X is Br, Cl or I to provide PP1-C(═O)NHCH 2 —R—C(Diox)CH 2 X;
b) converting the 1,3-dioxolane ring or 1,3-dioxane ring to a ketone to provide PP1-C(═O)NH—CH 2 —R—C(═O)CH 2 X; and
c) reacting the PP1-C(═O)NH—CH 2 —R—C(═O)CH 2 X with a second polypeptide bearing a cysteine residue.
11. The method of claim 10 , wherein the second polypeptide is a ubiquitin and the cysteine residue is present at the 6, 11, 27, 29, 33, 48 or 63 position of the ubiquitin.
12. The method of claim 10 , wherein the second polypeptide bears a reporter tag.
13. The method of claim 12 , wherein the reporter tag is selected from the group consisting of affinity tags, biotin and fluorophores.
14. The method of claim 12 , wherein the reporter tag is an affinity tag selected from the group consisting of HA-tag, His-tag, FLAG-tag, and Myc-tag.
15. The method of claim 10 , wherein the first polypeptide is a ubiquitin.
16. The method of claim 10 , wherein the first polypeptide is Ub 1-75 .
17. The method of claim 10 , wherein R is —CH 2 CH 2 —, n=2, and X is Br.
18. The method of claim 10 , wherein R is —CH═CH—, n=2 and X is Br.