Sensors and assays for ubiquitin or ubiquitin-like proteins

A method of determining an amount of a free ubiquitin protein in a sample, the method comprising:

• (a) contacting said sample for a period of time with a chimeric polypeptide that preferentially binds to said free ubiquitin protein as compared to a conjugated ubiquitin protein, wherein said chimeric polypeptide comprises:
• (i) a first polypeptide that binds a ubiquitin protein monomer and a second polypeptide that binds said ubiquitin protein monomer, wherein said first and second polypeptides bind non-overlapping regions of said ubiquitin protein monomer, wherein said first polypeptide binds to the C-terminus of said ubiquitin protein monomer and comprises a zinc finder binding domain of Isopeptidase T;
• wherein said second polypeptide binds to the ubiquitin hydrophobic patch of said ubiquitin protein monomer and comprises a ubiquitin binding domain selected from the group consisting of:
• Ubiquitin Associated domain (UBA), a Ubiquitin Interacting Motif (UIM), a double-sided ubiquitin-interacting motif (DUIM), a Motif Interacting with Ubiquitin (MIU), a coupling of ubiquitin conjugation to ER degradation (CUE), a Golgi-localized, Gamma-ear-containing, Arf-binding (GAT), a Jun kinase activation domain binding/Mpr1p and Pad1p N-termini (Jab 1/MPN), a Np14 a zinc finger (NZF), a ubiquitin-binding zinc finger (UBZ), a Ubiquitin binding surface (UBS), and a Ubiquitin binding motif (UBM);
• (ii) a first linker, wherein said first linker connects said first and second polypeptides;
• (b) determining:
• (i) an amount of said free ubiquitin protein bound to said chimeric polypeptide;
• or
• (ii) an amount of a free competitor ubiquitin protein bound to said chimeric polypeptide, wherein if step (b) (ii) is performed, the method further comprises step (c);
• (c) contacting said sample with said free competitor ubiquitin protein prior to step (b);
• wherein
• (i) said chimeric polypeptide,
• (ii) said free competitor ubiquitin protein, or
• (iii) each of said chimeric polypeptide and said free competitor ubiquitin protein further comprises a detectable label;
• thereby determining the amount of said free ubiquitin protein in said sample.