OCP1 and OCP2, the most abundant proteins in the cochlea, are evidently subunits of an SCF E3 ubiquitin ligase. Although transcribed from a distinct gene, OCP2 is identical to Skp1. OCP1 is equivalent to the F-box protein known as Fbs1, Fbx2, or NFB42 - previously shown to bind N-glycosylated proteins and believed to function in the retrieval and recycling of misfolded proteins. The high concentrations of OCP1 and OCP2 in the cochlea suggest that the OCP1-OCP2 heterodimer may serve an additional function independent of its role in a canonical SCF complex. At 25 degrees C, urea-induced denaturation of OCP1 is slow, but reversible. The data suggest that the protein possesses one or more disordered regions, a conclusion supported by analysis of the far-UV circular dichroism spectrum and the appearance of the (1)H, (15)N-HSQC spectrum. Thermal denaturation of OCP1 is irreversible, evidently due to formation of high molecular weight aggregates. Analysis with a kinetic model yields an estimate for the activation energy for unfolding of 49 kcal/mol. Urea denaturation data for OCP2 returns DeltaG(o) and m values of 6.2 kcal/mol and 1.5 kcal mol(-)(1) M(-1), respectively. In contrast to OCP1, thermal denaturation of OCP2 is reversible. In phosphate-buffered saline, at pH 7.40, the protein exhibits a DeltaH(vH)/DeltaH(cal) ratio of 1.69, suggesting that denaturation proceeds largely from the native dimer directly to the unfolded state. OCP1 and OCP2 associate tightly at room temperature. However, DSC data for the complex suggest that they denature independently, consistent with the highly exothermic enthalpy of complex formation reported previously. Study holds ProTherm entries: 25270, 25271, 25272, 25273 Extra Details: Buffer containing 1.0 mM tris(hydroxypropyl)phosphine Ubiquitin ligase; SCF complex; F-box protein; Organ of Corti; Protein stability; Calorimetry
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:55 p.m.
|Number of data points||9|
|Proteins||F-box protein 2|
|Assays/Quantities/Protocols||Experimental Assay: dHcal prot_conc:2.0 mg/mL ; Experimental Assay: Tm prot_conc:2.0 mg/mL ; Experimental Assay: dHvH ; Experimental Assay: m prot_conc:1.2-9.6 microM ; Experimental Assay: dG_H2O prot_conc:1.2-9.6 microM ; Experimental Assay: dHcal prot_conc:170 microM ; Experimental Assay: Tm prot_conc:170 microM ; Experimental Assay: m prot_conc:5 microM ; Experimental Assay: dG_H2O prot_conc:5 microM|
|Libraries||Mutations for sequence MDGDGDPESVGQPEEVSPEEACAEEACAEGDRPGEEEEEEQEELEEAAYLAELPEALLLRVLAQLPAAELVQACRLVCLRWKELVDGAPLWLLKCQQEGLVPEGCGEEEERDHWQRFYFLSKRRRNLLRNPCGEEDLEGWCDVEHGGDGWRVEEIPGDSGVEFPQDDSVRKYFASSFEWCRKAQVVDLQAEGYWEELLDTTQPAIVAKDWYSGRSDAGCLYELSVKLLSEHEDVLAEFNTGQVPVSPDSEEGGWTEISHTFTDYGPGVRFVRFEHGGQDAVYWKGWFGARVTNSSVWVEP|