Figure 2: S-nitrosylation of UCHL1 down regulates its catalytic activity and compromises ubiquitin binding.
From: S-nitrosylation of UCHL1 induces its structural instability and promotes α-synuclein aggregation
(a) Enzymatic activity of the nitrosylated UCHL1. UCHL1 and nitrosylated UCHL1 (2.5 nM) were incubated with Ub-AMC (450 nM). UV irradiation was used to check reversibility of UCHL1 activity after nitrosative modification. Hydrolysis was measured as an increase in fluorescence, indicating release of AMC, over time. (b) Values showing the rate of reaction catalyzed by control and nitrosylated UCHL1. (c,d) Sensograms correspond to the binding of UCHL1 (analyte) to an immobilized ubiquitin on CM5 biosensor chips (~2200 RU). (c) UCHL1 C90A and (d) nitrosylated UCHL1 C90A ranging from 0.078 μM to 50 μM were used. Inset represents steady state affinity curve and binding constant (KD) of UCHL1 C90A and nitrosylated UCHL1 C90A which were calculated to be 0.13 μM and 4.90 μM respectively using BIA evaluation software. Sensogram shown in different colors represent different concentrations (Red 50 μM, black 25 μM, purple 12.5 μM, cyan 6.25 μM, pink 3.12 μM, orange 1.56 μM and green 0.78 μM). (e) Values showing binding constant (μM) of nitrosylated and control UCHL1 C90A and TM.
