Characterization of Mitochondrial YME1L Protease Oxidative Stress-Induced Conformational State

Fluorescence Methods Applied to the Description of Urea-Dependent YME1L Protease Unfolding

“…Reference to Figure 3A demonstrates that F/F o from 0 to 4 M urea for apo YME1L can be described by a Gaussian function with maximum position at 2.34 ± 0.05 M urea, which steeply decreases to an average of F/F o = 0.99 ± 0.03 for [urea] > 4 M. Incubation in the presence of 20 µM ATP yields the same behavior, though with maximum F/F o observed at 2.59 ± 0.06 M urea ( Figure 3B). We have previously reported dissociation equilibrium constants for YME1L binding to nucleotide as 20 ± 7 µM and 74 ± 33 µM [30]. From this, we reason that the conditions presented in Figure 3B likely represent a mixture of bound and free YME1L ligation states.…”

“…All genes were synthesized and each cloned into the pET-24a(+) vector commercially by Genscript (Piscataway, NJ, USA). Soluble YME1L was expressed as the previously described cc-hex YME1L construct with the addition of an N-terminal His 6 -SUMO tag [29,30]. YME1L was purified as previously described [30] and protein concentration was determined prior to use in buffer H150 (25 mM HEPES (pH = 7.5 at 25 • C), 150 mM NaCl, 10% glycerol, 2 mM 2-mercaptoethanol, and 0.5 mM ZnCl 2 ) using extinction coefficients ε 280 = 3.29 × 10 4 M −1 cm −1 .…”

“…Soluble YME1L was expressed as the previously described cc-hex YME1L construct with the addition of an N-terminal His 6 -SUMO tag [29,30]. YME1L was purified as previously described [30] and protein concentration was determined prior to use in buffer H150 (25 mM HEPES (pH = 7.5 at 25 • C), 150 mM NaCl, 10% glycerol, 2 mM 2-mercaptoethanol, and 0.5 mM ZnCl 2 ) using extinction coefficients ε 280 = 3.29 × 10 4 M −1 cm −1 . Briefly, cc-hex YME1L was overexpressed in BL21(DE3)-competent Escherichia coli cells with an N-terminal His 6 -SUMO tag cleavable by treatment with the Ulp1 protease.…”

“…Nucleotide binding experiments were performed based on our previously reported protocol [30]. As shown schematically in Figure 5A, Syringes A and B contained 1 µM YME1L and 40 µM MANT-ATP, respectively, but in the presence of [urea] spanning from 0 to 4 M. All experiments were performed in buffer H150 lacking MgCl 2 under pseudo-first-order conditions with [MANT-ATP] in 40-fold excess of [YME1L].…”

“…[28]. Based on our previous work, the conformational behavior exhibited by a recombinant YME1L construct [29] depends on the concentration of available nucleotide [30]. Oxidative conditions are known to promote depletion of the nucleotide pool due to a decrease in mitochondrial respiratory function.…”