Figure 2: Engineering a soluble active YME1L protease.
From: Engineered AAA+ proteases reveal principles of proteolysis at the mitochondrial inner membrane
(a) Crystal structure of the cc-hex hexameric coiled coil (PDB ID: 3R3K) showing dimensions of the hexamer and schematic representation of the hexYME1L protease. (b) Migration profile of monomeric YME1L-AP and hexameric hexYME1L by size-exclusion chromatography showing observed and theoretical molecular weights for each species (observed/theoretical). (c) Rate of ATP hydrolysis by hexYME1L against increasing concentration of ATP. Lines are non-linear least-squares fits to the Hill version of the Michealis–Menten equation [v=kATPase/(1+KM/[ATP]n] (kATPase=42 ATPs min−1 enz6−1; KM=1.4 mM; n=2.6). (d) SDS–PAGE showing rapid degradation of β-casein (20 μM) by hexYME1L (1 μM). No degradation is observed in either the absence of ATP, the presence of the non-hydrolysable analogue ATPγS, or by a variant containing an ATPase abolishing mutation (hexYME1LE439Q) in the presence of ATP. (e) Loss of β-casein over time from degradation experiments in d. (f) Early time points (0–20 min) from e. Lines are linear fits used to calculate the initial rate of β-casein degradation by hexYME1L in the presence of ATP (0.49±0.08 molecules min−1 enz6−1). All data shown are from independent experiments and error bars indicate±s.e.m. (n=3).
