This suggests that both proteins use the classical Ser-His-Asp catalytic triad mechanism with serine acting as the nucleophile, histidine as the general acid-base, and aspartate helping to neutralize the charge forming on histidine during the catalysis.
To structurally assess the effect of these insertions and the identified critical differences between mtRF1 and mtRF1a, we built homology models for both mitochondrial proteins using the high-resolution structures of Thermus thermophilus RF1 bound to the ribosome in response to either a UAA [ 7] or UAG [ 8] stop codon as modeling templates.
Co-IP and denaturing co-IP were carried out for both endogenous and ectopically expressed proteins using the routine methods described in Supplementary data.
Distances calculated for both plastid and nuclear-encoded proteins using the Dayhoff and VT substitution matrices were larger than the average number of substitutions (i.e. calculated without substitution matrix), which shows that amino acids were most often substituted with similar residues, suggesting functional conservation.
We therefore decided to analyse the expression of 168 nuclear genes encoding mitochondrial proteins, using both the mitochondrial PCR array and the mitochondrial energy metabolism PCR array from SABiosciences (Qiagen) (results not shown).
Using this dataset, ligand and protein structures were setup using two different methods, (i), calculating Gasteiger charges on both the ligands and the proteins using AutoDockTools and (ii), calculating PM6 charges on both the ligands and the proteins using MOPAC2009 [70] on Docking Server (see Method section for details).
We attempted to re-solubilise both insoluble recombinant proteins using high concentrations of the denaturants urea or guanidinium hydrochloride followed by dialysis.
In the current study it was explored whether calculation of electrostatic potentials of both the ligands and the proteins using semi-empirical PM6 method increases docking accuracy.
The values for the fraction unfolded (αU) for both proteins were calculated using the following equation: 1where θF is the ellipticity value at 25 °C for completely folded or native protein, θt is the observed ellipticity value at any temperature between 25 and 90 °C, and θU is the ellipticity value at 90 °C for the completely denatured or unfolded state.
