Computational protocol: Physicochemical origin of high correlation between thermal stability of a protein and its packing efficiency: a theoretical study for staphylococcal nuclease mutants

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Protocol publication

[…] As indicated in , there are significantly many residues for which multiple side-chain conformation models are registered in the Protein Data Bank (PDB). Instead of generating the conformers arising from all the possible combinations of those side-chain models, we carefully adopt 8 representative conformers. A total of 8 structures are thus obtained for each of the wild type, four single mutants, and five multiple mutants. The N-terminal and C-terminal regions have not been determined in the X-ray crystallography. Since these regions should be flexible in both of the unfolded and folded states (i.e., their structural properties remain unchanged upon folding), they are not specifically modeled. The N-terminal region starts from Lys6 in 1EY0(WT), 1EYD(WT), and 1EY4(S59A) whereas it starts from Leu7 in the others. The coordinates assigned to the atoms of Lys6 are not very reliable because these atoms have relatively larger B-factors. For this reason and to impartially compare the wild type, four single mutants, and five multiple mutants in terms of the thermal stability, we cut Lys6 in the three PDB structures. It follows that we consider 135 residues ranging from Lys7 to Ser141. The proper protonated states of histidines are determined using the procedure “Calculate Protein Ionization and Residue pK” [] in Accelrys ( BIOVIA) Discovery Studio. This determination is necessitated so that the break of intramolecular hydrogen bonds can be avoided in the energy minimization described below.The folded structures thus obtained are slightly modified using the energy minimization by CHARMM [] to remove unrealistic overlaps of some constituent atoms. Since it is important to keep the original X-ray structures as strictly as possible, we put positional restraints in the harmonic form on all the heavy atoms and apply 100 steps of the steepest descent energy minimization followed by the adopted basis Newton-Raphson energy minimization which is continued until the energy change during the minimization cycle becomes smaller than or equal to 1.0×10−5 kcal/mol or until the number of steps reaches 200. The force constant for the positional restraints is M kcal/(mol·Å2) where M is the mass of each atom (for example, the force constant on a carbon atom is 12 kcal/(mol·Å2)). The minimization is performed by CHARMM [] with the help of MMSTB Toolkit [] using the CHARMM22 parameters [] with the CMAP correction [] and the GBMV implicit solvent model [,]. A quantity (e.g., the measure of the thermal stability ∑ depending on geometric characteristics of the folded and unfolded states) is calculated for the 8 structures and the average of the 8 values is adopted for the folded state. […]

Pipeline specifications

Software tools BIOVIA Discovery Studio, CHARMM
Application Small-angle scattering
Chemicals Hydrogen