Computational protocol: Structural and dynamical insight into thermally induced functional inactivation of firefly luciferase

Similar protocols

Protocol publication

[…] The complete three dimensional structure of Photinus pyralis luciferase (P08659 and EC 1.13.12.7) in free form was built by comparative homology modeling as the initial structure for further analysis by molecular dynamic simulations. Modeller [] was used for homology modeling to generate the 3D structure of each proposed model by satisfying spatial restraints imposed by the specified multiple sequence alignment. The sequence of Photinus pyralis mature luciferase (P08659) was fed to Basic Local Alignment Search Tool for Proteins (BLASTP) from National Center for Biotechnology Information (NCBI) to search against Protein Data Bank (PDB) by keeping its default algorithm parameters. To build the homology model of free luciferase structure, three incomplete structures of Photinus pyralis luciferase (1BA3, 1LCI and 3IEP PDB codes with 2.2, 2 and 2.1 Å resolution respectively) were taken as template structures and Modeller9v10 software simulation was used for model building. Modeller input alignment file was created manually via the alignment of three sequences produced by ClustalX program []. To guarantee sufficient conformational sampling, the generation of 100 models was supposed for satisfactory sampling. The selection of the best model was based on the DOPE assessment score. The best constructed model was then validated by PROCHECK [] for the quality of structures. [...] In order to refine and analyze the structural and dynamical behavior of the generated models by homology modeling, molecular dynamic simulation method was applied. All molecular dynamic simulations were carried out with the GROMACS simulation package version 4.5.4 [, ] using the Amber99SB force field parameters [] as implemented in GROMACS. The protonation state of the titratable groups of luciferase at pH 7.0 was determined after estimating their pKa values by H++ server (accessible at http://biophysics.cs.vt.edu/H++). Form 13 existing histidines, H310 and H419 were considered to be positive and the others were supposed to be neutral. Two sets of simulations were performed using the generated models in homology modeling as the initial structure: free luciferase at 298 K and free luciferase at 325 K (above the Tm of enzyme). For each simulation the enzyme was centered in periodic cubic box with edges of about 10 nm. The boxes were solvated with pure water using TIP4P-Ew water molecules []. To achieve a neutral simulation box, the net charge of the protein was neutralized by replacing water molecules with necessary Na+ ions. In order to remove bad atomic contacts, each solvated and neutralized system was subjected to energy minimization using the steepest descent algorithm until the maximum force was smaller than 500 kJ/mol.nm. After energy minimization, two separate position-restrained MD simulations were carried out. First, to set the atomic velocities and adjust the system temperature, an MD simulation was performed for 100 ps at desired temperature and in a constant volume condition (NVT). Second, to adjust pressure and densities, an MD simulation with 1 ns duration was carried out at constant temperature and pressure (NPT). In all of the NPT simulations, temperature and pressure were kept close to the intended values (298 K or 325 K and 1 bar) by using the modified Berendsen thermostat (V-rescale) [] and Berendsen barostat algorithm [] with τT = 0.1 and τP = 0.5 ps respectively. The LINCS algorithm was utilized to constrain all bonds []. A single-range cutoff was applied for calculation of the non-bonded interactions. The cutoff radius was set to 1.2 nm for both Coulombic and Lennard-Jones interactions. The Coulombic interactions of longer range were calculated by means of the particle-mesh Ewald (PME) algorithm []. After equilibration, all two sets of production MD simulations were carried out for 100 ns duration. Time steps of 2 fs duration were used and frames were collected every 2 ps.In order to evaluate the quality or sufficiency of conformational sampling of simulations, the production MD period was divided into four 25 ns parts and principal component analysis was performed on each sub-trajectory. Eigenvectors and eigenvalues were obtained from the diagonalization of the covariance matrices of the Ca atoms, and the principal components were generated by projecting the trajectories on the respective eigenvectors. The cosine content of the principal components was calculated to estimate whether the conformational fluctuations are connected with the potential (when the cosine content is close to 0) or with random diffusion (when the cosine content is close to 1) [].The collective and correlated motions of a protein can be detected through the analysis of molecular dynamic simulations via principal component analysis (PCA) or essential dynamic (ED) []. This is especially a useful method to compare the behavior of similar systems subjected to different conditions. The PCA or ED method is based on the construction of the covariance matrix of the coordinate fluctuations of CA atoms of enzyme along the trajectory after fitting to a suitable reference structure. After diagonalization of the covariance matrix, the information about the collective and correlated motion of protein was obtained from a few chosen eigenvectors and eigenvalues which account for the most variations in the system without much loss of information. […]

Pipeline specifications