Computational protocol: Computational and Functional Characterization of Angiogenin Mutations, and Correlation with Amyotrophic Lateral Sclerosis

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[…] The crystal structure of human Angiogenin (PDB: 1B1I) was used as the starting point. The heteroatoms (crystallographic waters and cofactor, CIT) were removed from the structure before simulation. The Angiogenin proteins containing D22G and L35P mutations were prepared in silico by replacing the target amino acid residues with the desired ones, while keeping the secondary structures intact. All simulations were performed using energy minimizations, followed by gradual heating of the system. Each system was initially minimized employing 2500 steps of steepest descent followed by 1000 steps of conjugate gradient minimization. Topology and parameter files for the protein were generated using the “ff99SB” force field . All hydrogen atoms were added using the Xleap tool of AMBER 11 . The system was solvated in an octahedral box of TIP3P water with ∼10 Å between the protein surface and the box boundary. The free protein was neutralized by adding Cl− counter ions for individual models. The SANDER module of AMBER 11 package was used for all the MD simulations . As described in our previous report , , all the MD simulations were performed for 100 ns in isothermal isobaric ensemble (NPT). MD simulations were carried out with periodic boundary conditions and using the Berendsen temperature coupling . The algorithm SHAKE was applied to fix all covalent bonds containing hydrogen atoms . The particle-mesh-Ewald (PME) method was used for treating the long-range electrostatic interactions. Simulation of Angiogenin with a tag containing the Glutathione-S-Transferase (GST) and hexa-histidine moieties was carried out in AMBER 11. The initial tagged structure was predicted and generated using the Bhageerath-H and I-TASSER protein structure prediction servers –. The lowest energy structure obtained from these prediction servers was used for a 50 ns long MD simulation. MD simulations were performed on a single graphics processing unit (GPU) card, installed at the Supercomputing Facility (http://www.scfbio-iitd.res.in/) of Indian Institute of Technology Delhi. [...] The cDNA for human ANG gene (369 bp) was amplified by PCR from the plasmid pCMV6-XL4 (OriGene) and eventually cloned in the BamHI and EcoRI restriction sites of the E. coli expression vector pGEX-6P-2 (GE-Healthcare), with a C-terminal hexa-histidine His-tag. The point mutations -D22G and L35P- were generated in ANG through site directed mutagenesis (Stratagene). Protein expression was induced in E. coli BL21 cells by addition of 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Sigma), at an A600 of 0.6. Cells were harvested 4 hours post-induction, and cell pellets were disrupted by the addition of lysozyme (Sigma), followed by sonication (Branson sonifier 250, Netherlands). Following centrifugation at 10,000 rpm for 1 hour, the Angiogenin-GST fusion proteins were recovered in the soluble fraction. Purification of wild-type and mutated proteins was performed through standard Ni-nitrilotriacetic acid (Ni-NTA) affinity-based purification procedure (Qiagen). Fractions containing eluted fusion proteins were pooled, dialyzed and concentrated, before being further purified through size-exclusion chromatography using a Superdex 75 10/300 GL column (GE Healthcare). The concentration of wild-type and mutant proteins was determined using an extinction coefficient of 54945 M−1 cm−1 at 280 nm (http://web.expasy.org/protparam/). […]

Pipeline specifications

Software tools Bhageerath-H, I-TASSER, ProtParam
Databases ExPASy
Applications Protein structure analysis, Protein physicochemical analysis
Organisms Homo sapiens
Diseases Amyotrophic Lateral Sclerosis, Genetic Diseases, Inborn