Computational protocol: Construction of Chimeric Dual-Chain Avidin by Tandem Fusion of the Related Avidins

Similar protocols

Protocol publication

[…] The predicted models of chimeric dual-chain fusion proteins were generated by exploiting the existing 3-D structures of dcAVD (PDB 2C4I), SA (PDB 1MK5), AVR2 (PDB 1WBI) and AVR4 (PDB 1Y53) as templates. The sequence forming the cp65-subunit of dcAVD (PDB 2C4I) was first replaced with a corresponding sequence from another protein that had its sequence reorganized equally (for details, see and ). The program Modeller 9v2 was then used to generate a homology model of the pseudodimer. Structural water molecules were included according to their template structures, and the following structures were also used to position the structural water molecules: PDB 1SLF, PDB 1AVE, and PDB 2AVI. The pseudotetrameric forms were then generated by positioning two pseudodimers by structural superimposition with BODIL . The generated homology models were subjected to molecular dynamics simulation using the program NAMD 2.6 and the CHARMM22 force field . First, the models (including structural waters extracted from PDB-files) were placed in a box filled with TIP3 waters (box size approximately 80 Å×80 Å×70 Å) using the SOLVATE algorithm in the program VMD 1.8.6 . Na+ or Cl− ions were added to neutralize the system. The complete systems contained 58377 (dcAVD/AVR4), 56803 (dcAVD/AVR2), and 53207 (dcAVD/SA) atoms. The systems were then subjected to minimization as follows: the first minimization was performed by fixing all the protein atoms and allowing water molecules to move according to minimization procedure implemented in NAMD for 4000 steps. The second 4000 step minimization was performed by releasing all the atoms in the system except Cα atoms. Finally, the system was minimized without constraints for 4000 steps.The minimized system was thermalized by increasing the temperature to 310 K in 31 ps with a Berendsen barostat (1 atm). This step was followed by the production of the coordinate trajectories under NPT conditions using the Berendsen barostat (1 atm) and the Berendsen thermostat at 310 K. The resulting data were analyzed using the programs VMD 1.8.7, PyMOL 1.3, and MS Excel 2010. RMSF calculations were run such that the Cα atoms of one peptide chain were first aligned, and RMSF (deviation over the last 10 ps) values were calculated for the Cα atoms of the aligned chain. RMSF values were taken every 5 ps for the last 3 ns of equilibration (600 time points) and averaged. The force-field interaction energies between domains of dcAVDs were calculated from the simulation trajectories by the NAMD 2.7 program using 5-ps time step. […]

Pipeline specifications

Software tools MODELLER, Bodil, NAMD, VMD, PyMOL
Applications Drug design, Protein structure analysis
Organisms Gallus gallus
Chemicals Biotin