We defined a procedure to generate the atomic coordinates of an artificial protein (SynCytb), recently synthesized using the template-assembled synthetic strategy. A four-helix bundle, binding two hemes, constitutes the catalytical heart of the photosynthetic cytochrome b6f (Cytb6f) complex and the respiratory cytochrome bc1 (Cytbc1) membrane-bound protein complex. The model structure mimics the central part of the Cytb subunit and reproduces a native-like function. It consists of a four-helix bundle, two hemes, and a cyclic decapeptide as a template. Since the X-ray structure is not yet solved, the structural elements of SynCytb were assembled from scratch using all known chemical and structural information available, thereby avoiding strain and atomic clashes. A great deal of work was done to generate the coordinates of this protein by a sophisticated stepwise modeling technique. The molecular mechanics was subsequently employed to fully relax the model structure by applying the stepwise constraint energy minimization. To validate the stability and robustness of the computer-generated model structure, SynCytb is tested by monitoring the conformational stability and fluctuations during a long-term molecular dynamics (MD) simulation and comparing the results with values obtained from the crystal structure of native Cytb. The RMSD (root-mean-square deviation) and RMSF (root-mean-square fluctuation) data from the native and artificial proteins MD simulations reveal very similar profiles over time, demonstrating a relative rigidness, stability, and strain-free of the final model structure. The results of the theoretical work presented in this chapter offer an extensive cheminformatics/bioinformatics analysis to elucidate and compare the structural details and heme conformations of both proteins. Specifically, the details related to redox cofactors and their close surroundings have been examined. We analyzed the relative orientation of axial histidines ligated to heme, the heme conformations, propionates orientations, and orientation of the tilted helical axes in native and SynCytó and compared them with available experimental data. The implemented analysis provides valuable information for further research and facilitates protein design and molecular modeling since the heme redox potential intrinsically and internally depends on the axial ligands' type, orientation, alignment, and basicity. The protein design presented here can be used to generate novel proteins with tailored structural and functional properties.
Dragan M. Popović (Sat,) studied this question.