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Differential tunnel-opening patterns were established in static structures of mammalian CYP450 isoforms and subsequently applied to identify tunnel-intersecting residues. The identified tunnel-intersecting residues permitted the subsequent construction of gating models via the identification of intra-protein interactions. We define 28 two-state gating models and 37 singlet gating-residue models. Our results reveal the preponderance of aromatic gating residues in CYP3A4 and CYP2A6, whereas we find a preponderance of polar/charged residues in CYP2C5. In CYP2C8 there is balanced presence of polar/charged and hydrophobic aliphatic residues in gating models, whilst in CYP2C9 there is balanced presence of all residue-types. These patterns suggest fast evolutionary dynamics for gating residues and we find that the average rate of evolution of gating residues in CYP2C5, CYP2C8, CYP2C9 and CYP2A6 is significantly faster than the average rate of evolution of the entire sequence. Our study identifies 67% of calculable gating models identified in the literature by molecular dynamics approaches and 92% of residues appearing in literature models appear in our models. However, only 6% of the models identified in this work had been previously-described in the literature. This suggests that our study has defined the most comprehensive list yet of tunnel-gating models in mammalian CYP450 and in doing so have created a benchmark for molecular dynamics approaches to the ligand-tunnelling problem in CYP450.

The study of the protein–protein interactions (PPIs) of unique ORFs is a strategy for deciphering the biological roles of unique ORFs of interest. For uniform reference, we define unique ORFs as those for which no matching protein is found after PDB-BLAST search with default parameters. The uniqueness of the ORFs generally precludes the straightforward use of structure-based approaches in the design of experiments to explore PPIs. Many open-source bioinformatics tools, from the commonly-used to the relatively esoteric, have been built and validated to perform analyses and/or predictions of sorts on proteins. How can these available tools be combined into a protocol that helps the non-expert bioinformaticist researcher to design experiments to explore the PPIs of their unique ORF? Here we define a pragmatic protocol based on accessibility of software to achieve this and we make it concrete by applying it on two proteins—the ImuB and ImuA’ proteins from Mycobacterium tuberculosis. The protocol is pragmatic in that decisions are made largely based on the availability of easy-to-use freeware. We define the following basic and user-friendly software pathway to build testable PPI hypotheses for a query protein sequence: PSI-PRED → MUSTER → metaPPISP → ASAView and ConSurf. Where possible, other analytical and/or predictive tools may be included. Our protocol combines the software predictions and analyses with general bioinformatics principles to arrive at consensus, prioritised and testable PPI hypotheses.

A number of molecular biology techniques are available to generate variants from a particular start gene for eventual protein expression. We discuss the basic principles of these methods in a repertoire that may be used to achieve the elemental steps in protein engineering. These include site-directed, deletion and insertion mutagenesis. We provide detailed case studies, drawn from our own experiences, packaged together with conceptual discussions and include an analysis of the techniques presented with regards to their uses in protein engineering.

In this work, a model for the interaction between CYP2B4 and the FMN domain of rat P450-oxidoreductase is built using as template the structure of a bacterial redox complex. Amino acid residues identified in the literature as cytochrome P450 (CYP)-redox partner interfacial residues map to the interface in our model. Our model supports the view that the bacterial template represents a specific electron transfer complex and moreover provides a structural framework for explaining previous experimental data. We have used our model in an exhaustive search for complementary pairs of mammalian CYP and P450-oxidoreductase (POR) charge clusters. We quantitatively show that among the previously defined basic clusters, the 433K-434R cluster is the most dominant (32.3% of interactions) and among the acidic clusters, the 207D-208D-209D cluster is the most dominant (29%). Our analysis also reveals the previously not described basic cluster 343R-345K (16.1% of interactions) and 373K (3.2%) and the acidic clusters 113D-115E-116E (25.8%), 92E-93E (12.9%), 101D (3.2%) and 179E (3.2%). Cluster pairings among the previously defined charge clusters include the pairing of cluster 421K-422R to cluster 207D-208D-209D. Moreover, 433K-434R and 207D-208D-209D, respectively the dominant positively and negatively charged clusters, are uncorrelated. Instead our analysis suggests that the newly identified cluster 113D-115E-116E is the main partner of the 433K-434R cluster while the newly described cluster 343R-345K is correlated to the cluster 207D-208D-209D.