Similarity Analysis Of The Blue Copper Protein Subfamilies With Respect To Their Molecular Interaction Properties

The molecular requirements necessary for the blue copper proteins to interact with their redox partners are analyzed. As highlighted in Section 1.2., the blue copper proteins are divided into subfamilies depending on their physicochemical, biological, and functional properties. What has not yet been assessed about these proteins is whether and to what extent they bind specifically to their redox partners and whether cupredoxins belonging to different subfamilies can substitute one for the other in binding to the same partners (23). Recognition of and interaction with a specific protein partner is commonly mediated by protein surface features, such as charge distribution. Therefore, comparison of the surface features of the different cupredoxins can be helpful in pinpointing the similarity and/or dissimilarity in their recognition properties and preferential binding mechanisms (26-28). The properties considered are the molecular electrostatic potential, which is important in biomolecular recognition at medium- and long-range distances, and hydrophobic potential, which is involved in short-range recognition, docking, and electron transfer. The analysis of these interaction properties was performed for a representative set of different cupredoxins selected from the available experimentally-determined structures (see Table 1).

2.1. Sequence and Structure Analyses

The first step for comparing different proteins belonging to the same superfamily is the analysis their primary structure (i.e., the amino acid sequence).

Preliminary hints for the analysis of the protein sequences were given by the alignment of all the selected cupredoxin sequences. This alignment was obtained with the CLUSTALX program (29) and is reported in Fig. 2. It shows clearly that the most conserved region among the different protein subfamilies is the Cu site (i.e., the residues liganding to the Cu atom).

A structure-based pairwise sequence alignment was then performed by the program MODELLER (30).

The alignment obtained for eight selected cupredoxins, which are representative of the different protein subfamilies studied, is reported in Fig. 3. The rational behind the use of structure-based sequence alignments rests in the fact that proteins belonging to different cupredoxin subfamilies often have very low sequence similarity (<20%) although they share the same fold and global archi-

Scheme 1.
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