PDB_NP_CONT

2.4. Surface characteristics

The overall hydrophobic contact area between residues of different monomers was calculated using the program PDB_NP_CONT (Drabløs, 1999) with the aid of a Perl script. The PDB_NP_CONT program calculates pairwise atom contact areas between apolar atoms using a set of 512 points located on a sphere around each atom. The sphere interaction radius of each atom is equal to the sum of the van der Waals radius of the atom type plus the radius of a water molecule. Then, for each atom, the closest interacting atom is found for every point that is not buried by other atoms of the same residue.

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Apolar contact area (ACA)

The apolar contact area is the area of contact between coupled non-polar atoms in a protein. It was calculated for different structural environments: nucleus, interface between monomers and protein surface. The program utilized was Pdb_np_cont [39], which calculates the area of contact between coupled non-polar atoms starting from a standard PDB file. Briefly, this method is based on the classification of points located on a sphere of interaction radius, surrounding each non-polar atom. The interaction radius is the van der Waals radius of each atom type, plus the radius of a water molecule. The output of this program was utilized to calculate the pairwise residue contact areas for every possible pair of residues belonging to the structures analysed.

Individual protein chains were used for analysis of the protein core, while analyses of the protein subunit interfaces were carried out with structures in their quaternary assembly. Only the 9 pairs of proteins possessing an oligomeric biological unit were used in this case. Protein interfaces consists of those residues making apolar contacts to another protein chain, as defined by Pdb_np_cont [38]. 

A residue was assigned to the structural environment "nucleus" if its relative solvent accessibility, calculated with the program NACCESS [40], is less than four different alternative thresholds (0%, 3%, 5% and 9%). A residue is instead assigned to the protein surface if its relative solvent accessibility is greater than an arbitrarily fixed threshold. To this end, four different alternative thresholds (25%, 40%, 55% and 70%) were used.

For each structural environment considered the per-residue mean apolar contact area was calculated by dividing the total apolar contact area by the number of residues belonging to that structural environment.

Conserved hydrophobic contacts (CHCs) were identified in each pair of homologous enzymes through the combined use of the web tools CE-MC [41] for calculating the structural alignment, SCR_FIND for identification of structurally conserved regions and CHC_FIND for the detection of the CHCs and their apolar contact area [42]. SCRs were defined as regions displaying a similar local conformation, with a mean positional RMSD of the equivalent α-carbon positions of the structures superposed ≤ 3.0 Å, lacking indels and composed of at least three consecutive residues. A CHC is defined as a conserved hydrophobic contact formed between two residues belonging to an SCR, in members belonging to a family or superfamily of proteins [42]. We considered only those contacts that are formed between residues distant at least three positions along the sequence and residues that are evolutionarily conserved according to the server CONSURF [43], i.e. belonging to the classes 7, 8 and 9. These three classes contain the most conserved amino acid positions from a total of nine equally sized categories of relative degree of conservation.

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