The hydrophobic patch of azurin (AZ) from Pseudomonas aeruginosa is an important recognition surface for electron transfer (ET) reactions. The influence of changing the size of this region, by mutating the C-terminal copper-binding loop, on the ET reactivity of AZ adsorbed on gold electrodes modified with alkanethiol self-assembled monolayers (SAMs) has been studied. The distance-dependence of ET kinetics measured by cyclic voltammetry using SAMs of variable chain length, demonstrates that the activation barrier for short-range ET is dominated by the dynamics of molecular rearrangements accompanying ET at the AZ-SAM interface. These include internal electric field-dependent low-amplitude protein motions and the reorganization of interfacial water molecules, but not protein reorientation. Interfacial molecular dynamics also control the kinetics of short-range ET for electrostatically and covalently immobilized cytochrome c. This mechanism therefore may be utilized for short-distance ET irrespective of the type of metal center, the surface electrostatic potential, and the nature of the protein-SAM interaction.

The C-terminal copper-binding loop in the ␤-barrel fold of the cupredoxin azurin has been replaced with a range of sequences containing alanine, glycine, and valine residues to assess the importance of amino acid composition and the length of this region. The introduction of 2 and 4 alanines between the coordinating Cys, His, and Met results in loop structures matching those in naturally occurring proteins with the same loop lengths. A loop with 4 alanines between the Cys and His and 3 between the His and Met ligands has a structure identical to that of the WT protein, whose loop is the same length. Loop structure is dictated by length and not sequence allowing the properties of the main surface patch for interactions with partners, to which the loop is a major contributor, to be optimized. Loops with 2 amino acids between the ligands using glycine, alanine, and valine residues have been compared. An empirical relationship is found between copper site protection by the loop and reduction potential. A loop adorned with 4 methyl groups is sufficient to protect the copper ion, enabling most sequences to adequately perform this task. The mutant with 3 alanine residues between the ligands forms a strand-swapped dimer in the crystal structure, an arrangement that has not, to our knowledge, been seen previously for this family of proteins. Cupredoxins function as redox shuttles and are required to be monomeric; therefore, none have evolved with a metal-binding loop of this length.loop modeling ͉ metalloproteins ͉ protein engineering ͉ protein folding ͉ strand-swapped dimerization L oops link the main secondary structure elements of all globular proteins and are invariably found at the molecular surface. These regions play essential roles by contributing to active sites and facilitating protein interactions. Surface loops can be determinants of protein interactions, and loops that disfavor association may help to maintain a protein as a monomer to enable rapid diffusion in a cell (1). Loop variations are therefore important for altering functionality without influencing a protein's core structure, and can also play a vital role in protein folding (2-4). Despite their importance, de novo loop design is only at a preliminary stage of development, and loop-forming amino acid sequences have not been established. The relationship between loop composition and structure is a feature of protein architecture that is currently poorly understood. Metalloproteins constitute Ϸ1/3 of structurally characterized proteins and it has been estimated that up to 50% of all proteins bind a metal ion (5). Those metalloproteins whose function necessitates an interaction with a protein partner invariably bind their cofactor via loop residues. The requirements for metal coordination by such loops provide additional constraints to these regions. Altering metal-binding loops can simultaneously tune the properties of a metal site and an important interaction surface.The ␤-barrel motif is one of the most widely occurring and the second most abundant...

No abstract