Structural and dynamical characterization of a biologically active unfolded fibronectin-binding protein from Staphylococcus aureus

A 130-residue fragment (D1-D4) taken from a fibronectin-binding protein of Staphylococcus aureus, which contains four fibronectin-binding repeats and is unfolded but biologically active at neutral pH, has been studied extensi vely by NMR spectroscopy. Using heteronuclear multidimensional techniques, the conformational properties of D1-D4 have been defined at both a global a nd a local level. Diffusion studies give an average effective radius of 26. 2 +/- 0.1 Angstrom, approximately 75% larger than that expected for a globu lar protein of this size. Analysis of chemical shift, (3)J(HN alpha) coupli ng constant, and NOE data show that the experimental parameters agree well overall with values measured in short model peptides and with predictions f rom a statistical model for a random coil. Sequences where specific feature s give deviations from these predictions for a random coil have however bee n identified. These arise from clustering of hydrophobic side chains and el ectrostatic interactions between charged groups. N-15 relaxation studies de monstrate that local fluctuations of the chain are the dominant motional pr ocess that gives rise to relaxation of the N-15 nuclei, with a persistence length of approximately 7-10 residues for the segmental motion. The consequ ences of the structural and dynamical properties of this unfolded protein f or its biological role of binding to fibronectin have been considered. It i s found that the regions of the sequence involved in binding have a high pr opensity for populating extended conformations, a feature that would allow a number of both charged and hydrophobic groups to be presented to fibronec tin for highly specific binding.