Relaxation of structural constraints during Amicyanin unfolding.

We study the thermal unfolding of amicyanin by quantifying the resiliency of the native state to structural perturbations. Three signatures characterizing stages of unfolding are identified. The first signature, lateral extension of the polypeptide chain, is calculated directly from the reported crystallographic data. Two other signatures, the radial displacement of each residue from Cu(II) and the angular spread in the chain as the protein unfolds, are calculated using crystallographic data in concert with a geometrical model we introduced previously (J.J. Kozak, H. B. Gray, R. A. Garza-López, J. Inorg. Biochem. 155(2016) 44-55). Particular attention is paid to the resiliency of the two beta sheets in amicyanin. The resiliency of residues in the near neighborhood of the Cu center to destabilization provides information on the persistence of the entatic state. Similarly, examining the resiliency of residues intercalated between structured regions (beta sheets, the alpha helix) provides a basis for identifying a "hydrophobic core." A principal focus of our study is to compare results obtained using our geometrical model with the experimental results (C. La Rosa, D. Milardi, D. M. Grasso, M. P. Verbeet, G. W. Canters, L. Sportelli, R. Guzzi, Eur. Biophy. J.30(8),(2002) 559-570) on the denaturation of amicyanin, and we show that our results support a classical model proposed by these authors.