Binding and orientation of ice nucleating proteins on hydrophilic and hydrophobic surfaces probed by photoelectron spectroscopies

Abstract

Specialized bacteria can effectively nucleate ice crystals using ice nucleating proteins (INPs) anchored to the cell surface. Biogenic freezing has several applications, from snow making to cryo-medicine and freeze/antifreeze materials. For biomimetic designs of INP analogs, it is important to understand how the proteins involved in the process bind to material surfaces. In this study, we determine the binding of a model INP to hydrophobic self-assembled monolayers (SAMs) as well as hydrophilic carboxyl-terminated SAMs. INPs are large proteins with more than 1200 amino acids and a long series of repeat units. Since full-length INPs are difficult to produce and handle, we have investigated a shorter model INP dubbed InaZ9R, which has nine repeat units and still folds into the hallmark beta-helix structure known from the full-length protein. Combining x-ray photoelectron spectroscopy and near-edge x-ray absorption fine structure spectroscopy, we find that InaZ9R form closely packed monolayers on both hydrophilic and hydrophobic surfaces. Angle-resolved nitrogen K-edge near-edge x-ray absorption fine structure spectra show a high degree of orientational order associated with the native β-sheet structure.