Hydration state of single cytochrome c monolayers on soft interfaces via neutron interferometry

Yeast cytochrome c (YCC) can be covalently tethered to, and thereby vectori ally oriented on, the soft surface of a mixed endgroup (e.g., -CH3/-SH = 6: 1, or -OH/-SH = 6:1) organic self-assembled monolayer (SAM) chemisorbed on the surface of a silicon substrate utilizing a disulfide linkage between it s unique surface cysteine residue and a thiol endgroup. Neutron reflectivit ies from such monolayers of YCC on Fe/Si or Fe/Au/Si multilayer substrates with H2O versus D2O hydrating the protein monolayer at 88% relative humidit y for the nonpolar SAM (-CH3/-SH = 6:1 mixed endgroups) surface and 81% for the uncharged-polar SAM (-OH/-SH = 6:1mixed endgroups) surface were collec ted on the NG1 reflectometer at NIST. These data were analyzed using a new interferometric phasing method employing the neutron scattering contrast be tween the Si and Fe layers in a single reference multilayer structure and a constrained refinement approach utilizing the finite extent of the gradien t of the profile structures for the systems. This provided the water distri bution profiles for the two tethered protein monolayers consistent with the ir electron density profile determined previously via x-ray interferometry (Chupa et al., 1994).