Exploring the orientation of a PAS-domain protein at model protein interfaces with distinct secondary-structure content across nano- and micro-scales

Abstract

While electrostatic interactions often drive protein adsorption at biointerfaces, the influence of the underlying secondary structure of the scaffold remains poorly understood. In this study, we compare the adsorption of photoactive yellow protein (PYP) onto three positively charged model protein surfaces: a rigid, planar β-sheet scaffold, a three-dimensional α-helical scaffold, and their (αβ) mixture, by using controlled pH and temperature adjustments to impose distinct secondary structures of poly-L-lysine. Using chiral and achiral vibrational sum-frequency generation (VSFG) and nano-FTIR spectroscopy, we demonstrate that the β-sheet surface promotes the formation of a well-defined monolayer with a signature β-scaffold peak at 1627 and 1680 cm⁻¹. In contrast, the α-helical surface induces a multilayering effect, resulting in a five-fold enhancement of the chiral signal and the emergence of a prominent broad feature at 1654 cm⁻¹, assigned to a chiral hydration network trapped within the protein assembly. Our results reveal that the 3D ‘brush-like’ topography of the helical surface provides superior orientational templating compared to the planar β-sheet, forcing the PYP molecules into a near-perfect vertical alignment. This work highlights that the conformational architecture of a biointerface is as critical as its chemical functionality in governing protein orientation and hydration at the nanoscale.