Insights into the role of protein molecule size and structure on interfacial properties using designed sequences

Abstract

Mixtures of a large, structured protein with a smaller, unstructured component are inherently complex and hard to characterize at interfaces, leading to difficulties in understanding their interfacial behaviours and, therefore, formulation optimization. Here, we investigated interfacial properties of such a mixed system. Simplicity was achieved using designed sequences in which chemical differences had been eliminated to isolate the effect of molecular size and structure, namely a short unstructured peptide (DAMP1) and its longer structured protein concatamer (DAMP4). Interfacial tension measurements suggested that the size and bulk structuring of the larger molecule led to much slower adsorption kinetics. Neutron reflectometry at equilibrium revealed that both molecules adsorbed as a monolayer to the air -water interface (indicating unfolding of DAMP4 to give a chain of four connected DAMP1 molecules), with a concentration ratio equal to that in the bulk. This suggests the overall free energy of adsorption is equal despite differences in size and bulk structure. At small interfacial extensional strains, only molecule packing influenced the stress response. At larger strains, the effect of size became apparent, with DAMP4 registering a higher stress response and interfacial elasticity. When both components were present at the interface, most stress-dissipating movement was achieved by DAMP1. This work thus provides insights into the role of proteins' molecular size and structure on their interfacial properties, and the designed sequences introduced here can serve as effective tools for interfacial studies of proteins and polymers.

Stabilizing and destabilizing protein surfactant-based foams in the presence of a chemical surfactant: Effect of adsorption kinetics

“…The observed adsorption kinetics of DAMP4 at pH 8.5 is consistent with the previous reported results of DAMP4 at pH 7.4 by Dwyer et al [24]. Results of Dwyer et al show that adsorption kinetics for DAMP4 at pH 7.4 is energy barrier-controlled, since the calculated experimental diffusion time constants are much larger than the theoretical values based on bulk diffusion.…”

“…Reflectivity data of all four cycles fitted to a monolayer with a thickness of 15.8 ± 1 Å. This thickness is consistent with a single layer of DAMP4 determined previously by neutron reflectometry [24]. This confirms that the DAMP4 molecules unfold at the interface as a linear a-helical structure as opposed to its four-helix bundle in the bulk solution [13].…”

“…corresponds to the equilibrium surface tension for each system. The gap between starting and ending surface pressure is related to the energy barrier to desorb adsorbed molecules during compression [24]. These gaps are 8.6 mN/m in the absence of Zn 2+ and 15.2 mN/m in the presence of Zn 2+ .…”

“…Small chemical surfactant molecules adsorb to the interface, within seconds, to achieve desired surface coverage and thus provide stable foams [20][21][22]. In contrast, due to their relatively large hydrodynamic molecular size and required structural changes at the interface, the adsorption of protein/peptide-based biosurfactants to interfaces normally takes tens of minutes, even hours to reach the equilibrium coverage [23,24]. In a study of a designed protein DAMP4 and its analogue DAMP1, Dwyer et al showed the adsorption of the larger molecule protein DAMP4 is accompanied with structural changes at the surface, and the adsorption kinetics is much slower than that of its smaller analogues DAMP1 [24].…”

Bioproduction and self-assembly of designer peptides

“…This was attributed to the requirement for rearrangement of the larger and more structured DAMP4. 195 There have been few studies of PA or β-sheet peptide assembly at air/oil-water interfaces, with most reports instead focusing on their assembly in solution (Sections 1.2.1 and 1.2.2).…”