Molecular Insights into the Physical Adsorption of Amphiphilic Protein PhaF onto Copolyester Surfaces

Abstract

Phasins are amphiphilic proteins located at the polymer−cytoplasm interface of bacterial polyhydroxyalkanoates (PHA). The immobilization of phasins on biomaterial surfaces is a promising way to enhance the hydrophilicity and supply celldirecting elements in bioinstructing processes. Optimizing the physical adsorption of phasins requires deep insights into molecular processes during polymer−protein interactions to preserve their structural conformation while optimizing surface coverage. Here, the assembly, organization, and stability of phasin PhaF from Pseudomonas putida at interfaces is disclosed. The Langmuir technique, combined with in situ microscopy and spectroscopic methods, revealed that PhaF forms stable and robust monolayers at different temperatures, with an almost flat orientation of its α-helix at the air−water interface. PhaF adsorption onto preformed monolayers of poly[(3-R-hydroxyoctanoate)-co-(3-R-hydroxyhexanoate)] (PHOHHx), yields stable mixed layers below π = ∼15.7 mN/m. Further insertion induces a molecular reorganization. PHOHHx with strong surface hydrophobicity is a more adequate substrate for PhaF adsorption than the less hydrophobic poly[(rac-lactide)-co-glycolide] (PLGA). The observed orientation of the main axis of the protein in relation to copolyester interfaces ensures the best exposure of the hydrophobic residues, providing a suitable coating strategy for polymer functionalization.

Phasin interactome reveals the interplay ofPhaFwith the polyhydroxyalkanoate transcriptional regulatory proteinPhaDinPseudomonas putida

“…Polyhydroxyalkanoates (PHA) are biodegradable polyesters synthesized from ( R )‐3‐hydroxyacyl‐CoA monomers, which can be produced from intermediates of carbon metabolism by diverse microorganisms and accumulate as intracellular insoluble granules (termed PHA granules or carbonosomes) (Rehm, 2010; Prieto et al ., 2016). Phasins are multifunctional proteins with the ability to associate to the surface of PHA granules in vivo and to PHA beads or films in vitro (Jendrossek, 2009; Maestro and Sanz, 2017; Tarazona et al ., 2019b). Although the exact composition of the carbonosome is unknown, phasins are recognized as the predominant granule associated proteins (GAPs) and make up approximately 2% of the total granule mass (Wieczorek et al ., 1995; Sznajder et al ., 2015).…”

Crystallization and degradation behaviour of multiblock copolyester blends in Langmuir monolayers

“…[9] Wide-angle X-ray scattering (WAXS) measurements were performed with a D8 Discover spectrometer (Bruker AXS, Karlsruhe, Germany) following a procedure described before. [9] Nuclear magnetic resonance spectroscopy (NMR) 1 H and 13 C NMR spectra were recorded using a DRX Avance 500 MHz spectrometer (Bruker, Rheinstetten, Germany) at room temperature in deuterated chloroform. Chemical shifts (δ) are reported in parts per million (ppm) relative to residual chloroform at δ 7.26 ppm for 1 H NMR spectra and δ 77.0 ppm for 13 C spectra.…”

“…[9] Nuclear magnetic resonance spectroscopy (NMR) 1 H and 13 C NMR spectra were recorded using a DRX Avance 500 MHz spectrometer (Bruker, Rheinstetten, Germany) at room temperature in deuterated chloroform. Chemical shifts (δ) are reported in parts per million (ppm) relative to residual chloroform at δ 7.26 ppm for 1 H NMR spectra and δ 77.0 ppm for 13 C spectra. Samples were dissolved in CDCl 3 at a concentration of 15 mg mL -1 .…”

Bioengineered Polyhydroxyalkanoates as Immobilized Enzyme Scaffolds for Industrial Applications

“…Another interesting class of PAPs, the phasins, are a type of amphipathic proteins that have several roles in controlling the structure and surface properties of PHA particles (Pieper-Fürst et al, 1994;Wieczorek et al, 1995). Notably, phasins have a high binding affinity to the outer surface of PHA particles in vivo and in vitro mediated by physical adsorption (Tarazona et al, 2019).…”