19 research outputs found
Dense Poly(ethylene glycol) Brushes Reduce Adsorption and Stabilize the Unfolded Conformation of Fibronectin
Polymer brushes, in which polymers are end-tethered densely to
a grafting surface, are commonly proposed for use as stealth coatings
for various biomaterials. However, although their use has received
considerable attention, a mechanistic understanding of the impact
of brush properties on protein adsorption and unfolding remains elusive.
We investigated the effect of the grafting density of poly(ethylene
glycol) (PEG) brushes on the interactions of the brush with fibronectin
(FN) using high-throughput single-molecule tracking methods, which
directly measure protein adsorption and unfolding within the brush.
We observed that, as grafting density increased, the rate of FN adsorption
decreased; however, surface-adsorbed FN unfolded more readily, and
unfolded molecules were retained on the surface for longer residence
times relative to those of folded molecules. These results, which
are critical for the rational design of PEG brushes, suggest that
there is a critical balance between protein adsorption and conformation
that underlies the utility of such brushes in physiological environments
Grafting Density Impacts Local Nanoscale Hydrophobicity in Poly(ethylene glycol) Brushes
Accumulated
single-molecule observations of a fluorescent solvatochromic
probe molecule were found to provide detailed local information about
nanoscale hydrophobicity in polymer brushes. Using this approach,
we showed that local hydrophobicity in poly(ethylene glycol) (PEG)
brushes was spatially heterogeneous and increased with the surface
grafting density of the polymer chains. These findings may provide
an explanation for prior observations of the denaturation of surface-adsorbed
proteins on PEG brushes with high grafting densities, which is believed
to influence protein-mediated cell–surface interactions. Moreover,
by employing the broad range of existing environmentally sensitive
fluorophores, this approach may potentially be used to characterize
nanoscale changes in a variety of physicochemical properties within
polymeric materials
Connecting Protein Conformation and Dynamics with Ligand–Receptor Binding Using Three-Color Förster Resonance Energy Transfer Tracking
Specific binding
between biomolecules, i.e., molecular recognition, controls virtually
all biological processes including the interactions between cells
and biointerfaces, both natural and synthetic. Such binding often
relies on the conformation of biomacromolecules, which can be highly
heterogeneous and sensitive to environmental perturbations, and therefore
difficult to characterize and control. An approach is demonstrated
here that directly connects the binding kinetics and stability of
the protein receptor integrin α<sub>v</sub>β<sub>3</sub> to the conformation of the ligand fibronectin (FN), which are believed
to control cellular mechanosensing. Specifically, we investigated
the influence of surface-adsorbed FN structure and dynamics on α<sub>v</sub>β<sub>3</sub> binding using high-throughput single-molecule
three-color Förster resonance energy transfer (FRET) tracking
methods. By controlling FN structure and dynamics through tuning surface
chemistry, we found that as the conformational and translational dynamics
of FN increased, the rate of binding, particularly to folded FN, and
stability of the bound FN−α<sub>v</sub>β<sub>3</sub> complex decreased significantly. These findings highlight the importance
of the conformational plasticity and accessibility of the arginine-glycine-aspartic
acid (RGD) binding site in FN, which, in turn, mediates cell signaling
in physiological and synthetic environments