Biofunctionalized Polymer Surfaces Exhibiting Minimal Interaction towards Immobilized Proteins

Abstract

Biomolecules exhibit remarkable sensitivity, selectivity and efficiency in their response to specific stimuli, which makes them extremely attractive targets for sensor applications.[1] Biosensor designs are often based on hybrid (biotic-abiotic) nanoscale interface technologies, in which biomolecules are immobilized on solid substrates. [2,3] The delicate structure of proteins requires coating of these surfaces with carefully designed films that interact only weakly with the protein, except through a strong tether for protein attachment, and thus preserve the functionally competent, properly folded conformation. The surface must not only resist interaction with the hydrophilic surface of the native protein structure, rather, for stability over long periods of times and/or under destabilizing conditions (extreme temperatures, cosolvents), the surface must also be inert towards hydrophobic protein moieties that become transiently or permanently exposed upon unfolding. Here we present a versatile surface preparation that matches exactly these requirements. It was specifically developed for single-molecule studies of protein folding, the intriguing process by which the linear polypeptide chain assumes its specific, functionally competent three-dimensional architecture. [4][5][6] Protein folding is an intrinsically heterogeneous process because a huge number of folding pathways on the free energy surface connect the myriad of unfolded conformations with the much smaller set of conformations belonging to the native state.[7] Förster resonance energy transfer (FRET) between a donor and acceptor fluorophore has been used extensively in equilibrium and time-resolved investigations of protein folding on large protein ensembles. [8][9][10] With the advent of single-molecule fluorescence spectroscopy, a technique has become available that enables us to examine protein folding pathways of individual protein molecules in real time, and to detect intermediate states and trajectories leading to misfolded structures, which have been implicated in diseases such as the spongiform encephalopathies (BSE, CJD, Alzheimer's disease).[11] The strong distance dependence of FRET can be exploited to observe the reconfiguration of a single polypeptide chain in real time. [12] To this end, a donor-acceptor pair of fluorescent dye molecules is attached to specific locations along the polypeptide chain so that the two dyes are in close proximity in the folded structure and further apart in the unfolded chain. Such experiments, performed on proteins diffusing freely in solution, [13,14] have already yielded insightful results. Free diffusion, however, limits the observation time to the transit time of the protein through the sensitive volume of the microscope, which is typically less than 1 ms. Longer observation time can be realized by fixing the molecules in space to within a volume smaller than the extent of the point-spread function of the microscope. Immobilization of proteins inside surface-bound lipid vesicles is an elegant approach, [15]...

Effects of surface tethering on protein folding mechanisms

“…The total protein-surface interaction potential is given by 10 . Here z i is the z coordinate of the ith residue; h is an energetic parameter equivalent to the value of h in the Lennard-Jones potential that models the attractive interactions between BB residues; and is the diameter of each monomer in the protein as well as the equilibrium length of the bonds between each monomer.…”

“…Additionally, they must have a negligible impact on the dynamics of the attached proteins. Nienhaus and coworkers (9,10) have developed and analyzed several types of surfaces, deemed minimally interacting, for the explicit purpose of performing experiments on individually tethered biomolecules. In particular, they have designed a star-shaped polyethylene glycol (PEG) surface that allows for the reversible folding and unfolding of attached proteins (10,11).…”

“…Nienhaus and coworkers (9,10) have developed and analyzed several types of surfaces, deemed minimally interacting, for the explicit purpose of performing experiments on individually tethered biomolecules. In particular, they have designed a star-shaped polyethylene glycol (PEG) surface that allows for the reversible folding and unfolding of attached proteins (10,11). Other groups have performed single-molecule experiments of biomolecules on a variety of surfaces, including BSA (12)(13)(14).…”

“…In the development of the chemically inert surfaces detailed above, researchers have taken care to ascertain that the stability and function of attached proteins are retained (10,12). However, it is not obvious that retaining protein function guarantees that folding is unchanged by tethering.…”

Single‐Molecule Fluorescence Studies of Protein Folding and Conformational Dynamics

“…As for Csp, sequential labeling was employed to equip RNAse H with Alexa Fluor 546 and Alexa Fluor 647 fluorophores at two engineered cysteines, but in contrast to the burst spectroscopy studies discussed above, Nienhaus and colleagues opted for single-molecule experiments with immobilized protein. 170,171 To prevent unspecific surface adsorption of the immobilized protein previously seen in another study, 127,139 the authors used chemically designed, biotinylated surface coatings prepared by spin coating (Figure 10). The surface coatings were made of ultrathin networks of isocyanate-terminated "star-shaped" poly(ethylene oxide) (PEO) molecules, cross-linked at their ends via urea groups to minimize the intertwining of a denatured polypeptide chain with the PEO polymer.…”