Theoretical studies of novel antifouling templates for specific capture of target proteins

GONZALEZ SOLVEYRA, ESTEFANIA; SZLEIFER, IGAL

San Luis

Encuentro; XLVIII Reunión Anual de la Sociedad Argentina de Biofísica; 2019

Sociedad Argentina de Biofísica

Single particle reconstruction (SPR) analysis of cryoelectron microscopy (cryoEM)data is a rapidly growing method for determining the near-atomic molecular structures of biological samples. Affinity grids have been the most widely used approach to address the sample preparation challenge. Unfortunately, this method suffers from the limitation of having the affinity ligands in a fixed orientation near the grid surface, leading topreferred orientations of the captured protein that limits the random orientations needed for SPR.In this work, our theoretical methods are focused on the rational design of multivalent rod-like affinity capture bottle-brush polymers that can be used to harvest the target protein from bulk solution. There are two major requirements that must be addressed: 1) that the rod-shaped template avoids non-specific protein adsorption (i.e., exhibitsantifouling properties) and 2) that the affinity ligand binds specifically to the target proteinwithout imposing a preferred orientation with respect to the template. To that end, we resort to a molecular theory (MT) that has been shown to provide accurate predictions for protein adsorption and ligand-receptor binding on surfaces and interfaces. We perform systematic studies of the surface modification of nanoconstructs of different geometry (planar, cylindrical, spherical) with PEG polymer brushes of variable chainlength and molecular architecture bearing end-modified affinity ligands. We explore the system parameters required for optimal fouling resistance of untagged proteins and specific capture of tagged proteins. The resistance of these constructs against nonspecific protein adsorption is studied using a non-tagged coarse-grained lysozyme model, while the binding of target proteins to the affinity rod-like templates is modeled using His-tag green fluorescent protein (His-GFP) as the targeted molecule.MT calculations provide the lysozyme adsorption isotherms (i.e., amount of adsorbed protein) and characterize the His-GFP ligand-receptor binding as a function of several design parameters, including polymer chain length, polymer architecture, affinity ligand surface density, cylinder size, and solution conditions (pH, lysozyme & His-GFP concentrations, ionic strength). We also explore the effects of using polymer mixtures consisting of shorter PEG chains (for increased antifouling performance) and longerPEG spacers end-functionalized with an affinity ligand (for specific targeting).