Creating Biomimetic Surfaces through Covalent and Oriented Binding of Proteins

Abstract

This manuscript describes a novel method for the biofunctionalization of glass surfaces with polyhistidine-tagged proteins. The main innovation of this methodology consists of the covalent binding between the nitrilotriacetic acid (NTA) moiety and the proteins, ensuring not only orientation, but also stability of the recombinant proteins on NTA-covered surfaces. In this work, as C-terminal polyhistidine tagged cadherin extracellular fragments have been used, this methodology guarantees the proper orientation of these proteins, by mimicking their insertion into cell plasma membranes. These biofunctionalized surfaces have been characterized by confocal microscopy, X-ray photoelectron spectroscopy, contact angle, and atomic force microscopy, showing a high density of cadherins on the glass surfaces and the stability of the linkage. The prepared materials exhibited a high tendency to promote cell spreading, demonstrating the functionality of the protein and the high utility of these biomaterials to promote cell adhesion events. Interestingly, differences in the cytoskeleton organization have been observed in cells adhering to surfaces with no cadherins or with nonoriented cadherins, in comparison to surfaces functionalized with well-oriented cadherins. This method, which allows the robust immobilization of polyhistidine tagged proteins due to their covalent binding and with a defined orientation, may also find particular usefulness in the making of protein biochips, for analysis of protein-protein interactions, as well as structural and single-molecule studies.

Well-defined biomimetic surfaces to characterize glycosaminoglycan-mediated interactions on the molecular, supramolecular and cellular levels

“…The interactions between biotin and SAv, or between Fc and Z domains, are strong enough for the surfaces to remain stable over many hours. Where required, such noncovalent yet rapid and highly specific interactions could be exploited for initial coupling to guide the subsequent formation of covalent bonds at desired sites with enhanced rates [59], thereby enhancing stability and further broadening the application range.…”

Pathogenic Neisseria meningitidis utilizes CD147 for vascular colonization

“…Force spectroscopy was performed between pilins and a CD147-functionalized surface. CD147-Fc-His or control ALCAM-1-Fc-His chimera were deposited on a glass surface using a nitrilotriacetic acid that ensured covalent and oriented binding with the protein 54 . MBP-pilins and control proteins (MBP, cyclophilin) were coated on AFM tips using established protocol known to keep protein functionality intact as previously published 55,56 .…”

Cobalt(III)‐Mediated Permanent and Stable Immobilization of Histidine‐Tagged Proteins on NTA‐Functionalized Surfaces

“…To increase the affinity between the His‐tag and the Ni 2+ –NTA group, molecules with multiple NTA groups and protein with longer (i.e., His10‐tag) or multiple His‐tags have been developed. Furthermore, strategies that preform the complex with the His‐tag and Ni 2+ –NTA complex and then establish a covalent bond in a second step through photochemical reactions or less specific NHS/ N ′‐(3‐dimethylaminopropyl)‐ N ‐ethylcarbodiimide (EDC) or epoxide chemistry have been suggested . Nonetheless, these approaches require complex syntheses of special small molecules or lack high specificity.…”