Controlling Protein Surface Orientation by Strategic Placement of Oligo-Histidine Tags

Abstract

We report oriented immobilization of proteins using the standard hexahistidine (His6)-Ni2+:NTA (nitrilotriacetic acid) methodology, which we systematically tuned to give control of surface coverage. Fluorescence microscopy and surface plasmon resonance measurements of self-assembled monolayers (SAMs) of red fluorescent proteins (TagRFP) showed that binding strength increased by 1 order of magnitude for each additional His6-tag on the TagRFP proteins. All TagRFP variants with His6-tags located on only one side of the barrel-shaped protein yielded a 1.5 times higher surface coverage compared to variants with His6-tags on opposite sides of the so-called β-barrel. Time-resolved fluorescence anisotropy measurements supported by polarized infrared spectroscopy verified that the orientation (and thus coverage and functionality) of proteins on surfaces can be controlled by strategic placement of a His6-tag on the protein. Molecular dynamics simulations show how the differently tagged proteins reside at the surface in “end-on” and “side-on” orientations with each His6-tag contributing to binding. Also, not every dihistidine subunit in a given His6-tag forms a full coordination bond with the Ni2+:NTA SAMs, which varied with the position of the His6-tag on the protein. At equal valency but different tag positions on the protein, differences in binding were caused by probing for Ni2+:NTA moieties and by additional electrostatic interactions between different fractions of the β-barrel structure and charged NTA moieties. Potential of mean force calculations indicate there is no specific single-protein interaction mode that provides a clear preferential surface orientation, suggesting that the experimentally measured preference for the end-on orientation is a supra-protein, not a single-protein, effect.

Elasticity‐Dependent Fast Underwater Adhesion Demonstrated by Macroscopic Supramolecular Assembly

“…Thea bove modulus-dependent MSA phenomenon is interpreted by the theory of multivalency, [23][24][25][26][27][28][29] which describes the enhancement of the binding forces between large interfaces common in biological systems, [24][25][26] such as interactive virus/cell, cell/cell, proteins,o ra rtificial intervesicular interaction systems. [27][28][29] With as imilar surface chemistry,t he key to realizing multivalencyi st he substrate deformability,which determines the molecular motility of the interactive moieties.…”

Elasticity‐Dependent Fast Underwater Adhesion Demonstrated by Macroscopic Supramolecular Assembly

“…The above modulus‐dependent MSA phenomenon is interpreted by the theory of multivalency, which describes the enhancement of the binding forces between large interfaces common in biological systems, such as interactive virus/cell, cell/cell, proteins, or artificial intervesicular interaction systems . With a similar surface chemistry, the key to realizing multivalency is the substrate deformability, which determines the molecular motility of the interactive moieties .…”

Orthogonal supramolecular protein assembly on patterned bifunctional surfaces

“…Our recent studies on the oriented assembly of H 6 -tagged monomeric variants of an Entacmaea quadricolor red fluorescent protein, TagRFP, on Ni(II)NTA-functionalized surfaces, yielded association binding constants varying across two orders of magnitude for variants containing one (K = 3 Â 10 6 M À1 ), two (4 Â 10 7 M À1 ) or three (3 Â 10 8 M À1 ) H 6 -tags. 21 Depending on the location of the H 6 -tags on the protein, surface coverage and protein orientation could be controlled. 21 However, key to the present work is the insight that the reversibilities of the different variants' binding show considerable differences, depending on their respective number of H 6 -tags.…”

“…21 Depending on the location of the H 6 -tags on the protein, surface coverage and protein orientation could be controlled. 21 However, key to the present work is the insight that the reversibilities of the different variants' binding show considerable differences, depending on their respective number of H 6 -tags. The larger the number of H 6 -tags the more stringent the conditions necessary to reverse the binding.…”

“…Binding of TagRFP with a single H 6 -tag to Ni(II)NTA surfaces was reversed by washing with mere buffer, binding of TagRFP via two H 6 -tags required washing with buffers containing competitors such as imidazole and EDTA, to be reversibly or irreversibly broken, resp., while triply H 6 -tagged TagRFP resisted complete removal from Ni(II)NTA surfaces, even after extensive washing in the presence of competitors. 21 Therefore, we selected for the current study two of the above-mentioned TagRFP variants, specifically, with one (1H 6 TagRFP) and three (3H 6 TagRFP) H 6 -tags (Chart 1, for more details see Chart S1, ESI †). These TagRFP variants were engineered to contain one H 6 -tag at the N-terminus, in the case of 1H 6 TagRFP.…”