Exploring of protein – protein interactions at the solid – aqueous interface by means of contact angle measurements

https://doi.org/10.1016/j.colsurfb.2016.02.005Get rights and content

Highlights

  • His6-VC1-RAGE domain was bond to Cu(II) or Ni(II) complexes with dipyrromethene.

  • The contact angle method was suitable for study of RAGE-S100B interactions.

  • The Ni(II)-dipyrromethene was superior for binding His6-VC1-RAGE.

Abstract

In this article we present the results of the studies on interactions between the VC1 domain of the Receptor for Advanced Glycation End Products (RAGE) and its ligand, the S100B protein, performed by contact angle measurements. Histidine-tagged (His6) VC1-RAGE domain was covalently bonded to Cu(II) or Ni(II) complexes with dipyrromethene (DPM) self-assembled on gold surface. The method based on the theory of van Oss was used for the purpose of determining the Lifshitz–van der Waals (γLW) component as well as the electron acceptor—electron donor (the Lewis acid–base, γ+–γ) parameters of the VC1-RAGE-S100B complex. Moreover, the surface free energies of the interactions between the VC1 domain attached to the surface and the ligand present in the aqueous phase were determined. The specificity of the VC1- RAGE interactions with the ligand studied was also proved.

Introduction

The immobilization of proteins on solid substrates has recently attracted much attention [1], [2], [3]. Many studies are related to the exploration of new materials for protein adsorption or methods of uniform and controlled protein immobilization. Highly efficient immobilization of biomolecules on the surface, retaining their full activity and stability, is a crucial step in biosensing technology [4], [5], [6].

Special attention has been paid to immobilization of histidine (His6)-tagged proteins onto substrates coated with metal cations chelated by nitrilotriacetic acid (NTA), iminodiacetic acid (IDA) or pentetic acid (DPTA) [7], [8], [9], [10], [11], [12], [13], [14], [15], [16]. This reaction is based on the high affinity of six histidines (called His-tag) to divalent metal cations: Ni2+, Cu2+, Co2+ or Zn2+ and formation of coordination bonds between the histidine imidazole ring and the metal cations. The main advantages of this strategy are controlled protein immobilization and good homogeneity of the adsorbed layer [17]. Furthermore, the His-tag could be genetically incorporated into any recombinant proteins without damage of native and biologically active structure [18].

A method for the creation of a redox-active complex based on Cu(II) ions with dipyrromethene (DPM) on the surface of gold electrodes has been recently developed [19], [20]. Moreover, this new system was applied for specific immobilization of His-tagged proteins on the surface of a gold electrode [21].

The dipyrromethenes are fully conjugated flat bipyrrolic molecules, which upon deprotonation form strong chelates with metal ions, such as Ni2+, Cu2+, Zn2+ or Co2+, just to name of few [22]. These complexes are neutral with regard to the electric charge [22]. Using the surface plasmon resonance technique, we have demonstrated that the binding capacity of the His-tagged Janus kinase 2 protein to the DPM-Cu(II) complex is comparable to the Biacore NTA-chip based on a NTA-modified dextran layer [11]. Thus, it has been proved that a DPM-Cu(II) complex having strong affinity towards His-tagged proteins is suitable for the creation of bio-functional surfaces.

The interactions between the Receptor of Advanced Glycation End Product (RAGE) and its specific ligands, such as amyloid-β or S100B protein were studied using bi-functional redox active layers [12], [13], [15], [21]. RAGE is a member of immunoglobulin (Ig) superfamily, composed of a V-type immunoglobulin (Ig) domain and two C-type Ig domains (C1 and C2). This receptor is involved in cellular signalling events upon binding of variety of ligands, such as a family of S100 proteins, amyloid-β or glycated proteins. RAGE interacts with a diverse set of ligands is associated with specific diseases, such as diabetes, Alzheimer or cancer [23], [24].

The data gained by contact angles measurements associated with the van Oss approach [25], [26], [27], [28], [29], [30] are frequently used for the calculation of Lifshitz–van der Waals (LW) and Lewis electron acceptor (acid)/electron donor (base) surface tension components of bacteria [31], [32], [33] or proteins adsorbed on solid substrates [34], [35], [36], [37]. Although this approach can help to explain the mechanism of interaction of bacteria or proteins with the surface and the changes in their structure upon adsorption, there are not many reports on ligand-receptor interactions studied using this system.

Here, we have undertaken a study on the determination of the surface energy components (polar and apolar) of VC1-RAGE and its complex with S100B protein based on contact angle measurements with water, ethylene glycol and diiodomethane used as tested liquids. His-tagged VC1-RAGE was attached to Ni(II) or Cu(II) complexes with a dipyrromethene deposited on the surface of gold.

Section snippets

Materials

The dipyrromethene-SH (DPM) was synthesized by Prochimia Surfaces Company (Poland). N-Acetylocysteamine (NAC), dichloromethane, copper(II) acetate, nickel(II) acetate, ethylene glycol, diiodomethane, NaCl, TRIS, CaCl2, were obtained from Sigma-Aldrich (Poland). Methanol was purchased from POCH (Poland). All aqueous solutions were prepared with deionized and charcoal-treated water (resistivity of 18.2  cm) obtained with a Mili-Q reagent grade system (Millipore, Bedford, MA). His6-VC1-RAGE domain

Characterization of DPM-Cu(II) and DPM-Ni(II) SAMs deposited on the surface of gold substrates and determination of the components of the surface energy

The gold substrates were cleaned using a UV/ozone chamber. Such a treatment creates a very hydrophilic gold surface. The droplet of water spreads out on it and the contact angle was almost unmeasurable. The successive modification steps caused decreasing hydrophilicity of the gold substrates. As a result, the contact angles using water, ethylene glycol and diiodomethane became possible to measure (Scheme 1).

In the first step, the procedure of deposition of a mixed SAM containing a

Conclusions

It has been proved that the contact angle method is a suitable technique for controlling the successive steps of modification of gold substrates and exploring the interactions between the protein attached to the gold substrate and the protein present in the aqueous solution. The complex of dipyrromethene with Ni(II) was more suitable for the binding of the His-tagged VC1 domain of RAGE in comparison to the complex of dipyrromethene with Cu(II). The contact angle method allows also checking the

Acknowledgements

This work was supported by statutory funds of the Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences, Olsztyn, Poland. We would like to thank L. Zhukova from the Institute of Biochemistry and Biophysics, Polish Academy of Sciences in Warsaw, Poland for providing samples of the VC1 domain and S100B protein.

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