Immobilization of His-tagged kinase JAK2 onto the surface of a plasmon resonance gold disc modified with different copper (II) complexes

Highlights

• New sensing SPR platforms based on Cu(II) complexes with dipyrromethene and pentetic acid thiol ligands were developed.

• Kinetic and thermodynamic analysis proved stable immobilization of His-tagged kinase JAK2 on new sensing platforms.

• Binding capacity for new SPR sensing platforms were comparable to NTA-modified dextran layers.

Abstract

New surface plasmon resonance (SPR) sensing platforms which consists of copper (II) complexes of a pentetic acid thiol ligand (DPTA-Cu(II)) and of a thiol derivative of dipyrromethene (DPM-Cu(II) created on the surface of gold SPR disc were applied to oriented immobilization of His-tagged Janus kinase 2 (GST-His₆-JAK2). This method is based on the covalent bond formation between histidine from a His-tag chain of a protein and Cu(II) centres from the complexes. The kinetic and thermodynamic parameters of the oriented immobilization of GST-His₆-JAK2 protein to DPTA-Cu(II) and DPM-Cu(II) complexes attached to the Au surface of a SPR disc were discussed.

Introduction

Janus kinases (JAK) are non-receptor tyrosine kinases. The JAK family in mammals consists of four members: JAK1, JAK2, JAK3 and TYK2 (tyrosine kinase 2) [1]. They are multidomain proteins with a molecular weight of about 120–140 kDa. Catalytic domains of these kinases, located at the C-terminus of the protein sequence are functional tyrosine kinases. JAKs have a so-called ‘activation loop’ that regulates kinase activity and is a major site of autophosphorylation [2]. The JAK kinases are involved in the JAK/STAT pathway, which is the major signalling cascade downstream from cytokine, chemokine and growth factor receptors. Stimulation of cells with a cytokine results in activation of JAKs, which phosphorylate and activate STATs, promoting their dimerization and nuclear translocation where they regulate transcription of STAT-dependent genes. Under normal physiological conditions the ligand-dependent activation of the JAK/STAT signalling is transient and tightly regulated [3]. Abnormal JAK/STAT signalling pathways may cause higher activation of JAK2 which can promote the expression of important oncogenes and has been implicated in tumorigenesis of the primary mediastinal B-cell lymphomas (PMBL) and other hematopoietic cancers. Therefore, kinases can be important targets of molecular therapy directed against these diseases.

The most commonly used method to determine Janus kinase activity are radioisotope methods [4], phosphor-specific antibodies [5], surface plasmon resonance [6] and fluorescence resonance energy transfer-based systems [7].

In order to explore the kinase – potential drug interactions we have developed electrochemical biosensors to study the interaction between recombinant histidine-tagged Rio1 protein with small molecule inhibitors [8]. Recombinant rHis₆-Rio1 protein was covalently immobilized to the NAC-N(IDA-like)-Cu(II) complex on the surface of gold electrodes.

Here, we present the work on biosensors based on the principle of surface plasmon resonance (SPR), which have emerged among the most widely used label free detection tools for the study of biomolecular interactions such as protein–protein [9], [10], antibody-antigen [11], [12] and receptor–ligand [13], [14]. SPR offers the ability for direct measuring of interactions in real time, which allows the quantitative determination of kinetic and thermodynamic parameters [15].

Oriented immobilization of proteins on a solid surface is a fundamental interest in the study of the interaction between receptors and ligands [16], [17], [18], [19]. The His-tag chemistry causes the correct orientation of the protein. The His-tag typically consists of five or six consecutive His residues added to the C- or N-terminus of the protein [20]. The most popular systems for oriented immobilization of His-tagged proteins are complexes of nitrilotriacetic acid (NTA) or iminodiacetic acid (IDA) with transition metal ions (Ni²⁺, Cu²⁺) [20], [21], [22], [23], [24], [25], [26], [27], [28]. Nitrilotriacetic acid forms a tetradentate chelate with Ni²⁺ and Cu²⁺, which is able to bind His-tagged proteins via coordinated bonds [29]. A coordinative complex with a binding affinity of K_D equal to 1 µM is simply formed between one molecule of NTA and two imidazole rings on His-tagged protein [20], [27]. Dextran-based SPR chips modified with covalently linked NTA molecules are commercially available and have been used for the detection of the complex between Me²⁺−NTA and His-tagged proteins [20], [24], [26], [30].

Searching for new methods for oriented immobilization of His-tagged proteins, we developed a new systems based on copper (II) complexes with a pentetic acid thiol ligand (DPTA-Cu(II)) and a thiol derivative of dipyrromethene (DPM-Cu(II)). These two complexes were deposited on the surface of the gold electrode destined for electrochemical measurements [8], [31], [32], [33]. The main goal of the electrochemical study was the development of the analytical tools suitable for determination of interactions between His-tagged proteins, immobilized on the surface copper(II) complexes, and their ligands/inhibitors [8], [31], [32], [33]. The thiol derivative of DPM immobilized on the gold surface is suitable for Cu (II) complexation [34], [35]. The complex between DPM-Cu(II) and histidine chains was described as of the distorted square-planar type [36]. The pentetic acid thiol ligand (DPTA) is a derivative of diethylenetriaminepentaacetic acid (H₅DPTA). The molecule of H₅DPTA can provide eight potential binding/coordinating centres [37]. Siddiqi et al. studied the complexes of H₅DPTA with metal ions such as Cr²⁺, Mn²⁺, Co²⁺, Ni²⁺ and Cu²⁺ [38]. The spectrophotometric data suggest a hexacoordinate distorted octahedral environment around the metal ion. The two available binding sites in Me²⁺-H₃DPTA complexes can be used to form a bond with imidazole units from histidine tags.

In this work, a strategy toward oriented immobilization of the His-tagged protein (GST-His₆-JAK2) to the SPR sensing platforms based on new copper (II) complexes with the pentetic acid thiol ligand DPTA-Cu(II) and the thiol derivative of dipyrromethene DPM-Cu(II), as well as the kinetic and thermodynamic parameters of these processes are reported.