1 Introduction
1.2 Analytical methods for characterisation of protein-ligand interactions
A large number of bioanalytical methods have been developed over the years for structure characterisation and determination of protein-ligand complexes. These methods have provided a pool of information regarding protein structure and function.
Each of these approaches has its strengths and weaknesses, especially with regard to the sensitivity and specificity [1, 2, 10]. A summary of analytical methods applied for identification and characterisation of protein-ligand interactions is summarised in Table 1. These methods enable the definition and analysis of protein structure, both in native form as well as while interacting with molecular partners, and some thermodynamic characteristics.
The primary structure of proteins is crucial since the specific amino acid sequences determine structural characteristics of the proteins such as the formation of the disulphide bridges and accessibility to post-translational modifications. Tandem mass spectrometry fragmentation directly enables the identification of amino acid sequences and consequently the elucidation of unknown protein sequences. The protein terminal groups can be determined by N- or C-terminal analysis, while partial sequences can be determined by chemical or enzymatic degradation in combination with Edman-sequencing [43, 44].
Approaches to determine secondary structure elements of proteins are spectroscopic methods, such as IR spectroscopy [45] and circular dichroism spectroscopy (CD) [46, 47]. CD spectroscopy is frequently used for proteins with α-helical structure, while IR spectroscopy is employed for the characterisation of proteins with ß-sheet or ß-turn structures.
Table 1: Analytical methods applied for structural and thermodynamic protein characterisation and identification.
Analytical methods Ref. Characteristics
Edman sequencing [43, 44]
Circular Dichroism (CD) [46, 47]
IR Spectroscopy [45]
HDX [48-51]
Nuclear Magnetic Resonance (NMR) [52, 53]
X-Ray co-crystalography [54-56]
Mass Spectrometry (MS) [57-59]
Epitope mapping [60-63]
Bio-Layer Interferometry [81, 82]
Analytical Ultracentrifugation (AUC) [8, 83, 84]
Fluorescence Resonance Energy Transfer
(FRET) [85]
Nuclear magnetic resonance (NMR) and X-ray crystallography methods are applied for the characterisation of the tertiary and quaternary structure in proteins.
High resolution 2D and 3D NMR provides structure analysis in solution and information about the dynamics of protein molecules. However, this method is limited to small proteins (<30 kDa). Previous studies [58] have shown tertiary structure-selective modification of charged residues as an efficient approach for the structural characterisation of proteins; X-ray crystallography and mass spectrometry have been shown to be complementary analytical tools for defining precisely chemically modified structures.
Amide hydrogen/deuterium exchange mass spectrometry has become in recent years a powerful method for high-resolution analysis of protein dynamics, structure and function [48-51]. Hydrogen/deuterium exchange approaches can provide information that augments and refines information derived from high-resolution structural studies, and can provide detailed information on native protein structure when structural information is unavailable. Structural studies using mass spectrometry coupled with hydrogen/deuterium exchange can be carried out in a number of physiologically relevant contexts, including ligand binding, self-association, and conformational switching. Advancements in other techniques such as Raman spectroscopy also hold promise for use in high-resolution and high-throughput protein structure and dynamics studies [48-50, 89].
Chemical crosslinking is based on formation of covalent bonds between different molecules (intermolecular) or parts of a molecule (intramolecular) and has been successfully applied in protein-ligand interactions analysis in combination with mass spectrometry [90, 91] as a tool for structure determination [92]. It is a fast procedure with low material consumption offering the opportunity to gain insight into 3D structures of proteins or protein complexes under native conditions. The aim of performing intramolecular chemical crosslinking of a protein is to get information on its three dimensional structure [93], whereas the approach of intermolecular crosslinking is focused on the elucidation of interaction between different protein molecules [66].
Thermodynamic approaches enable the determination of specific thermodynamic and energetic parameters of protein-ligand interactions (e.g.
equilibrium constants, kinetic constants and binding energies). Most of the analytical methods used in determination of thermodynamic characteristics of protein-ligand interactions are applied on a surface at which one of the binding partners is immobilised. Several types of biosensors, such as Surface Plasmon Resonance (SPR), Surface Acoustic Wave (SAW), Quartz Crystal Microbalance (QCM) and Bio-Layer Interferometry (BLI), have been applied in the analysis of protein-ligand interactions.
Each of these bioaffinity techniques has its strengths and weaknesses with regard to the detection approach. However, all biosensors independent of the detection method are measuring the same type of binding curve (Figure 2).
Figure 2: Association and dissociation curve of a protein-ligand interaction. A protein binds to the covalently immobilised ligand during sample injection, resulting in an increase in signal (response). At the end of the injection, the sample is replaced by a continuous flow of buffer, and the decrease in signal now reflects dissociation of the protein [3].
One of the first biosensors introduced was the surface plasmon resonance (SPR) biosensor. The SPR method measures changes in the resonant angle of a laser
limitation that it is difficult to detect protein from blood or serum, and on cell surfaces [70-72].
Another optical label free technology, Bio-Layer Interferometry (BLI), analyses the interference pattern of white light reflected from two surfaces: a layer of immobilised protein on the biosensor tip and an internal reference layer. Any change in the number of molecules bound to the biosensor tip causes a shift in the interference pattern that can be measured in real-time. The binding between an immobilised ligand on the biosensor tip surface and an analyte in solution produces an increase in optical thickness at the biosensor tip which results in a wavelength shift, ∆λ. This wavelength shift is a direct measure of the change in thickness of the biological layer [81, 82].
The Quartz Crystal Microbalance (QCM) technology is a sensitive approach capable of measuring changes in mass at the molecular level. An applied AC-potential causes the quartz crystal to vibrate at a low resonance frequency. The frequency will change as molecules bind to the immobilised ligand on the crystal surface and this frequency change is used to characterise label-free molecular interactions in real time.
In addition to measuring the frequency, the dissipation can be measured and is related to the protein viscoelastic properties [67-69].
In the early 19th century, the first use of the surface acoustic wave (SAW) technology was reported by the brothers Paul-Jacques and Pierre Curie who discovered the piezoelectric effect [94] and by Lord Rayleigh who described surface waves by examining earthquakes [95]. In 1979 the first gas sensing application was perfomed based on Rayleigh waves with a sensitive polymer layer [73-76]. The surface acoustic wave created and used for the detection of protein-ligand interaction is a mechanical acoustic wave, called a Love wave. The development and characterisation of a Love wave based biosensor was described by Schlensog et. al.
[77]. The Love waves travel along the surface of a piezoelectric crystal and the interdigital transducers (IDTs) deposited on the surface of the piezoelectric substance guide the waves between two electrodes. The wave changes in both, phase and amplitude, in response to proteins binding to an immobilised ligand on the crystal surface. The piezoelectric crystals can be made to vibrate at a specific high frequency
with the application of an electrical signal, e.g. 150 MHz [79]. These oscillations are mechanical waves that travel through the bulk matter. Their frequency is dependent on the electrical frequency applied to the crystal as well as the crystals mass. Therefore, when the mass increases due to binding of proteins, the oscillation frequency changes and the resulting change can be measured electrically and used to determine the additional mass of the crystal. This is the function principle of a QCM [67]. If the oscillation is confined in a thin layer on the surface of the crystal, one can speak of SAW [78-80, 96]. Different types of acoustic waves can be employed in a SAW device, but Love waves [77] offer particularly high sensitivities due to the confinement of the acoustic energy to the sensing surface. The Love waves are in fact horizontally polarized guided waves [97].
All biosensors enable the determination of kinetics data such as on- and off-rates and a KD value. The differences between all biosensors relate to the detection and type of applications. QCM is somewhat less sensitive compared to SAW due to the low frequency used for detection, SPR is limited to gold surfaces and only BLI provides high-throughput analysis capability.
Enzyme-linked immunosorbent assay (ELISA) is a biochemical technique used to detect the presence, and determine the affinity of an antibody or an antigen in a sample. Performing an ELISA involves at least one antibody with specificity for a particular antigen. The sample with an unknown amount of antigen or antibody is immobilised on a solid support (usually a polystyrene microtiter plate) either non-specifically (via adsorption to the surface) or non-specifically (via capture by another antibody or a sample-specific antigen). After the sample is immobilised, the detection antibody is added, forming a complex with the antigen. The detection antibody can be covalently linked to an enzyme or can itself be detected by a secondary antibody that is linked to an enzyme through bio-conjugation. Between each step, the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are not specifically bound. After the final wash step, the plate is developed by adding an enzymatic substrate to produce a visible signal which indicates the quantity
Fluorescence Resonance Energy Transfer (FRET) is a fluorescence based approach in which the photon energy is transferred from an excited fluorophore labelled protein (the donor) to another fluorophore labelled protein (the acceptor) when both are located within close proximity (1-10 nm). By using fluorescence digital imaging microscopy, one can visualise the location of green fluorescent proteins within a living cell and thereby follow the time course of the changes in FRET corresponding to cellular events at a millisecond time resolution. The observation of such dynamic molecular events in vivo provides vital insight into the action of biological molecules [85].
Analytical ultracentrifugation (AUC) is a classical method for the characterisation of interactions between purified proteins in solution. Protein complexes can be characterised with regard to their stoichiometry and the thermodynamic binding constants of complex formation. Sedimentation techniques can distinguish among multiple coexisting complexes of different stoichiometries and also provide information on self-associated properties and on mixed self- and hetero-association [8, 83].
For all these bioaffinity techniques, the combination of different approaches for the simultaneous structural identification, characterisation and kinetics determination of protein-ligand complexes has not yet been previously reported. The direct instrumental combination of a biosensor technique with mass spectrometry has been first developed in the present dissertation.