Enzyme solid-state support assays: a surface plasmon resonance and mass spectrometry coupled study of immobilized insulin degrading enzyme
Solid-support based assays offer several advantages that are not normally available in solution. Enzymes that are anchored on gold surfaces can interact with several different molecules, opening the way to high throughput array format based assays. In this scenario, surface plasmon resonance (SPR) and mass spectrometry (MS) investigations have often been applied to analyze the interaction between immobilized enzyme and its substrate molecules in a tag-free environment. Here, we propose a SPR-MS combined experimental approach aimed at studying insulin degrading enzyme (IDE) immobilized onto gold surfaces and its ability to interact with insulin. The latter is delivered by a microfluidic system to the IDE functionalized surface and the activity of the immobilized enzyme is verified by atmospheric pressure/matrix assisted laser desorption ionization (AP/MALDI) MS analysis. The SPR experiments allow the calculation of the kinetic constants involved for the interaction between immobilized IDE and insulin molecules and evidence of IDE conformational change upon insulin binding is also obtained.
KeywordsSolid-state assay Surface plasmon resonance Mass spectrometry Insulin degrading enzyme Conformational change
Insulin degrading enzyme (IDE) (Farris et al. 2003) is a zinc metalloprotease, able to degrade several different substrates besides insulin (e.g., β-amyloid) that are involved in many pathological conditions such as Alzheimer’s disease (AD) and Parkinson’s disease (PD) (Vepsäläinen et al. 2007; Kurochkin and Goto 1994; Blomqvist et al. 2004). Very recently, the structures of human IDE in complex with four different substrates have been reported and some insights into the interaction mechanism have also been given (Shen et al. 2006). IDE has a buried catalytic site in the structure and access to this chamber is kinetically controlled by a closed–open conformational switch, so IDE probably conforms to a complex kinetic model where catalysis does not lead automatically to product release. Instead, an additional step is required in which the protease opens up to allow the products to escape and a “latch” system has been proposed in order to explain the experimental results obtained so far for this enzyme (Leissring and Selkoe 2006). Recent developments on IDE conformational changes suggest that, because of the extensive interaction between N- and C-terminal domains, IDE could exist in its closed conformation without requiring the binding energy contributed by its substrate (Im et al. 2007). Moreover, it has been reported that in solution IDE exists as a mixture of monomers, dimers, and tetramers and the equilibrium between the different forms is concentration-dependent, with the dimer the more active form (Song et al. 2003). Very recently, (unpublished data) we have also found that the various IDE oligomeric forms produce different insulin fragmentation patterns. The latter can also be altered by the presence of other molecules such as ubiquitin (Grasso et al. 2008).
In this scenario, although the exact molecular mechanism of the interaction between IDE and insulin is still unclear, it is likely that IDE has an open conformation in the active state and a closed one when is inactive or is bound to its substrates. Similar behaviors have been observed in the past for other enzymes which undergo a hinge twist motion between two separate domains (Sharff et al. 1992). Although optical and spectroscopic techniques, like CD, fluorescence, NMR, and X-ray scattering, are routinely used to investigate the conformational state of proteins in solution or crystal (Drobny et al. 2003; Andrade et al. 2004), a valid contribution toward a better understanding of IDE-substrates interaction mechanism is also expected from alternative experimental approaches such as solid-state assays. The development and optimization of an immunocapture-based assay for the specific measurement of IDE activity in brain tissue homogenates has already been described (Miners et al. 2008). However, such method requires a fluorescent tag to be present in the substrate and the investigation of the proteolytic action of the enzyme is limited to the only cleavage site where the fluorescent tag is attached.
Surface plasmon resonance (SPR) is able to detect variation in physical parameters (for instance, dielectric constant) caused by volume changes of surface bound proteins and it is usually applied to study biomolecular interactions in real time to obtain kinetics parameters (Homola 2006). In 1998, the first investigation of pH-induced structural transitions of immobilized proteins by SPR was reported (Sota et al. 1998). This was the first description of a correlation between resonance angle shifts and conformational changes of immobilized proteins, opening the way to several indirect observations of protein conformational changes using SPR (Kim et al. 2005; Kang et al. 2006; Geitmann and Danielson 2004).
Our group has already shown the advantages offered by coupling SPR and mass spectrometry (MS) for studying a certain class of enzymes (Grasso et al. 2005, 2007a) in a solid-state format. However, in the latter cases no insights into the molecular mechanism of the interaction were given and the analysis was limited to the calculation of enzyme activity or the identification of the immobilized biomolecules (Grasso et al. 2006).
In order to overcome the above mentioned limitations, in this paper we propose an SPR-MS combined experimental approach that gives an insight into the molecular mechanism of the interaction between an enzyme and one of its substrate. The proposed method is able to detect if the interacting immobilized enzyme undergoes a conformational change upon substrate binding. For this reason, the interaction between IDE and insulin molecules is here scrutinized. We immobilized IDE onto a gold substrate by the amino coupling approach and used atmospheric pressure/matrix assisted laser desorption ionization (AP/MALDI)-MS to monitor the activity of the anchored biomolecules. The latter are arrayed in a spatially resolved manner by coupling the SPR imaging (SPRI) technique with a home-made microfluidic system, allowing the calculation of the kinetic constants involved for the IDE-insulin interaction. The proposed SPR experimental approach produces evidences of the enzyme conformational changes upon insulin binding, in accordance with the view that active substrate-free IDE is in its open conformation (Im et al. 2007). In this way, the study of the monomeric form of the wild type enzyme interacting with one of its natural substrates is feasible and insight into kinetic details of the interaction between immobilized IDE and insulin is provided.
Materials and methods
IDE, his-tag, rat, and recombinant from Spodoptera frugiperda was purchased from Calbiochem. Insulin from bovine pancreas, phosphate buffer solution (PBS), α-cyano-4-hydroxycinnamic acid (CHCA), trifluoro acetic acid (TFA), acetonitrile (C2H3N), ethanol solution, ethanolamine-HCl 1M, guanidine-HCl 8M, sucrose and dithiobis (N) succinimidyl propionate (Lomant’s reagent) were all purchased from Sigma-Aldrich, while ZipTipSCX pipette tips were from Millipore, and dithiol tethers SPT-0013 and SPT-0014C were purchased from Sensopath. Gold substrates (GWC Technologies, USA) were obtained by thermally evaporating a gold layer (450 Å) onto SF-10 glass slides (Schott, USA). Chromium (50 Å) was used as the adhesion layer.
Surface plasmon resonance
Kinetics parameters obtained by fitting the sensorgrams recorded for the interaction between immobilized IDE and insulin according to the conformational model described in the text (see Fig. 5)
1.0 (±0.3) × 103 M−1 s−1
1.3 (±0.2) × 10−2 s−1
2.4 (±0.2) × 10−3 s−1
1.5 (±0.7) × 10−3 s−1
PDMS microfluidic channels fabrication
The evaluation of the kinetic constants for biomolecular interactions by SPR experiments can be affected by diffusion problems (D’Agata et al. 2008). In order to estimate the contribution of diffusion to the kinetic parameters obtained, km is normally used to describe the diffusion of the protein to the surface and it can be related to the diffusion coefficient of the protein. The dimensions of the above described microchannels ensured that, at the flow rate used in our SPRI experiments for the interaction between IDE and insulin (100 µl min−1), the value of km describing the diffusion of insulin to the gold surface was 1.4 × 108 M−1 s−1. The latter is well above the rate constants values reported in Table 1 and therefore the results obtained are not affected by diffusion problems.
AP/MALDI-MS experiments were carried out, using a Finnigan LCQ Deca XP PLUS (Thermo Electron Corporation, USA) ion trap spectrometer which was fitted with a MassTech Inc. (USA) AP/MALDI PDF-source. The latter consists of a flange containing a computer-controlled X–Y positioning stage and a digital camera, and is powered by a control unit that includes a pulsed nitrogen laser (wavelength 337 nm, pulse width 4 ns, pulse energy 300 µJ, repetition rate up to 10 Hz) and a pulsed dynamic focusing (PDF) module that imposes a delay of 25 μs between the laser pulse and the application of the high voltage to the AP/MALDI target plate. Laser power was attenuated to about 55%. The target plate voltage was 1.8 kV. The ion trap inlet capillary temperature was 220°C. Capillary and tube lens offset voltages of 30 and 15 V, respectively, were applied. Other mass spectrometer parameters were as follows: multipole 1 offset at −3.75 V, multipole 2 offset at −9.50 V, multipole RF amplitude 400 V, lens at −24.0 V and entrance lens at −88.0 V. Automatic gain control (AGC) was turned off and instead the scan time was fixed by setting the injection time to 220 ms and using five microscans. Although there is the risk of loosing resolution, the latter experimental conditions were chosen as sensitivity was the main goal in all MS experiments carried out on the immobilized enzyme.
Results and discussion
Insulin fragments detected from the supernatant solution in contact with the IDE functionalized surface
Calculated peaks (m/z)
Experimental peaks (m/z)
A(1–13) + B(1–9)
A(1–14) + B(1–9)
The above described SPRI approach is applied to verify the conformational change of immobilized enzyme molecules occurring upon substrate binding, giving an insight into the IDE-insulin interaction mechanism. In fact, it is important to highlight that the MS results showed that the enzyme is mainly immobilized on the surface as a monomer and therefore the conformational change observed by applying our SPRI approach refers only to this specific oligomeric form of IDE, conferring an advantage to the use of the solid-supported coupled SPRI-MS approach over other solution-based investigation tools. Particularly, we confirmed a model where, in the absence of substrate, the inactive resting state of monomeric IDE is in a closed conformation, since substrates cannot gain access to the catalytic chamber when the protease is in this state. Only when the enzyme switches to its open conformation the substrate molecules can properly interact with IDE (see Table 1 for the rate constants values). Thus, the closed conformation is critical for regulating the catalytic cycle of monomeric IDE while a conformational switch to the open state needs to occur if the enzyme is to carry out its catalytic activity.
A solid-support based assay that allows a multiplexed approach to study IDE-insulin interaction was proposed. We immobilized IDE molecules on gold surfaces and monitored their activity by AP/MALDI-MS, demonstrating the ability of the anchored monomeric form of the enzyme to cleave insulin molecules. SPRI experiments subsequently were carried out on the immobilized enzyme molecules, producing evidence of a conformational change of IDE upon insulin binding. Kinetic parameters were calculated and SPRI experiments based on different ratios of insulin concentrations and contact times have also been carried out to give an insight into the IDE-insulin interaction. Particularly, the results confirmed a model where the closed conformation is the inactive resting state of monomeric IDE while the open conformation is its active state that switches to a closed state upon substrate binding. According to this model IDE should shrink its hydrodynamic radius upon insulin binding.
We thank MIUR (FIRB RBNE03PX83, RBIN04L28Y) and “EURAMY: Systemic Amyloidoses in Europe”, 037525 (LSHM-CT-2006-037525) for partial financial support.
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