4.5.1 Fluorometric assay (III)
The binding affinity of the unconjugated small molecules to avidin variants was determined utilizing intrinsic fluorescence originating from the aromatic amino acid residues (mainly tryptophan) of avidin and the fluorescence quenching caused by ligand binding. In brief, protein samples of 100nM were excitated at 280nm, and the emission was collected at 350nm using a QuantaMaster Spectrofluorometer (Photon Technology International, Inc.) with 2nm slits. The assay was performed in a quartz cuvette with stirring at 25 °C. The ligand was added to the protein sample in small aliquots, and fluorescence intensity was monitored after a short incubation. First experiments were performed with ligand concentrations of between 66nM–30µM, and repeated for protein-ligand pairs with a Kd value less than 500nM using smaller amounts of the ligand (6nM–50µM) added to the protein sample. The dissociation constant (Kd) was determined from the resulting quenching curve using a GraphPad Prism (GraphPad Software, Inc.). The data were fitted to a quadratic equation for tight binding interactions, which takes ligand depletion and non-specific binding into account. For biotin-binding measurements, up to 350nM protein samples were used in order to achieve a reasonable Bmax value.
4.5.2 Surface plasmon resonance (I)
The binding kinetics of steroid-binding avidin variants, sbAvd-1 and sbAvd-2, was analyzed with a BIAcore X optical biosensor (Biocore, Uppsala, Sweden).
Testosterone-BSA was coupled to the carboxymethylated dextran layer of a sensor chip using EDC/NHS-chemistry (1000 RU, 40 µl/min flow rate). The binding of sbAvd-1, sbAvd-2, and wt Avd (as a negative control) samples on testosterone-BSA-coated chips was measured using different protein concentrations and the kinetic constants were determined using the BiaEvaluation software according to the manufacturer’s instructions. Testosterone-binding was more closely detected by measuring binding in the presence of varying concentrations (0.75–50µM) of free testosterone. To evaluate the binding specificity of the steroid-binding avidin variants, the binding of sbAvd-1 and sbAvd-2 to the testosterone-BSA surface was competed against free steroid hormone molecules (testosterone, DHEAS, androstendione, estradiol, and DHT (Steraloids Inc.)) and free biotin (Sigma-Aldrich).
The biotin-binding kinetics were determined by preparing a second sensor chip coupled with biotin (~130RU). However, it is important to note that the determination of the bound mass is not very accurate in the case of small molecules because the immobilization can change the physicochemical properties of the surface.
4.5.3 Protein Microplate Assay (I, III)
The ligand-binding specificity of selected avidin forms was determined with a microplate assay. In the assays, wells of MaxiSorp F96 microplates (Nunc) were coated by applying 100µl of PBS containing 500ng of BSA- or HSA-conjugated ligands (listed in Table 5) at +37 °C for 2 hours. As negative controls, carrier proteins HSA (from VTT Technical Research Center of Finland, Espoo) or BSA (Sigma-Aldrich) were used. After blocking and washing the wells, purified proteins (~100 ng/well) in measurement buffer were added. In case the proteins were preincubated beforehand with biotin, 10µM D-biotin (Sigma-Aldrich) was used. The avidin variants bound to the wells were detected using polyclonal anti-avidin (University of Oulu) as a probe. After incubation with AP-conjugated coat anti-rabbit IgG Aldrich) and applying phosphatase substrate solution (1mg/ml pNPP (Sigma-Aldrich) in 1M diethanolamine pH 9.8 with 0.5mM MgCl ), A was measured with
4.5.4 Molecular Dynamics (MD) Simulations (III)
The antidin sbAvd-1 homology model (based on [PDB:1VYO] (Repo et al., 2006)) was prepared, and testosterone was docked into the biotin-binding pocket using GOLD (Hornak et al., 2006). Testosterone was examined only in a binding orientation where it enters the binding pocket with its 17-OH group ahead, since the testosterone-conjugate used in biopanning is coupled to its carrier protein via the 3-keto oxygen. Tetrameric sbAvd-1 with four bound testosterone molecules was solvated in TIP3P water, and the system charge was neutralized with chloride ions, resulting in a total of 52,833 atoms. The Amber ff99SB force field (Jones et al., 1997) was used for the protein, and for testosterone the GAFF force field (Wang et al., 2004), with the simulation run in NAMD 2.9 (Phillips et al., 2005) using a time step of 1fs. A 50-ns MD simulation was produced at 300K and atmospheric pressure using the Langevin temperature and pressure controls. Analyses and figures were prepared using VMD 1.9.1 (Humphrey et al., 1996) and Pymol 1.7 (Shrödinger, LLC).
4.5.5 Interaction analysis by Molecular Recognition Force Spectroscopy (I) The interaction of sbAvd-1 and sbAvd-2 with testosterone and biotin was studied also by molecular recognition force spectroscopy (MRFS) by our collaborators in the laboratory of Professor Peter Hinterdorfer at Johannes Kepler University Linz (Linz, Austria). The proteins were covalently bound to modified mica sheets via lysines as previously described (Kamruzzahan et al., 2004). The testosterone was covalently attached to an AFM tip via a flexible spacer, heterobifunctional Fmoc-PEG-NHS crosslinker, as previously described (Wildling et al., 2009). All MRFS experiments were performed on a Pico SPM I (Agilent Technologies, Santa Clara, CA). All the used, modified cantilevers had nominal spring constants between 10-30pN/nm (Veeco Instruments, Santa Barbara, CA), and the effective spring constants were determined by the thermal noise method (Hutter and Bechhoefer, 1993; Butt and Jaschke, 1995). Force-distance cycles were completed using a z-range of 200-300nm.
Sweep durations were adjusted between 0.25–4s. During one data set of 1000 force-distance curves, the lateral tip position was changed (a few hundred nm) about every 100 curves to ensure that the binding events were statistically reasonable. To prove the specificity of the binding, the ligand-binding sites of proteins were blocked by
~1h incubation with free testosterone (200nM) added to the measuring solution.
5 RESULTS AND DISCUSSION
Based on the original papers (I–III) and unpublished material.