RAFT polymers for protein recognition

Summary A new family of linear polymers with pronounced affinity for arginine- and lysine-rich proteins has been created. To this end, N-isopropylacrylamide (NIPAM) was copolymerized in water with a binding monomer and a hydrophobic comonomer using a living radical polymerization (RAFT). The resulting copolymers were water-soluble and displayed narrow polydispersities. They formed tight complexes with basic proteins depending on the nature and amount of the binding monomer as well as on the choice of the added hydrophobic comonomer.

an ice-water bath. Sodium hydroxide solution (50%, 50.4 g, 0.63 mol) was added dropwise over 60 min while the temperature was kept below 25 °C. The solution was then stirred overnight. Water (200 mL) was added, followed by concentrated HCl (30 mL) with vigorous stirring and nitrogen purging for 30 min. The yellow solid was filtered, washed with water and dried under vacuum. Yield: 2.66 g (21%). 1  Typical synthesis of RAFT copolymers S10: NIPAM (1.488 g, 13.1 mmol) and sodium methacrylate (0.142 g, 1.3 mmol) were dissolved in methanol (20 mL) in a polymerization tube fitted with a septum cap ( Figure 1). The mixture was degassed by bubbling nitrogen through it for 30 min. S,S'-Bis(α,α'-dimethylα"-acetic acid)-trithiocarbonate (8) (74.4 mg, 0.26 mmol) and azo initiator V-50 (23.8 mg, 0.08 mmol) were then added and the mixture was heated to 60 °C for 48 h. The solvent was evaporated, the residue dissolved in THF and precipitated into pentane, then left to dry to give a pale yellow, odorous powder. 1   0.26 mmol) and azo initiator V-50 (23.8 mg, 0.08 mmol) were then added and the mixture was heated to 60 °C for 48 h. The solvent was evaporated, the residue dissolved in THF and precipitated into pentane, then left to dry to give a pale yellow, odorous powder. 1

GPC
GPC measurements were carried out with Polymer Laboratories PL-gel mixed-B columns (10 mm particle size, 100 -10 6 Å pore size, effective MW range 10 3 -10 7 g mol -1 , column length 3×30 cm, fitted with guard columns) and a refractive index detector. Samples were injected through a Rheodyne 7725i injection port with a 200 μL loop. The column was calibrated with a series of poly(ethylene glycol)/poly(ethylene oxide) standards using aqueous sodium nitrate (0.2 M)/sodium hydrogen orthophosphate (0.1 M) as eluent. Samples were made up at concentrations of approx. 2.0 mg mL -1 and filtered prior to injection. Elution time for GPC runs was 30 min. A typical analysis is shown in Figure 2.
A stock solution of protein was prepared in pH 7.0 phosphate buffer; a typical concentration of a protein stock solution was 1.02 × 10 -5 mol L -1 for cytochrome C. A solution of 20 mg of S5 polymer in 0.5 mL of protein stock solution was used as the sample solution for the titration. A solution of microgel in buffer solution (20 mg in 0.5 mL) served as the blank solution.
A reference cell was filled with 2.5 mL of phosphate buffer in a quartz cuvette. The sample cuvette was filled with 2.5 mL of protein stock solution. Equal aliquots of sample solution and blank solution were titrated into the sample cuvette and the blank cell, respectively, to maintain the same concentration of polymer in each cuvette. UV-vis spectra were recorded after the solution had equilibrated during 15 min. of stirring. Titrations were carried out at room temperature (20 °C).
The UV-vis absorbance vs wavelength data was pasted into a spreadsheet and the second derivative was calculated according to the Savitsky-Golay [7][8][9] method with the help of a macro [9]. Since the second derivative of the absorbance is insensitive to light scattering, this gave a clearer representation of the changes in the UV-vis spectrum of the protein that occurred during binding of the protein to the RAFT copolymer.  Figure 5: Top: second-derivative UV-vis spectra observed during a titration of a stock solution of S10CH15 into a solution of cytochrome C. Below: corresponding isotherm for complex formation between S10CH15 and cytochrome C; the drawn curve represents the calculated isotherms for 1:1 binding, whereas the filled diamonds are experimental values.

ITC titration
ITC titrations were performed with micro-calorimeter VP-ITC at 25 °C. The initial volume of the solution in the cell was 1.4211 mL. 10 mM Hepes (pH 7) was used as the buffer. In all the titrations, protein solutions were used as hosts in the cell and polymer solutions as ligands.
The dilution effect of the polymers were measured as reference and subtracted from the titrations before the calculation. Two kinds of evaluation methods incorporated in the software were used to get the best fittings and smallest errors: one set of sites and sequential binding with 2 binding sites. The macroscopic binding constant, change of enthalpy and entropy are given here. For every pair, two titration curves are shown. The left curve is calculated with the concentration of the polymer, and the right one is calculated with the concentration of the functional monomer.

B20CH15 vs. Histone
Sequential binding two sites

B20CH15 vs. Lysozyme
Sequential binding two sites