Modulation of Biointeractions by Electrically Switchable Oligopeptide Surfaces: Structural Requirements and Mechanism

Understanding the dynamic behavior of switchable surfaces is of paramount importance for the development of controllable and tailor-made surface materials. Herein, electrically switchable mixed self-assembled monolayers based on oligopeptides have been investigated in order to elucidate their conformational mechanism and structural requirements for the regulation of biomolecular interactions between proteins and ligands appended to the end of surface tethered oligopeptides. The interaction of the neutravidin protein to a surface appended biotin ligand was chosen as a model system. All the considerable experimental data, taken together with detailed computational work, support a switching mechanism in which biomolecular interactions are controlled by conformational changes between fully extended (“ON” state) and collapsed (“OFF” state) oligopeptide conformer structures. In the fully extended conformation, the biotin appended to the oligopeptide is largely free from steric factors allowing it to efficiently bind to the neutravidin from solution. While under a collapsed conformation, the ligand presented at the surface is partially embedded in the second component of the mixed SAM, and thus sterically shielded and inaccessible for neutravidin binding. Steric hindrances aroused from the neighboring surface-confined oligopeptide chains exert a great influence over the conformational behaviour of the oligopeptides, and as a consequence, over the switching efficiency. Our results also highlight the role of oligopeptide length in controlling binding switching efficiency. This study lays the foundation for designing and constructing dynamic surface materials with novel biological functions and capabilities, enabling their utilization in a wide variety of biological and medical applications.

N(CH 2 CH 3 ) 3 . For the preparation of the mixed oligopeptide:TEGT SAMs, solutions of the oligopeptide (0.1 mM ) and TEGT (0.1 mM ) were prepared in HPLC EtOH containing 3% (v/v) N(CH 2 CH 3 ) 3 , and mixed at different volume ratios. Subsequently, the clean gold substrates were immersed in the mixed solution for 12 h to form the mixed SAMs on the gold surfaces. The substrates were rinsed with HPLC EtOH, an ethanolic solution containing 10% (v/v) CH 3 COOH, and UHP H 2 O and dried under a stream of N 2 . Note that the mixed SAMs were deposited in the presence of N(CH 2 CH 3 ) 3 to prevent the formation of hydrogen bonds between the NH 2 functional groups of the bound thiolate peptide on Au surface and that of free thiol peptide in the bulk solution. [2]

Electrochemical Surface Plasmon Resonance (SPR)
SPR switching experiments were performed with a Reichert SR7000DC Dual Channel Spectrometer (Buffalo, NY, USA) at 25°C using a three-electrode electrochemical cell and a Gamry PCI4/G300 potentiostat. The SAMs prepared on Reichert Au sensor chips served as the working electrode, the counter electrode was a Pt wire, and a standard calomel electrode (SCE) was used as the reference electrode. Prior to the neutravidin binding studies, the sensor chips were equilibrated with degassed PBS, followed by application of either + 0.3 V, − 0.4 V or open circuit conditions for 10 min while passing degassed PBS through the electrochemical cell at a flow rate of 100 μL min − 1 . While still applying a potential, neutravidin (500 μ L, 37 μ g mL − 1 ), was injected over the sensor chip surface for 10 s at 1500 μL min − 1 and then 30 min at 8 μL min − 1 (the decrease in flow rate from 1500 to 8 μ L min − 1 ensures that sufficient exposure time is provided for binding to occur between the biotin on the surface and neutravidin in solution). In order to remove any unbound neutravidin, the sensor chips were washed with degassed PBS for 10 s at a flow rate of 1500 μL min − 1 , followed by 20 min at a flow rate of 100 μL min − 1 while still applying a potential to the chips. The averages and standard errors reported were determined from at least three different SPR measurements.

X-ray photoelectron spectroscopy (XPS)
XPS spectra were obtained on the VG Escalab 250 instrument based at University of Leeds EPSRC Nanoscience and Nanotechnology Facility, UK. XPS experiments were carried out using a monochromatic Al K α X-ray source (1486.7 eV) and a take-off angle of 15°. High-resolution scans of N (1s) and S (2p) were recorded using a pass energy of 150 eV at a step size of 0.05 eV. Fitting of XPS peaks was performed using the Avantage V 2.2 processing software. Sensitivity factors used in this study were: N (1s), 1.73; S (2p), 2.08; Au (4f 7/2), 9.58; Au (4f 5/2), 7.54. The averages and standard errors reported were determined from at least four different XPS measurements.

Force field test
Since the conformational switching of biotin-nKC chains mainly results from the rotation of the C-C bonds, the energy scan for biotin-4KC molecule with different C1-C2-C3-C4 dihedrals (, Fig. S3) was carried out by both force field methods and density functional theory (DFT) calculations with the B3LYP functional and 6-31G(d) basis set. Three kinds of force fields, cvff, compass and pcff were tested. The result is shown in Figure S3. The cvff force field shows the best performance. Although it overestimates the energies compared to the DFT result, it displays the right shape of the energy curve.
In contrast, both compass and pcff force fields result in a significant deviation from the DFT result. So the cvff force field was adopted throughout our simulations.

Computational details
Five layers of gold atoms cut from the Au(111) surface were adopted to model the gold substrates used in the experiment and they were fixed during the simulations. All MD simulations were performed in the canonical (NVT) ensemble using the cvff force field. The temperature was set to 298 K by using the Andersen thermostat. [3] The equations of the motion were integrated using the velocity Verlet algorithm [4] with the time step of 1fs. The atomic charges for the biotin-nKC molecules were updated every 100ps by DFT calculations, at the M06-2X/6-31G(d,p) level of theory. The Discover module in the Materials Studio package [5] was employed to run all the MD simulations. All DFT calculations were carried out with the Gaussian 09 program package. [6] 7. MD simulation snapshots for biotin-4KC and biotin-6KC under different electric fields Figure S4. The conformational change of bioin-4KC under different electric fields, along with the MD simulation snapshots.