Single Molecule Force Spectroscopy and Molecular Dynamics Simulations as a Combined Platform for Probing Protein Face-Specific Binding

Abstract

Biomolecular interactions frequently occur in orientation-specific manner. For example, prior nuclear magnetic resonance spectroscopy experiments in our lab have suggested the presence of a group of strongly binding residues on a particular face of the protein ubiquitin for interactions with Capto MMC multimodal ligands ("Capto" ligands) (Srinivasan, K.; et al. Langmuir 2014, 30 (44), 13205-13216). We present a clear confirmation of those studies by performing single-molecule force spectroscopy (SMFS) measurements of unbinding complemented with molecular dynamics (MD) calculations of the adsorption free energy of ubiquitin in two distinct orientations with self-assembled monolayers (SAMs) functionalized with "Capto" ligands. These orientations were maintained in the SMFS experiments by tethering ubiquitin mutants to SAM surfaces through strategically located cysteines, thus exposing the desired faces of the protein. Analogous orientations were maintained in MD simulations using suitable constraining methods. Remarkably, despite differences between the finer details of experimental and simulation methodologies, they confirm a clear preference for the previously hypothesized binding face of ubiquitin. Furthermore, MD simulations provided significant insights into the mechanism of protein binding onto this multimodal surface. Because SMFS and MD simulations both directly probe protein-surface interactions, this work establishes a key link between experiments and simulations at molecular scale through the determination of protein face-specific binding energetics. Our approach may have direct applications in biophysical systems where face- or orientation-specific interactions are important, such as biomaterials, sensors, and biomanufacturing.

Identification of preferred multimodal ligand‐binding regions on IgG1 FC using nuclear magnetic resonance and molecular dynamics simulations

“…The simulation box dimensions were 8.5 nm × 10 nm × 13 nm, allowing for a buffer of roughly 1.5 nm on each side of the protein. The F C was prevented from rotating in each simulation by restraining a single alpha carbon buried in the center of each of the four F C domains using a harmonic potential with a spring constant of 40,000 kJ/mol/nm 2 (Srinivasan et al, 2017). This allowed the use of a rectangular simulation box without risking the protein interacting with itself through periodic boundary conditions.…”

“…Yu et al (2015) employed coarse‐grained simulations to investigate the preferred binding orientation of lysozyme on an HCIC surface at different ligand densities under a range of salt concentrations. Our lab has been actively involved in studying the preferred binding regions of small proteins in MM CEX systems by employing protein libraries (Chung et al, 2010), MD simulations (Banerjee et al, 2017; Freed et al, 2011; Parimal et al, 2015, 2017), atomic force microscopy (Srinivasan et al, 2017), and nuclear magnetic resonance (NMR) (Chung et al, 2010; Holstein et al, 2012, 2013; Srinivasan et al, 2014).…”

Single-molecule force spectroscopy: A facile technique for studying the interactions between biomolecules and materials interfaces

“…Srinivasan et al investigated the face-specific binding of proteins with the Capto MMC multimodal ligands by SMFS ( Figure 5a) [74]. For this aim, they utilized the Capto ligands to modify the Au-coated AFM tip to form a SAM on tip, and used two proteins with exposed "preferred" (UBQ S20C) and without (UBQ A46C) binding faces to modify the Au surface.…”

Identification of Preferred Multimodal Ligand Binding Regions on IgG1 FC using Nuclear Magnetic Resonance and Molecular Dynamics Simulations

“…The simulation box dimensions were 8.5nm x 10nm x 13nm, allowing for a buffer of roughly 1.5nm on each side of the protein. The F C was prevented from rotating in each simulation by restraining a single alpha carbon buried in the center of each of the four Fc domains using a harmonic potential with a spring constant of 40,000kJ/mol/nm 2 (Srinivasan et al, 2017). This allowed the use of a rectangular simulation box without risking the protein interacting with itself through periodic boundary conditions.…”

“…Yu et al employed coarse-grained simulations to investigate the preferred binding orientation of lysozyme on a HCIC surface at different ligand densities under a range of salt concentrations (Yu, Liu, & Zhou, 2015). Our lab has been actively involved in studying the preferred binding regions of small proteins in MM CEX systems by employing protein libraries (Chung, Hou, et al, 2010), MD simulations Freed, Garde, & Cramer, 2011;Parimal, Garde, & Cramer, 2015, 2017, Atomic Force Microscopy (AFM) (Srinivasan et al, 2017) and Nuclear Magnetic Resonance (NMR) Holstein, Chung, et al, 2012;Holstein, Parimal, McCallum, & Cramer, 2013;Srinivasan, Parimal, Lopez, McCallum, & Cramer, 2014).…”