Measuring Ligand Binding Kinetics to Membrane Proteins Using Virion Nano-oscillators

Abstract

Membrane proteins play vital roles in cellular signaling processes and serve as the most popular drug targets. A key task in studying cellular functions and developing drugs is to measure the binding kinetics of ligands with the membrane proteins. However, this has been a long-standing challenge because one must perform the measurement in a membrane environment to maintain the conformations and functions of the membrane proteins. Here, we report a new method to measure ligand binding kinetics to membrane proteins using self-assembled virion oscillators. Virions of human herpesvirus were used to display human G-protein-coupled receptors (GPCRs) on their viral envelopes. Each virion was then attached to a gold-coated glass surface via a flexible polymer to form an oscillator and driven into oscillation with an alternating electric field. By tracking changes in the oscillation amplitude in real-time with subnanometer precision, the binding kinetics between ligands and GPCRs was measured. We anticipate that this new label-free detection technology can be readily applied to measure small or large ligand binding to any type of membrane proteins and thus contribute to the understanding of cellular functions and screening of drugs.

Roles of entropic and solvent damping forces in the dynamics of polymer tethered nanoparticles and implications for single molecule sensing

“…This nding also indicates that entropy, rather than damping, should be the dominant factor for the nano-oscillators developed by us previously. 8,16,30 Hydrodynamic boundary effects…”

“…9 These technologies have provided valuable information on molecules, such as DNA conformation changes, [10][11][12] DNA-protein interactions, [13][14][15] and proteinbiomarker interactions. 4,16,17 Central to all these technologies is the dynamics of the systems, which is controlled by the entropic force associated with conformational changes of the molecule [18][19][20] and solvent damping force exerted on the molecule and the probe. 8,[21][22][23] Despite the importance, the interplay of the two forces and their dependence on the size of the probe, length of the molecule, viscosity of the solvent and time scale of the dynamics remain to be understood.…”

Future perspective: high-throughput construction of new ultrasensitive cytokine and virion liquid chips for high-throughput screening (HTS) of anti-inflammatory drugs or clinical diagnosis and treatment of inflammatory diseases

“…Therefore, studies on the ligand-membrane protein interactions associated with an intracellular antiinflammatory mechanism are very meaningful for the development of recombinant virion and cytokine chips for clinical inflammatory disease detection and therapy ( Fig. 2b) [20,[22][23][24][25][26].…”

“…With a similar constructive method, a total of 332 (98.5%) GPCR open reading frames (ORFs) of a total of 337 non-odorant GPCRs were subcloned into the UL27 locus (gB) of the herpes virus genome and used to fabricate the VirD-GPCR array for high-content study of their pharmacological activity and preferred drug targets (Fig. 3) [20,[23][24][25][26]. Because the molecular drug-receptor interactions on these recombinant virions will trigger charge distribution and electron transition to produce electrochemical signaling, IFNAR2 and IL10RA membrane proteins can be applied with detection of electrical signals for HTS of anti-inflammatory molecular drugs (Fig.…”

“…Different shear forces such as pulsating (PS) and oscillatory shear force (OS) can also be loaded to gain variable binding kinetics to characterize the drug activity (Fig. 3) [6,20]. TheNLRP2 gene can be knocked down by ShRNA lentivirus for high-throughput construction of a cytokine solution chip…”