Molecular Spring Constant Analysis by Biomembrane Force Probe Spectroscopy

Summary

A biomembrane force probe (BFP) is an in situ dynamic force spectroscopy (DFS) technique. BFP can be used to measure the spring constant of molecular interactions on living cells. This protocol presents spring constant analysis for molecular bonds detected by BFP.

Abstract

A biomembrane force probe (BFP) has recently emerged as a native-cell-surface or in situ dynamic force spectroscopy (DFS) nanotool that can measure single-molecular binding kinetics, assess mechanical properties of ligand-receptor interactions, visualize protein dynamic conformational changes and more excitingly elucidate receptor mediated cell mechanosensing mechanisms. More recently, BFP has been used to measure the spring constant of molecular bonds. This protocol describes the step-by-step procedure to perform molecular spring constant DFS analysis. Specifically, two BFP operation modes are discussed, namely the Bead-Cell and Bead-Bead modes. This protocol focuses on deriving spring constants of the molecular bond and cell from DFS raw data.

Introduction

As a live-cell DFS technique, BFP engineers a human red blood cell (RBC; Figure 1) into an ultrasensitive and tunable force transducer with a compatible spring constant range at 0.1-3 pN/nm^1,2,3. To probe ligand-receptor interaction, BFP enables DFS measurements at ~1 pN (10^-12 N), ~3 nm (10^-9 m), and ~0.5 ms (10^-3 s) in force, spatial, and temporal resolution^4,5. Its experimental configuration consists of two opposing micropipettes, namely the Probe and t....

Protocol

1. Obtain Analyzable DFS Events

1. Start the experiment in the software (e.g., LabVIEW VI) for the BFP control and parameter setting (Figure S1A).
2. Observe the repetitive probe bead-target bead/cell touches in the software for BFP Monitor (Figure S1B).
3. Test and achieve the adhesion frequency ≤ 20% within the first 50 touches by tuning the impingement force and contact time, by which it ensures that ≥ 89% of DFS adhesion event are mediated by single.......

Discussion

In summary, we have provided a detailed data analysis protocol for preprocessing the DFS raw data and deriving molecular spring constants in the BFP Bead-Bead and Bead-Cell analysis modes. Biomechanical models and equations required for determining molecular and cellular spring constants are presented. Albeit different integrins are studied, the k_mol measured by the Bead-Bead mode and the Bead-Cell mode possesses significant range differences (Figure 3A vs.

Materials

Name	Company	Catalog Number	Comments
3-Mercaptopropyltrimethoxysilane (MPTMS)	Uct, Specialties, llc	4420-74-0	Glass bead functionalization
Anhy. Sodium Phosphate Dibasic (Na2HPO4)	Sigma-Aldrich	S7907	Phosphate buffer preparation
BFP data acquisition VI	LabVIEW		BFP control and parameter setting
BFP data analysis VI	LabVIEW		BFP raw data analysis
Biotin-PEG3500-NHS	JenKem	A5026-1	RBC biotinylation
Borosilicate Glass beads	Distrilab Particle Technology, Netherlands	9002	Glass bead functionalization
Bovine serum albumin	Sigma-Aldrich	A0336	Ligand functionalization
Camera VI	LabVIEW		BFP monitoring
D-glucose	Sigma-Aldrich	G7021	Tyrode’s buffer preparation
Hepes	Sigma-Aldrich	H3375	Tyrode’s buffer preparation
MAL-PEG3500-NHS	JenKem	A5002-1	Glass bead functionalization
Potassium Chloride (KCl)	Sigma-Aldrich	P9541	Tyrode’s buffer preparation
Sodium Bicarbonate (NaHCO3)	Sigma-Aldrich	S5761	Carbonate/bicarbonate buffer preparation; Tyrode’s buffer preparation
Sodium Carbonate (Na2CO3)	Sigma-Aldrich	S2127	Carbonate/bicarbonate buffer preparation
Sodium Chloride (NaCl)	Sigma-Aldrich	S7653	Tyrode’s buffer preparation
Sodium Phosphate Monobasic Monohydrate (NaH2PO4•H2O)	Sigma-Aldrich	S9638	Phosphate buffer preparation
Streptavidin-Maleimide	Sigma-Aldrich	S9415	Glass bead functionalization