Torque spectroscopy of single proteins and membranes

We are developing torsion profiling techniques for protein and membrane characterization and for nanomechanical studies on structure-function relationships [1,2]. The technique is based on an in-plane rotating magnetic field that applies a torque onto superparamagnetic particles. The angular orientation of the particles is tracked using fluorescent labels which are imaged in a TIRF or a confocal microscope.

In one part of our research we use the magnetic torque spectrometer to investigate the torsional properties of individual protein pairs that consist of an antibody and its antigen. Single-protein resolution is attained by sufficiently diluting the concentration of immobilized proteins [3]. The goal is to be able to relate the nanomechanical properties of the protein complex with its structure and to relate the functional properties (e.g. binding strength) to the torsional deformation. In addition we study the influence of molecules in solution (e.g. surfactants) on the torsional properties of protein-pairs.

We also apply torque spectroscopy for the study of cell membranes and more recently also for the study of fluid-fluid interfaces. In the cell experiments, we combine magnetic particle actuation with rotational and translational particle tracking to simultaneously measure the mechanical stiffness of live cells in three rotational and two translational directions. When using particles that bind via integrins to the cell membrane and the subjacent cortex, we measured an isotropic stiffness and a characteristic power-law dependence of the shear modulus on the applied frequency [4]. We have applied the same technique to measure the translational and rotational stiffnesses of phagocytic cups as a function of time [5]. The measured evolution of stiffness reveals a characteristic pattern with a pronounced peak preceding the finalization of uptake. This approach is a novel way to measure the progression of emerging phagocytic cups and their mechanical properties in situ and in real time.

References

[1] Torsion Stiffness of a Protein Pair Determined by Magnetic Particles,  X.J.A. Janssen, J.M. van Noorloos, A. Jacob, L.J. van IJzendoorn, A.M. de Jong, M.W.J. Prins, Biophys. J. 100, 2262-2267 (2011).
[2] Torsion Profiling of Proteins Using Magnetic Particles, A. van Reenen, F. Gutiérrez-Mejía, L.J. van IJzendoorn, M.W.J. Prins, Bioph. J. 104, 1073-1080 (2013)
[3] Quantification of protein-ligand dissociation kinetics in heterogeneous affinity assays, A. Jacob, L.J. van IJzendoorn, A.M. de Jong, and M.W.J. Prins, Anal. Chem. 84, 9278-9294 (2012)
[4] Probing the cell membrane by magnetic particle actuation and Euler angle tracking, M. Irmscher, A. M. de Jong, H. Kress, and M. W. J. Prins, Biophys. J. 102, 698–708 (2012).
[5] A method for time-resolved measurements of the mechanics of phagocytic cups, M. Irmscher, A.M. de Jong, H. Kress, M.W.J. Prins, J. R. Soc. Interface 10, 20121048- (2013).