Genomes to Life Contractor-Grantee Workshop III
February 6-9, 2005, Washington, D.C.
Technology Development and Use
Imaging, Molecular, and Cellular Analysis
95
Direct Determination of Affinity in Individual Protein-Protein Complexes in Mono and Multivalent Configurations Using Dynamic Force Spectroscopy
Todd A. Sulchek1, Kevin Langry1, Raymond W. Friddle1, Timothy V. Ratto1, Sally DeNardo2, Huguette Albrecht2, Michael Colvin1, and Aleksandr Noy1,*(noy1@llnl.gov)
1Lawrence Livermore National Laboratory, Livermore, CA and 2University of California, Davis, CA
Our laboratory at LLNL has been developing techniques for direct determination of the energy landscapes for biological molecule interactions. Interactions between proteins drive a vast variety of cellular events, and direct determination of the strength of these interactions is important to the efforts in understanding cellular metabolism and high-throughput characterization of protein complexes. Recent advances in single biological molecule manipulation and measurement have enabled direct measurements of interaction forces between individual biological molecules. We have been using atomic force microscopy (AFM) to determine energy barriers and kinetic parameters for the dissociation of individual protein-protein complexes.
We used the atomic force microscope (AFM) to measure the binding forces between single molecule mucin1 (Muc1) protein and an antibody screened against Muc1. Muc1 is overexpressed on cell surfaces in a number of human cancers. Our collaborators at the UC Davis Cancer Center use antibodies to Muc1 as the targeting mechanism for delivery of radioimmunotherapeutic drugs, which consist of several such antibodies tethered to a common radioactive payload. Direct determination of binding affinities for mono and multivalent configurations of such drugs is critical for their optimization.
Our measurements utilized the proteins linked to the surfaces of the AFM tip and sample by flexible tethers (Figure 1). This is a versatile and general approach that spatially separates specific interactions and allows quick rejection of non-specific binding events. We have confirmed measurement of specific interactions by blocking it in a competition assay. Moreover, we were able to identify and discriminate between single and multiple rupture events by monitoring the interaction force and the nature of the tether stretch.
Measurements of the binding strength as the function of the bond loading rate (dynamic force spectra) allowed us to determine energy barriers, thermodynamic off-rates and the distance to the transition state for simultaneous dissociation of one, two, and three protein-protein pairs (Figure 2). Remarkably, the dynamic force spectra for single and multiple bonds show very similar slopes corresponding to the bond width for individual protein complex. These experimental observations confirm the theoretical prediction for unbinding of molecular bonds in parallel configuration. We also show that although our measured bond strength scales linearly with the number of molecule pairs, multivalent configuration leads to a precipitous decrease in the thermodynamic off-rates for the complex dissociation. Finally, we will discuss approaches for performing these measurements in high-throughput manner for potential end-line characterization of protein complexes and affinity tags.
Figure 1: Schematic of the measurement setup. (A) gold coated tip, (B) thiol surfactant, (C,E) PEG tethers, (D) Muc1 antibody and Muc1 peptide complex.
Figure 2: A dynamic force spectrum showing rupture events for one (☐,☐), two (◊), and three (∆) bonds. The blue square points (☐) correspond to stepwise ruptures of single bonds in quick succession, and red square points (☐) correspond to individual single bond rupture events. The least squares line fits predict the thermodynamic off-rates of 7∙10-3 s-1, 7∙10-5 s-1, and 4∙10-9 s-1 for the rupture of one, two, and three bonds respectively.
This work was funded by the LLNL LDRD program.
* Presenting author
