In this work we propose a joint project between the Erickson and Chen labs at Cornell University to develop a new approach to study the weak protein-protein interactions that govern intracellular metal and metal co-factor transport at the single molecule level. The approach involves the use of """"""""Optically Resonant NanoTweezers"""""""" which we demonstrated during preceding exploratory R21 grant are capable of trapping proteins as small as a few nanometers, breaking through a long established barrier in optical physics. In addition to developing comprehensive information on the protein interaction dynamics for copper ion and vitamin B12 trafficking, through this program we will develop two general NanoTweezer based protocols for a quantitative single molecule florescence quenching assay (smFQ) and a single molecule florescence resonant energy transfer assay (smFRET) that can be applied to numerous other biophysical problems. Safe trafficking of metal ions and metal-containing cofactors inside cells to avoid toxicity is mediated by metallochaperones which deliver these reactive species to their target destinations while protecting them from adventitious reactions. Abnormal function of this transport pathway can lead to diseases such as Wilson disease, Menkes disease, and familial amyotrophic lateral sclerosis. Despite its importance, very limited quantitative information is available on the biophysical mechanisms that enable this safe transfer or cause it to break down. A major difficulty in obtaining this information is the lak of a single molecule analysis tool which can simultaneously: (1) capture and suspend small molecules in free solution for an indefinite period time (2) effectively """"""""concentrate"""""""" the set of molecules of interest to a point where weak protein-protein interactions can be studied and (3) allow rapid modulation of the external environmental conditions. One potential method by which the above goals could be achieved is through the use of optical tweezers. Fundamentally however, existing optical confinement techniques are limited by diffraction which places a lower bound on the size of dielectric target which can be trapped to about 100nm. With the optically resonant nanotweezer technology we have shown that this force can be enhanced 1000's of times so as to trap proteins (including the Wilson disease proteins used here) as small as a few nanometers. In this proposal, we show how we can adapt this technology to (1) non-invasively capture and suspend individual macromolecules in free solution (2) guide additional molecules to the capture region so that interactions can be observed and (3) maintain captured particles in position while the suspending solution is changed. When applied to intracellular metal transport these capabilities can speed up the process for discovering how metalochaperones respond to different environmental conditions and ultimately what leads to the pathologies listed above.

Public Health Relevance

Metal ions and metal containing cofactors are essential nutrients that can also be toxic if their concentration exceeds the physiological limit or their presence in the cell is mis-regulated. Abnormal function of metal transport molecules can lead to diseases such as Wilson disease, Menkes disease and familial amyotrophic lateral sclerosis. In this work we propose to develop a new approach that can help to understand the function of a series of proteins which control the intracellular trafficking of these essential cofactors.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Nanotechnology Study Section (NANO)
Program Officer
Lewis, Catherine D
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Cornell University
Engineering (All Types)
Schools of Engineering
United States
Zip Code
Chen, Tai-Yen; Cheng, Yu-Shan; Huang, Pei-San et al. (2018) Facilitated Unbinding via Multivalency-Enabled Ternary Complexes: New Paradigm for Protein-DNA Interactions. Acc Chem Res 51:860-868
Santiago, Ace George; Chen, Tai-Yen; Genova, Lauren A et al. (2017) Adaptor protein mediates dynamic pump assembly for bacterial metal efflux. Proc Natl Acad Sci U S A 114:6694-6699
Yang, Feng; Chen, Tai-Yen; Krzemi?ski, ?ukasz et al. (2016) Single-molecule dynamics of the molecular chaperone trigger factor in living cells. Mol Microbiol 102:992-1003
O'Dell, Dakota; Schein, Perry; Erickson, David (2016) Simultaneous Characterization of Nanoparticle Size and Particle-Surface Interactions with Three-Dimensional Nanophotonic Force Microscopy. Phys Rev Appl 6:
Schein, Perry; O'Dell, Dakota; Erickson, David (2016) Dynamics of an optically confined nanoparticle diffusing normal to a surface. Phys Rev E 93:062139
Chen, Tai-Yen; Jung, Won; Santiago, Ace George et al. (2015) Quantifying Multistate Cytoplasmic Molecular Diffusion in Bacterial Cells via Inverse Transform of Confined Displacement Distribution. J Phys Chem B 119:14451-9
Martell, Danya J; Joshi, Chandra P; Gaballa, Ahmed et al. (2015) Metalloregulator CueR biases RNA polymerase's kinetic sampling of dead-end or open complex to repress or activate transcription. Proc Natl Acad Sci U S A 112:13467-72
Kang, Pilgyu; Schein, Perry; Serey, Xavier et al. (2015) Nanophotonic detection of freely interacting molecules on a single influenza virus. Sci Rep 5:12087
Schein, Perry; Ashcroft, Colby K; O'Dell, Dakota et al. (2015) Near-field Light Scattering Techniques for Measuring Nanoparticle-Surface Interaction Energies and Forces. J Lightwave Technol 33:3494-3502
Schein, Perry; Kang, Pilgyu; O'Dell, Dakota et al. (2015) Nanophotonic force microscopy: characterizing particle-surface interactions using near-field photonics. Nano Lett 15:1414-20

Showing the most recent 10 out of 13 publications