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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM106420-03
Application #
8776721
Study Section
Nanotechnology Study Section (NANO)
Program Officer
Lewis, Catherine D
Project Start
2013-01-01
Project End
2016-12-31
Budget Start
2015-01-01
Budget End
2015-12-31
Support Year
3
Fiscal Year
2015
Total Cost
$289,862
Indirect Cost
$99,862
Name
Cornell University
Department
Engineering (All Types)
Type
Schools of Engineering
DUNS #
872612445
City
Ithaca
State
NY
Country
United States
Zip Code
14850
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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
Schein, Perry; O'Dell, Dakota; Erickson, David (2016) Dynamics of an optically confined nanoparticle diffusing normal to a surface. Phys Rev E 93:062139
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:
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
Chen, Tai-Yen; Santiago, Ace George; Jung, Won et al. (2015) Concentration- and chromosome-organization-dependent regulator unbinding from DNA for transcription regulation in living cells. Nat Commun 6:7445
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

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