Probing Transient Copper Chaperone−Wilson Disease Protein Interactions at the Single-Molecule Level with Nanovesicle Trapping

Jaime J. Benítez,† Aaron M. Keller,† Patrick Ochieng,‡ Liliya A. Yatsunyk,§ David L. Huffman,*‡ Amy C. Rosenzweig,§ and Peng Chen*†;
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, Department of Chemistry, Western Michigan University, Kalamazoo, Michigan 49008, and Departments of Biochemistry, Molecular Biology, and Cell Biology and of Chemistry, Northwestern University, Evanston, Illinois 60208
J. Am. Chem. Soc., 2008, 130 (8), pp 2446–2447
DOI: 10.1021/ja7107867
Publication Date (Web): February 5, 2008
Copyright © 2008 American Chemical Society
†

 Cornell University.

,
‡

 Western Michigan University.

,
§

 Northwestern University.

,
*

In papers with more than one author, the asterisk indicates the name of the author to whom inquiries about the paper should be addressed.

, pc252@cornell.edu, ; , david.huffman@wmich.edu

Abstract

Abstract Image

Transient metallochaperone−target protein interactions are essential for intracellular metal trafficking but challenging to study at both the ensemble and the single-molecule level. Here we report using nanovesicle trapping to enable single-molecule fluorescence resonance energy transfer (smFRET) studies of transient interactions between the copper chaperone Hah1 and the fourth metal-binding domain of its target protein, the Wilson disease protein (WDP). We were able to monitor their interactions in real time one event at a time, capture distinct protein interaction intermediates, resolve intermediate interconversion dynamics, and quantify both the interaction kinetics and thermodynamics in the absence of copper. The study exemplifies the ability of nanovesicle trapping in combination with smFRET for studying weak protein interactions and provides insight into how Hah1 and WDP may collaborate to mediate copper transfer inside cells.

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History

  • Published In Issue February 27, 2008
  • Received December 3, 2007

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