pubs.acs.org/Langmuir © 2010 American Chemical Society
Application of HaloTag Protein to Covalent Immobilization of Recombinant Proteins for Single Molecule Force Spectroscopy Yukinori Taniguchi and Masaru Kawakami* School of Materials Science, Japan Advanced Institute of Science and Technology (JAIST), 1-1 Asahidai, Nomi, Ishikawa 923-1292, Japan Received April 27, 2010. Revised Manuscript Received June 2, 2010 We have developed the HaloTag system for the covalent immobilization of polyproteins onto a mica substrate for single molecule force spectroscopy using the atomic force microscope. A recombinant fusion polyprotein of titin I27 with HaloTag7 protein was produced, and the covalent and site-specific attachment on a HaloTag-ligand-modified mica surface was confirmed by force-extension measurements. Two mechanical unfolding intermediates of HaloTag7 protein were found by contour length analysis. This tethering method allows site-specific covalent immobilization of a protein that complements the standard method utilizing thiol-gold interaction, thus facilitating force-extension measurements for cysteine-containing proteins.
Introduction Over the past decade, single-molecule force spectroscopy using atomic force microscopy (AFM) has emerged as a powerful tool for investigating the mechanical and dynamical property of many proteins. In typical mechanical unfolding experiments of proteins, a tandemly arranged multidomain protein (herein a “polyprotein”) is tethered between the substrate and cantilever to apply a stretching force.1,2 One of the critical issues in this type of experiment is how to immobilize such a protein onto the substrate and cantilever. Nonspecific interactions (physical adsorption) have been extensively employed to attach a tandem protein to the substrate. In this case, however, for successful pulling measurements, different parts of the polyprotein have to adhere strongly to both the substrate and the cantilever but also not generate artifacts in the force-extension profile due to nonspecific protein-substrate/tip interactions. Balancing these competing effects is challenging and may be one of the reasons for the low yield (the relative frequency of useable data in terms of number of approach-retract cycles). This problem is partly solved by the chemical attachment of proteins at one end onto the substrate. The most common method is the formation of a gold-thiol bond between the gold-coated substrate and the sulfur atom of the side chain of a cysteine residue introduced to one terminus of the polyprotein. Gold-thiol bonds form spontaneously and are strong enough to study mechanical unfolding events of proteins (its mean rupture force is ∼1.4 nN).3 However, the gold-thiol method is problematic for proteins which have intrinsic cysteine residues in their amino acid sequence as these may form competing gold-thiol attachment points, complicating the analysis of resultant force-extension profiles. To avoid this, intrinsic cysteine residues are substituted with other amino acids (e.g., *To whom correspondence should be addressed. E-mail: kmasaru@ jaist.ac.jp. Telephone & fax: þ81-761-51-1593. (1) Carrion-Vazquez, M.; Oberhauser, A. F.; Fowler, S. B.; Marszalek, P. E.; Broedel, S. E.; Clarke, J.; Fernandez, J. M. Mechanical and chemical unfolding of a single protein: a comparison. Proc. Natl. Acad. Sci. U.S.A. 1999, 96 (7), 3694-3699. (2) Brockwell, D. J. Probing the mechanical stability of proteins using the atomic force microscope. Biochem. Soc. Trans. 2007, 35 (Pt 6), 1564-1568. (3) Grandbois, M.; Beyer, M.; Rief, M.; Clausen-Schaumann, H.; Gaub, H. E. How strong is a covalent bond? Science 1999, 283 (5408), 1727-1730.
Langmuir 2010, 26(13), 10433–10436
serine), which might affect the thermodynamic stability4 or the catalytic activity/function of the protein. Indeed, many biologically important proteins have cysteine residues,5,6 and they have barely been studied by force spectroscopy. Other strong, sitespecific, and cysteine-independent immobilization methods are thus required. High affinity noncovalent biological interactions such as His:Ni.nitrilotriacetic acid, GST:glutathione, and biotin: (strept)avidin are very effective in specifically immobilizing proteins onto substrates under standard solution conditions. However, such methods are unsuitable for many mechanical unfolding measurements because application of force onto these interactions hugely increases their off rates (the rupture force of such tags is
