Halotag Tethers to Study Titin Folding at the Single Molecule Level

Ionel Popa, Ronen Berkovich, Jorge Alegre-Cebollada, Jaime Andres Rivas-Pardo, Julio M. Fernandez

Research output: Contribution to journalArticlepeer-review


Single molecule force spectroscopy techniques have become important tools to study properties of proteins that operate under force; a common occurrence in Biology. Lack of specific attachment of single molecules to the force probe limits the measuring time and often leads to tethering at random places along a protein substrate. Here we present a technique based on polyprotein engineering and HaloTag attachment that is capable of avoiding these limitations. This method shows full-length polyprotein unfolding, high pick-up yield and detachment forces of ~2 nN. Compared to other covalent attachments, this method shows a specific signature, given by the partial unfolding of HaloTag. We find that placing the HaloTag at the N-end of the construct shows an unfolding contour length of 66 nm and a mechanical strength of ~131 pN. Placing the HaloTag at the C-end of the construct exposes to force a more stable part of the protein, which shows an increased mechanical strength of
~491 pN and a contour length increment of 27 nm. We use HaloTag covalent attachment to study the folding of I27, a model protein from human titin. We expose I27 polyprotein constructs to successive cycles of high and low force, which unfolds and refolds the component protein domains. Covalent attachment greatly expands the tethering time of a polyprotein construct and allows
for the measuring of the unfolding and folding rates from a single trace. Furthermore, repeated unfolding and refolding of the same polyprotein reveals subtle effects such as folding intermediates and slow oxidation of cysteines, which affects the mechanical stability of titin. This method opens a new approach to study protein folding at the human timescale, where proteins such as titin are slowly turned over after several days and to determine their energy landscape.
Original languageEnglish GB
JournalBiophysical Journal
Issue number2
StatePublished - 1 Jan 2014


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