Contour length and refolding rate of a small protein controlled by engineered disulfide bonds

Sri Rama Koti Ainavarapu, Jasna Brujić, Hector H. Huang, Arun P. Wiita, Hui Lu, Lewyn Li, Kirstin A. Walther, Mariano Carrion-Vazquez, Hongbin Li, Julio M. Fernandez

    Research output: Contribution to journalArticlepeer-review

    Abstract

    The introduction of disulfide bonds into proteins creates additional mechanical barriers and limits the unfolded contour length (i.e., the maximal extension) measured by single-molecule force spectroscopy. Here, we engineer single disulfide bonds into four different locations of the human cardiac titin module (I27) to control the contour length while keeping the distance to the transition state unchanged. This enables the study of several biologically important parameters. First, we are able to precisely determine the end-to-end length of the transition state before unfolding (53 Å), which is longer than the end-to-end length of the protein obtained from NMR spectroscopy (43 Å). Second, the measured contour length per amino acid from five different methods (4.0 ± 0.2 Å) is longer than the end-to-end length obtained from the crystal structure (3.6 Å). Our measurement of the contour length takes into account all the internal degrees of freedom of the polypeptide chain, whereas crystallography measures the end-to-end length within the "frozen" protein structure. Furthermore, the control of contour length and therefore the number of amino acids unraveled before reaching the disulfide bond (n) facilitates the test of the chain length dependence on the folding time (τF). We find that both a power law scaling τF ∝ nλ with λ = 4.4, and an exponential scaling with n0.6 fit the data range, in support of different protein-folding scenarios.

    Original languageEnglish (US)
    Pages (from-to)225-233
    Number of pages9
    JournalBiophysical journal
    Volume92
    Issue number1
    DOIs
    StatePublished - Jan 2007

    ASJC Scopus subject areas

    • Biophysics

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