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

¹ Columbia University
² University of Illinois at Chicago
³ Columbia university
⁴ Instituto Cajal, Consejo Superior de Investigaciones Cientificas
⁵ University of British Columbia

^* To whom correspondence should be addressed. E-mail: sra2104{at}columbia.edu.

Submitted on June 17, 2006
Revised on August 5, 2006
Accepted on 13 September 2006

	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 prior to unfolding (53 Å), which is longer than the end-to-end length of the protein obtained from NMR spectroscopy (43 Å). Secondly, 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, while 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 prior to reaching the disulfide bond (n) facilitates the test of the chain length dependence on the folding time (_F). We find that both a power law scaling _F~n with = 4.4, and an exponential scaling with n^0.6 fit the data range, in support of different protein folding scenarios.

Key Words: disulfide engineering, force spectroscopy, mechanical unfolding, protein folding, single molecule, titin I27

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