Molecular Calipers for Highly Precise and Accurate Measurements of Single-Protein Mechanics

Abstract

Single-molecule atomic force spectroscopy (AFM) has evolved into a powerful technique toward elucidating conformational changes in proteins when exposed to applied force. AFM technologies that are currently available allow for precise measurements of proteins length changes during conformational transitions. However, because of systematic errors in piezo calibration as well as errors originating from fitting experimental data using a worm-like chain model of polymer elasticity, high-precision measurements of length changes do not necessarily translate into highly accurate measurements of length changes, resulting in uncertainty in obtaining structural information about protein conformational changes. Actually achieving highly precise and accurate force spectroscopy measurements remains a challenge. Here, we report a protein caliper method that eliminates systematic errors that occur during single-protein force spectroscopy measurements, and thus achieves highly precise and accurate length change measurements in protein mechanics studies. To do this, a series of loop elongation variants of the small protein GB1, which differ by 2, 5, 10, 15, and 24 amino acid residues, were engineered. Differential measurements of amino acid residue length obtained from different AFM setups result in a precise measure of the length of a single amino acid residue, which varies within different AFM setups because of systematic error between individual AFM piezoelectric calibrations. The measured length of a single amino acid residue from a given AFM setup is then used as a caliper for the given setup to eliminate systematic error, leading to highly accurate and precise measurements of the number of amino acid residues that are involved in a conformation change of a polypeptide chain. We further developed a more precise, robust, and model-free method to determine the apparent size of single amino acid residues and conformational changes of proteins. This method improves the accuracy of single protein force spectroscopy measurements, providing an accurate means of measuring force-induced protein conformational changes.

Reversible Unfolding and Folding of the Metalloprotein Ferredoxin Revealed by Single-Molecule Atomic Force Microscopy

“…To investigate the mechanical unfolding mechanism of aFd, we first constructed the Cys-aFd-GL15-Cys heterodimer, and used maleimide–thiol coupling chemistry to construct the polyprotein (aFd-GL15) n for single-molecule AFM experiments . GL15 is a loop insertion variant of the small model protein GB1 in which a stretch of 15 flexible residues is inserted into the second loop of GB1. , The signatures of the mechanical unfolding of GL15 are a contour length increment (Δ L c ) of ∼23 nm and a unfolding force of ∼150 pN at a pulling speed of 400 nm/s, as detailed in our previous work . The well-characterized GL15 domain serves both as a “fingerprint domain” for detecting single-molecule force spectrum for (aFd-GL15) n , as well as an internal force caliper in the (aFd-GL15) n polyprotein .…”

“…GL15 is a loop insertion variant of the small model protein GB1 in which a stretch of 15 flexible residues is inserted into the second loop of GB1. , The signatures of the mechanical unfolding of GL15 are a contour length increment (Δ L c ) of ∼23 nm and a unfolding force of ∼150 pN at a pulling speed of 400 nm/s, as detailed in our previous work . The well-characterized GL15 domain serves both as a “fingerprint domain” for detecting single-molecule force spectrum for (aFd-GL15) n , as well as an internal force caliper in the (aFd-GL15) n polyprotein . Stretching the (aFd-GL15) n polyprotein allowed us to stretch aFd from its N- and C-termini.…”

Engineering protein polymers of ultrahigh molecular weight via supramolecular polymerization: towards mimicking the giant muscle protein titin

“…18,33 Fitting the sawtooth peaks to the worm-like chain (WLC) model of polymer elasticity clearly revealed two populations of unfolding events: blue events with a contour length increment (DLc) of $18 nm, and events colored in red with a DLc of $28 nm, corresponding to the unfolding of the oxidized reconstituted GL5CC and I27 domains, respectively. 32,33,35 The observed number of unfolding force peaks of GL5CC and I27 domains in a force-extension curve is oen more than 13, indicating that the stretched protein polymer is at least a septamer of (G C -I27-G N ) n . These observations indicated that the stretched protein polymers are indeed continuous protein polymers made of tandem repeats of Representative force-extension curves of the G C -I27-G Nbased protein polymers (200 mM G C -I27-G N was used in the polymerization).…”

“…G N and G C are two fragments from GL5CC we constructed previously. 30,32 GL5CC is split into two fragments at residue 42: G N is the N-terminal fragment (residues 1-42) and G C is the Cterminal fragment (residues 43-61). Both G N and G C were amplied with the template of GL5CC-27w34f through polymerase chain reaction (PCR).…”

“…[26][27][28][29][30] This non-covalent "GB1" fold has a dissociation constant K d of $9 Â 10 À6 M. 26 In our previous work, we engineered a loop elongation variant of GB1, termed GL5CC, where 5 residues were inserted into the unstructured loop and residues 42/44 were mutated to cysteines. 31,32 When split into fragments G N (residue 1-42) and G C (residues 43-61), G N and G C can reconstitute into GB1's native fold (Fig. 1A).…”