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. 2015 Aug 3;112(33):10389–10394. doi: 10.1073/pnas.1504625112

Fig. 2.

Fig. 2.

Single molecule force experiments of the NBD by optical tweezers. (A) Optical trap assay. The protein is tethered to the beads by two DNA handles containing a corresponding epitope (biotin, green hexagon; digoxygenin, brown hexagon). The connection between the DNA and protein is realized by the modification of the two cysteine residues of the protein by the single-stranded DNA–maleimide oligonucleotide complementary to the DNA handle overhang. The 1 μm-sized functionalized (α-digoxygenin, purple square; streptavidin, light blue square) glass beads are trapped in the highly focused laser beam. One of the beams is reflected by the steerable mirror, which enables pulling and stretching of a single protein. (B) Force–extension curves of a single NBD domain (the apo-form). The protein–DNA construct was stretched (black curve, 1 kHz filtered) and relaxed (purple curve, 1 kHz filtered) at a constant velocity of 20 nm/s. The trace depicts two parts corresponding to the stretching of the DNA handles and a sudden rip at 34 pN corresponding to the unfolding of the protein. The dashed lines correspond to a WLC fit to the data yielding the contour-length increase of 134 nm. (C) A magnification of the force–extension trace. The unfolding phase of the protein reveals transient populations of the intermediates (black squares; for details, see Single Molecule Force Experiments on the NBD of the DnaK Chaperone). (D) Contour-length transformation of the force–extension data. Shown are multiple unfolding phases. (E) Contour-length increase versus time for the wild-type (black) and insert variants (purple for K183-Insert, red for A290-Insert, and gray for D364-Insert). The insert variants have an additional increase in the contour length (∼7 nm) due to insertion of a highly flexible 20 aa large loop. The plus sign (+) indicates an increase in the length at a particular position. (F) Comparison of the unfolding phases between different protein forms: apo, MgATP, and MgADP. The conditions were 50 mM Tris⋅HCl, 150 mM KCl, 5 mM MgCl2, and 1 mM ATP/ADP, pH 7.8. (G) Contour-length increase versus time for the wild-type (black) and insert variants (purple for K183-Insert, red for A290-Insert, and gray for D364-Insert) under holo-conditions (1 mM ATP, 5 mM MgCl2).