The molten globule state is unusually deformable under mechanical force

Abstract

Recently, the role of force in cellular processes has become more evident, and now with advances in force spectroscopy, the response of proteins to force can be directly studied. Such studies have found that native proteins are brittle, and thus not very deformable. Here, we examine the mechanical properties of a class of intermediates referred to as the molten globule state. Using optical trap force spectroscopy, we investigated the response to force of the native and molten globule states of apomyoglobin along different pulling axes. Unlike natively folded proteins, the molten globule state of apomyoglobin is compliant (large distance to the transition state); this large compliance means that the molten globule is more deformable and the unfolding rate is more sensitive to force (the application of force or tension will have a more dramatic effect on the unfolding rate). Our studies suggest that these are general properties of molten globules and could have important implications for mechanical processes in the cell.

Fig. 1.

Experimental setup (A) and structure of holomyoglobin (B). (A) The protein was tethered between two polystyrene beads through functionalized dsDNA attached to the protein at the engineered cysteine residue, thereby determining the axis along which the force was applied. One bead is held on a pipette tip by suction, and a dual beam counterpropagating optical trap manipulates the other. By monitoring the bead in the trap, the force on the tether and the relative extension of the tether were measured. (B) The structure of holomyoglobin (Protein Data Bank IDcode 1BZ6). The regions that are thought to be structured in the molten globule state are highlighted in red. The arrows indicate the pulling axis for the N/C variant (in green) and the 53/C variant (in blue) with the end-to-end distance between the pulling points as determined from the structure in parentheses.

Fig. 2.

Force-ramp traces of the N/C and 53/C variant at pH 7 and pH 5. Force-ramp experiments are depicted showing the force as a function of the trap position with the pulling traces shown in blue and the relaxation traces shown in red. Traces from the N/C variant are shown in A and C at pH 7 and pH 5, respectively. Traces from the 53/C variant are in B and D at pH 7 and pH 5, respectively. Histograms are shown of the unfolding (in blue) and refolding force (in red) distributions.

Fig. 3.

Force-jump experiments on the N/C and 53/C variant of apomyoglobin. (A) A sample trace of a force-jump unfolding experiment depicting the force and trap position as a function of time at the high force after the jump from the low force. After waiting several seconds to ensure complete refolding to the native state, the trap position was moved increasing the tension on the tether to the desired higher force and maintained constant by feedback. (B and C) Linear fit of the natural log of the unfolding rate constants as a function of force are shown for the N/C (B) and 53/C (C) variants. The distance to the transition state is determined from the slope of the lines using Bell’s model (Eq. 1).

Fig. 4.

Constant trap position experiments on the N/C and 53/C variants. Typical 1-s trace of a constant trap position experiment with the force averaged down to 1,000 Hz (in blue) for the N/C variant (A) and the 53/C variant (C). The inferred trajectory of the molecule at 500 Hz is shown in red. The accompanying histogram is of the force measured over 1 min depicting the two observed populations. Linear fits of the natural logarithm of the rate constants as a function of force are shown for the N/C (B) and 53/C (D) variants. The distance to the transition state is determined from the slope of the lines using the Bell’s model.