Measurement of the effect of force on the helix-coil transition using hybrid smFRET-magnetic tweezers
The alpha helix is the predominant element of secondary structure in proteins and is involved in a number of force-dependent biological processes. Despite its importance, direct measurements of its response to force have not been made. Investigations of the mechanical properties of biomolecules usually rely on force spectroscopic techniques such as optical tweezers or atomic force spectroscopy, but these techniques have failed to detect the unfolding/folding signature of the alpha helix, presumably because the cooperativity of the helix-coil transition is broad and the expected extension change is small.
This research describes the development of an instrument combining magnetic tweezers and total internal reflection fluorescence microscopy (TIRF) with the goal of capturing the effect of force on an isolated alpha helix. Changes in extension are reported by single-molecule Förster resonance energy transfer (smFRET) between small-molecule fluorophores conjugated near the middle of such a helix. This technique provides an extremely sensitive probe of conformation that is unaffected by the DNA handles typically used in optical tweezers experiments and is amenable to both nucleic acid and protein constructs. Calibration is performed and verified using the known worm-like chain (WLC) force-extension behavior of DNA, its overstretching transition, and the opening and closing of a previously characterized DNA hairpin.
Using this instrument, the alpha helix is observed to unfold cooperatively and reversibly from 11 pN to 17 pN, indicating rapid equilibrium between the helix and coil states. This contrasts with the behavior of an unstructured polypeptide that exhibits a steady, non- cooperative FRET decrease across the 1-30 pN range of applied force. The behavior of the unstructured polypeptide is well described by a WLC model, reformulated here in terms of root-mean square displacement. The helix-prediction algorithm AGADIR, when modified to incorporate the effect of force and a WLC model of the unfolded state, successfully explains the observed folding signature of the alpha helix.