Force-dependent unfolding dynamics of spectrin R16: Resolving experimental contradiction and unveiling model consistency

Abstract Spectrin domains, characterized by a distinctive triple helix structure, are crucial in physiological processes, particularly in maintaining membrane shape and crosslinking cytoskeletons. Previous research on the 16th domain of α -spectrin repeats (R16) has yielded conflicting results: bulk experiments showed an unfolding rate approximately two orders of magnitude faster than the zero-force result extrapolated from single-molecule force spectroscopy experiments using atomic force microscopy (AFM). To address this discrepancy, we investigated the folding and unfolding rates of R16 across a broader range of forces using magnetic tweezers (MT). Our findings reveal that AFM results at higher forces cannot be directly extrapolated to the low-force regime due to a nonlinear relationship between force and the logarithm of the unfolding rate. We demonstrated that two-dimensional model, structural-elastic model, and two-pathway model can all effectively explain the experimental data when they capture the core physics of the short unfolding distance at low forces. Our study provides a more comprehensive understanding of the unfolding dynamics of the spectrin domain, resolves previous contradictory experimental results, and highlights the common basis of different theoretical models.