Examining the Influence of Linkers and Tertiary Structure in the Forced Unfolding of Multiple-Repeat Spectrin Molecules

The unfolding pathways of multiple-repeat spectrin molecules were examined using steered molecular dynamics (SMD) simulations to forcibly unfold double- and triple-repeat spectrin molecules. Although SMD has previously been used to study other repeating-domain proteins, spectrin offers a unique challenge in that the linker connecting repeat units has a definite secondary structure, that of an α-helix. Therefore, the boundary conditions imposed on a double- or triple-repeat spectrin must be carefully considered if any relationship to the real system is to be deduced. This was accomplished by imposing additional forces on the system which ensure that the terminal α-helices behave as if there were no free noncontiguous helical ends. The results of the SMD simulations highlight the importance of the rupture of the α-helical linker on the subsequent unfolding events. Rupture of the linker propagates unfolding in the adjacent repeat units by destabilizing the tertiary structure, ultimately resulting in complete unfolding of the affected repeat unit. Two dominant classes of unfolding pathways are observed after the initial rupture of a linker which involve either rupture of another linker (possibly adjacent) or rupture of the basic tertiary structure of a repeat unit. The relationship between the force response observed on simulation timescales and those of experiment or physiological conditions is also discussed

Extending a Spectrin Repeat Unit. II: Rupture Behavior

A spectrin repeat unit was subject to extension using cyclic expansion nonequilibrium molecular dynamics. Periodic boundary conditions were used to examine the effects of the contiguous α-helical linker on the force response. The measured force-extension curve shows a linear increase in the force response when the spectrin repeat unit is extended by ∼0.4 nm. After that point, the force response peaks and subsequently declines. The peak in the force response marks the point where the spectrin repeat unit undergoes a change in its material properties from a strongly elastic material to a mostly viscous one, on the timescales of the simulations. The force peak is also correlated with rupture of the α-helical linker, and is likely the event responsible for the peaks in the sawtooth-pattern force-extension curves measured by atomic force microscopy experiments. Rupture of the linker involves simultaneously breaking approximately four hydrogen bonds that maintain the α-helical linker. After this initial rupture, the linker undergoes simple helix-to-coil transitions as the spectrin repeat unit continues to be extended. The implications of linker rupture in the interpretation of unfolding and atomic force microscopy experiments are also discussed

Extending a Spectrin Repeat Unit. I: Linear Force-Extension Response

Nonequilibrium molecular dynamics simulations were used to calculate the elastic properties of a spectrin repeat unit. A contiguous α-helical linker was constructed by employing periodic boundary conditions, allowing a novel scheme for evaluating the thermodynamic force as a function of extension. By measuring the force-extension response under small extensions, spectrin was observed to behave primarily as an elastic material with a spring constant of 1700 ± 100 pN/nm. The implications of this spring constant, in terms of the properties of the spectrin tetramer, are also discussed