Unfolding a Linker between Helical Repeats Vanessa Ortiz 1,2,3 , Steven O. Nielsen 1,3 , Michael L. Klein 1,3 and Dennis E. Discher 2,3 * 1 Center for Molecular Modeling Department of Chemistry University of Pennsylvania Philadelphia, PA 19104, USA 2 Biophysical Engineering Laboratory, Department of Chemical and Biomolecular Engineering, University of Pennsylvania, Philadelphia PA 19104, USA 3 Laboratory for Research on the Structure of Matter, University of Pennsylvania, Philadelphia PA 19104, USA In many multi-repeat proteins, linkers between repeats have little secondary structure and place few constraints on folding or unfolding. However, the large family of spectrin-like proteins, including a-actinin, spectrin, and dystrophin, share three-helix bundle, spectrin repeats that appear in crystal structures to be linked by long helices. All of these proteins are regularly subjected to mechanical stress. Recent single molecule atomic force microscopy (AFM) experiments demonstrate not only forced unfolding but also simultaneous unfolding of tandem repeats at finite frequency, which suggests that the contiguous helix between spectrin repeats can propagate a cooperative helix-to-coil transition. Here, we address what happens atomistically to the linker under stress by steered molecular dynamics simulations of tandem spectrin repeats in explicit water. The results for a-actinin repeats reveal rate-dependent pathways, with one pathway showing that the linker between repeats unfolds, which may explain the single-repeat unfolding pathway observed in AFM experiments. A second pathway preserves the structural integrity of the linker, which explains the tandem-repeat unfolding event. Unfolding of the linker begins with a splay distortion of proximal loops away from hydrophobic contacts with the linker. This is followed by linker destabilization and unwinding with increased hydration of the backbone. The end result is an unfolded helix that mechanically decouples tandem repeats. Molecularly detailed insights obtained here aid in understanding the mechanical coupling of domain stability in spectrin family proteins. q 2005 Elsevier Ltd. All rights reserved. Keywords: spectrin repeats; steered molecular dynamics; mechanical unfolding; alpha-helical linkers; cooperative domain unfolding *Corresponding author Introduction Multi-domain proteins prove to be a dominant fraction of the proteome, 1 but the physical roles of linkers between domains as domain separators and/or connectors are not always clear. Multi- repeat proteins have now proven accessible to elegant single molecule experiments with atomic force microscopy (AFM) 2 and optical trapping, 3 which allow the study of domain unfolding and folding 4 while lending insight into linker stability. The various immunoglobulin domains of the cytoskeletal protein titin 2,3 as well as related fibronectin-III domains 5,6 unfold under force one domain at a time. The unfolding force clearly depends on the extension or loading rate, 7 but there is no significant dependence on the linkers between domains. 8 In contrast, a much more cooperative coupling of domain unfolding under force is exhibited by spectrin family proteins. 9 Spectrin, dystrophin, a-actinin, and related pro- teins share a serial repeat structure (called a spectrin repeat or domain), bind actin filaments, and clearly contribute resilience to the cell and its mem- brane. 10–13 Evidence for dynamic dissociation of spectrins under cell stress is just emerging, 14 as is the possibility of spectrin unfolding in situ. 15 The characteristic spectrin repeats fold into a bundle of three antiparallel a-helices, 12,16–20 with any two adjacent repeats linked by an w8–9 nm helix that extends from one domain to the next. 12,18–21 Even where non-helical linkers have been predicted by various secondary structure algorithms, the con- necting linker is helical in crystal structures of tandem repeats. 21,22 Forced unfolding of spectrin repeats occurs at five to ten times smaller forces 9,23,24 than the forces measured for unfolding 0022-2836/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. Abbreviations used: AFM, atomic force microscopy; SMD, steered molecular dynamics. E-mail address of the corresponding author: discher@seas.upenn.edu doi:10.1016/j.jmb.2005.03.086 J. Mol. Biol. (2005) 349, 638–647