Abstract
Protein extensibility appears to be based broadly on conformational changes that can in principle be modulated by protein-protein interactions. Spectrin family proteins, with their extensible three-helix folds, enable evaluation of dimerization effects at the single molecule level by atomic force microscopy. Although some spectrin family members function physiologically only as homodimers (e.g. alpha-actinin) or are strictly monomers (e.g. dystrophin), alpha- and beta-spectrins are stable as monomeric forms but occur physiologically as alpha,beta-heterodimers bound laterally lengthwise. For short constructs of alpha- and beta-spectrin, either as monomers or as alpha,beta-dimers, sawtooth patterns in atomic force microscopy-forced extension show that unfolding stochastically extends repeats approximately 4-5-fold greater in length than native conformations. For both dimers and monomers, distributions of unfolding lengths appear bimodal; major unfolding peaks reflect single repeats, and minor unfolding peaks at twice the length reflect tandem repeats. Cooperative unfolding thus propagates through helical linkers between serial repeats (1, 2). With lateral heterodimers, however, the force distribution is broad and shifted to higher forces. The associated chains in a dimer can stay together and unfold simultaneously in addition to unfolding independently. Weak lateral interactions do not inhibit unfolding, but strong lateral interactions facilitate simultaneous unfolding analogous to serial repeat coupling within spectrin family proteins.
Highlights
As cytoskeletal proteins, spectrin superfamily proteins play important roles in cell organization and membrane mechanics [3, 4]
Associate head-to-head as tetramers that cross-link actin is especially crucial to the resilience of the red cell membrane in circulation
The actinin homodimer has a homogeneous affinity throughout its laterally associated chain, and its association constant is much greater compared with the spectrin heterodimer
Summary
Spectrin superfamily proteins play important roles in cell organization and membrane mechanics [3, 4]. Like red cell spectrin, ␣-actinin, with its four homologous repeats, associates laterally and cross-links actin, imparting stability to structures that range from focal adhesions to Z-lines of myotubes [5]. Other AFM studies have elaborated RNA hairpin interactions [16] and insulin monomer-monomer association/dissociation interactions [17], none to date have explicitly examined the inhibiting effects, if any, of protein dimerization on domain unfolding. Spectrin dimerization is predicated on the typical range of protein interactions: hydrophobic sequestration, hydrogen bonding, and most importantly electrostatic interactions between paired triple-helical bundles [13] We probe these dimeric interactions through their effects on flexibility and unfolding. By a thorough statistical analysis, we conclude that strong lateral interactions lead to coupled unfolding of laterally adjacent repeats, whereas weak lateral interactions are surprisingly neutral in their effects on the force to unfold a repeat
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