Tubulin Nanorings

Abstract

Biological systems routinely produce nanoscopic molecular structures with considerably less dispersion in size and shape than encountered in most manufactured materials. Indeed, Biological structures are frequently and essentially monodisperse. An example of this uniformity, combined with an intriguing geometry, is the nanometer-scale protein nanorings produced by interaction of the protein tubulin with certain hydrophobic tri-, tetra-and pentapeptides originally extracted as natural products from marine biosystems. Different peptides produce different sized nanorings, but we focus on those produced by binding to tubulin of the cyclic depsipeptide cryptophycin. The nanorings that form upon binding of this ligand show a sharp mass distribution indicating that the nanorings are made of 8 tubulin dimers of 100 kDa.In this submission, we demonstrate how a combination of fluorescence correlation spectroscopy, dynamic light scattering, electron microscopy, analytical ultracentrifugation, small-angle neutron scattering, and modeling is applied to reveal interactions of tubulin and cryptophycin in solution and to characterize their structures. We find that the cryptophycin-tubulin nanorings (~25 nm diameter) are single-walled, appear rigid, are composed of 8 tubulin dimers in a single closed ring, and are stable upon dilution to nanomolar concentrations.Similar studies with a different peptide, the linear pentapeptide dolastatin 10, demonstrated that binding of this peptide to tubulin produces larger nanorings (14 tubulin dimers, ~45 nm diameter rings), with slightly different properties. The ability to adjust the ring size with different peptides, and produce uniform nanorings with properties that differ slightly between size classes, makes the tubulin-peptide ring structures an appealing structural system.

Interdimeric Curvature in Tubulin–Tubulin Complexes Delineates the Microtubule-Destabilizing Properties of Plocabulin

“…Electron microscopy evidence has shown that plocabulin and its analogous compound PM050489 bind to unpolymerized tubulin and induce the in vitro formation of single-wall tubulin rings composed of 7 ± 1 dimers with average outer diameters of ∼25 nm. Similar single-wall ring assemblies have been found in tubulin complexes with other potent MT-destabilizing agents such as the cryptophycin peptide. − On the other hand, rhizoxin binding leads to nonpolymerizable tubulin forms that do not undergo aggregation, whereas vinblastine site ligands promote the assembly of long helical tubulin filaments . Other polymeric structures can also be assembled from tubulin, including large double-wall rings (∼50 nm) from ligand-free tubulin. , Based on this information, it is relevant to evaluate whether MD simulations predict the conformational changes that will lead to oligomeric ring assemblies from plocabulin–tubulin complexes.…”