Hydrodynamics of Nanoscopic Tubulin Rings in Dilute Solutions Hace `ne Boukari, 1, * Ralph Nossal, 1 Dan L. Sackett, 1 and P. Schuck 2 1 Laboratory of Integrative and Medical Biophysics, NICHD, Bethesda, Maryland 20892, USA 2 Division of Bioengineering and Physical Science, ORS/OD, National Institutes of Health, Bethesda, Maryland 20892, USA (Received 28 January 2004; published 26 August 2004) We combine fluorescence correlation spectroscopy and sedimentation velocity measurements to probe the hydrodynamic behavior of tubulin dimers and nanoscopic tubulin rings. The rings are rigid, have circular geometry, and are monodisperse in size. We use the high-precision ratio of the sedimentation coefficients and that of the translational diffusion coefficients to validate theories for calculating the hydrodynamic properties of supramolecular structures. DOI: 10.1103/PhysRevLett.93.098106 PACS numbers: 87.15.–v, 87.64.–t, 87.68.+z Various biological entities have a well-defined toroidal, or ringlike, shape when in aqueous solution. Examples are bacteriophage DNA condensed with multivalent cations [1,2], hexameric protein complexes involved in DNA replication [3,4], assemblies of RNA binding proteins [5], tubulin-containing ring structures [6–8], and dyna- min oligomers [9]. In this Letter we focus on nanoscopic protein ring polymers that form when -tubulin dimers (“Tu”MW100 kDa) bind cryptophycin molecules (“Cr”  MW  350 Da). These tubulin ring polymers have several attributes that can be exploited for testing the validity of hydrodynamic theories of supramolecular structures: namely, well-defined circular geometry, very narrow dispersity in structure, high rigidity, and good stability against dilution [6,7,10,11]. Crucial to this study is the finite number of the subunits (tubulin) that com- poses the closed ring polymers, unlike the open-ended supramolecular structures such as microtubules and actin filaments. We combine measurements of these rings and their protein subunits, obtained by two independent tech- niques: fluorescence correlation spectroscopy (FCS) [10,12–15] and sedimentation velocity analytical ultra- centrifugation (SV) [16]. We show precise quantitative agreement between the ratios of the diffusion coefficients of these supramolecules and those of their sedimentation coefficients. More importantly, we make use of the pre- cision of the ratios to test the applicability of theoretical analyses that relate the structure of nanoscopic objects of complex shape to their hydrodynamic properties. The -tubulin dimer, which is the basic building block of tubulin polymers, is a protein composed of two subunits differing in sequence but having very similar shape (width: 4.6 nm; height: 4.0 nm; depth: 6.5 nm) and molecular weight [17]. Tubulin is ubiquitously found in eukaryotic cells and is the principal constituent of micro- tubules, which are primary components of several criti- cally important cytoskeletal structures [18]. However, cryptophycin, a cyclic depsipeptide obtained from cya- nobacteria [7], causes significant depolymerization of microtubules and induces, instead, the formation of closed rings (  24:8 nm diameter [6,10]). The rings con- tain eight tubulin dimers (see Fig. 1 inset) and, for the solution conditions in this investigation, only individual tubulin dimers and closed rings seem to be present. FCS and SV measurements yield, respectively, the dis- tributions of the hydrodynamic diameters and sedimen- tation coefficients of the polymeric structures in the cryptophycin-tubulin solutions. These can be compared FIG. 1. Distributions of the sedimentation coefficients deter- mined by inversion of SV centrifugation profiles measured on tubulin and tubulin-cryptophycin solutions at 25000 rpm and 50 000 rpm, respectively, (Svedberg  10 13 sec). Inset shows an averaged image of cryptophycin-tubulin rings from cryo- electron microscopy [6]. The box encloses one tubulin dimer and the bar represents 10 nm. VOLUME 93, NUMBER 9 PHYSICAL REVIEW LETTERS week ending 27 AUGUST 2004 098106-1 0031-9007= 04=93(9)=098106(4)$22.50 098106-1