Factors implicated in determining the structure of zinc tubulin‐sheets: Lateral tubulin–tubulin interaction is promoted by the presence of zinc

Torre, Javier de la; Villasanté, Alfredo; Corral, Javier; Ávila, Jesús

doi:10.1002/jsscb.380170208

Cited by 12 publications

(6 citation statements)

References 30 publications

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“…This technique has been used for tubulin sheets containing MAPs (16) but was unsuitable for MAPs-free sheets because of the interaction of an anionic tubulin domain (perhaps the carboxyl-terminal domain) with the mica support. (c) Several micrographs were screened by optical diffraction (13), and the pattern obtained for one of them is indicated (circle). Optical diffraction of catalase crystals in the same grids was done as an internal control of dimensions.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Involvement of the carboxyl-terminal domain of tubulin in the regulation of its assembly.

Serrano¹,

Torre²,

Maccioni³

et al. 1984

Proc. Natl. Acad. Sci. U.S.A.

244

205

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Limited proteolysis of phosphocellulose-purifled tubulin with subtilisin resulted in cleavage of both a and ,B tubulin subunits, with the formation of two major fragments (S., and Sp, 48 kDa) and a small peptide (4 kDa) containing the carboxyl-terminal region of tubulin. Interestingly, tubulin cleaved under the present conditions showed an increased ability to assemble into large polymers in the absence of MAPs and under conditions that do not promote assembly of undigested tubulin-i.e., low magnesium concentrations and the absence of taxol and polyalcohols. The critical concentrations for the subtilisin-cleaved tubulin assembly was similar to that of MAPs-promoted tubulin assembly. Assembly product from subtilisin-cleaved tubulin consisted mainly of protofiament bundles, hooked polymer, and open tubules, structures showing equatorial and longitudinal spacings of 50 and 40 A, respectively. The existence ofjunctions between polymer walls indicates that the carboxyl-terminal removal facilitates polymer-polymer interactions. These results, together with previous studies on the involvement of the carboxyl-terminal domain of tubulin in its interaction with MAP-2, suggest a regulatory role for this domain in tubulin assembly. Thus, in general terms the tubulin molecule can be analyzed as a protein containing two essential domains with functional significance, one domain playing a major role in self-association and the other (the carboxyl-terminal moiety) playing a regulatory role in modulating the interactions responsible for selfassociation.Brain tubulin prepared by cycles of assembly-disassembly contains other proteins that are designated microtubule-associated proteins (MAPs). Tubulin stripped of MAPs is unable to assemble except under certain conditions far removed from the physiological ones. Such conditions involve solvents containing glycerol, dimethyl sulfoxide, or polyethylene glycol, and usually the presence of high concentrations of magnesium (5-10 mM) or the addition of taxol or polycations, which may substitute for the requirements of MAPs (e.g., see ref. 1). Recently it has been shown that the binding of one of these proteins to tubulin is through a cationic domain of MAP-2 (2), which binds to the anionic domain present in the carboxyl terminus of the a and ,3 polypeptides of the tubulin molecule (3,4). In this report we show that the removal of the carboxyl-terminal portion of the purified tubulin by limited proteolysis with subtilisin results in a molecule that can self-assemble in the absence of MAPs or taxol and at protein concentrations similar to those required for undigested tubulin in the presence of MAPs. The resulting polymeric structures are mainly hooked microtubules, folded sheets, and protofilament bundles. MATERIAL AND METHODSPurification of Tubulin. Tubulin from pig brain was prepared by temperature-dependent cycles of assembly-disassembly by the procedure of Shelanski et al. (5) and was stored as pellets at -70'C. Immediately before use, the pellets were resuspended in 0.1 M 2-(N...

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Section: Resultsmentioning

confidence: 99%

“…analyzed by electrophoresis in NaDodSO4/polyacrylamide slab gels (7-12.5%) by the procedure of Laemmli (10) (12). Furthermore, assembled samples were placed on carbon/collodioncoated grids and stained with 1% (wt/wt) uranyl acetate as described and observed in a Jeol lOOB electron microscope (13).…”

Section: Methodsmentioning

confidence: 99%

Involvement of the carboxyl-terminal domain of tubulin in the regulation of its assembly.

Serrano¹,

Torre²,

Maccioni³

et al. 1984

Proc. Natl. Acad. Sci. U.S.A.

244

205

View full text Add to dashboard Cite

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“…Microtubules are the most common and important form of polymeric tubulin, in which the protein is arranged into rodlike protofilaments, which are associated laterally to form a hollow tube. However, in vitro tubulin has demonstrated the unique ability to polymerize into a wider variety of lesser known morphologies, including rings, ribbons, macrotubules, and sheets. − Many of these polymeric structures have been important in developing an understanding of the tubulin and microtubule structure, for example, identifying factors influencing the straight versus bent conformation of protofilaments − or the solution of the tubulin crystal structure . The present work focuses on immobilization of these two-dimensional (2D) crystals on solid supports for study with scanning probe microscopy and the resultant structural changes induced by substrate effects.…”

Section: Introductionmentioning

confidence: 99%

“…The assembly of tubulin into 2D crystals induced by the presence of zinc was first reported in 1976 and has been the subject of numerous studies over the past 40 years. − Although they have no known function in vivo, tubulin sheets have been essential in the study of microtubules. Because there are only minor differences in the tubulin structure in sheets compared to microtubules and because the protofilaments are nearly identical in both structures, sheets constitute a convenient and appealing system that has been exploited to study both dimeric and polymeric tubulin.…”

Section: Introductionmentioning

confidence: 99%

Structural Changes in Tubulin Sheets Caused by Immobilization on Solid Supports

et al. 2018

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In the presence of zinc, the protein tubulin assembles into two-dimensional sheets that are a useful model system for the study of both tubulin and microtubule structure. Tubulin sheets present an ideal protein structure for study with atomic force microscopy because they contain a two-dimensional crystalline protein lattice and retain many of the structural features of tubulin and microtubules. However, high-resolution imaging requires nonperturbative immobilization onto an appropriate imaging substrate. In this report, several substrates commonly used for scanning probe microscopy are evaluated for their ability to effectively immobilize tubulin sheets: mica, gold, highly ordered pyrolytic graphite, and carbon-coated electron microscopy grids. We hypothesize that the different intermolecular interactions presented by these substrates will affect the morphology of adsorbed tubulin sheets as well as the amount of other contaminating adsorbates. Tubulin sheets were successfully imaged on all of these substrates and structural characterization is reported. The most consistent results were obtained on carbon-coated electron microscopy grids, which preserved fine structural features of the sheets and had the least amount of contamination from the adsorption of unpolymerized tubulin. Images of tubulin sheets obtained with atomic force microscopy also compare favorably with published electron micrographs of sheets produced using similar procedures. This work demonstrates the importance of assessing substrate effects when studying two-dimensional protein crystals and identifies suitable substrates for immobilizing tubulin sheets.

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“…Lung tubulin polymers were examined by electron microscopy as described previously (de la Torre et al, 1981).…”

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confidence: 99%

Quantitative determination, isolation and characterization of pig lung tubulin

Diez¹,

Little²,

Ávila³

1984

Biochemical Journal

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Tubulin from pig lung was quantitatively determined, isolated and characterized. It accounted for about 0.3-0.4% of the total soluble protein of pig lung, as measured by colchicine binding or radioimmunoassay. Purified tubulin was obtained by several cycles of polymerization and depolymerization in the presence of dimethyl sulphoxide and 2H2O as stabilizing agents. The proteolytic cleavage patterns of the lung tubulin subunits closely resembled those of other mammalian cytoplasmic tubulin subunits, such as those of brain and kidney. However, the pattern of lung isotubulins on isoelectric focusing differed substantially from that of brain isotubulins . These differences did not appear to be the result of major lung tubulin post-translational modifications, since approximately the same pattern of isotubulins was found for the tubulin synthesized by lung poly(A)-containing mRNA in a reticulocyte system in vitro.

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Factors implicated in determining the structure of zinc tubulin‐sheets: Lateral tubulin–tubulin interaction is promoted by the presence of zinc

Cited by 12 publications

References 30 publications

Involvement of the carboxyl-terminal domain of tubulin in the regulation of its assembly.

Involvement of the carboxyl-terminal domain of tubulin in the regulation of its assembly.

Structural Changes in Tubulin Sheets Caused by Immobilization on Solid Supports

Quantitative determination, isolation and characterization of pig lung tubulin

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