A new method for fibrous protein analysis illustrated by application to tubulin microtubule polymerisation and depolymerisation

Marrington, Rachel; Seymour, Mark; Rodger, Alison

doi:10.1002/chir.20305

Cited by 34 publications

(33 citation statements)

References 30 publications

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“…This is currently hindered by the quality of the absorbance data and the significant contribution made by scattering (which does not follow a simple wavelengthpower law). [21,22] It should be noted, however, that the situation in reality is almost certainly a mixture of binding modes. Something like the one proposed may be the dominant one.…”

Section: Resultsmentioning

confidence: 95%

The Binding of Single‐Stranded DNA and PNA to Single‐Walled Carbon Nanotubes Probed by Flow Linear Dichroism

Rajendra

Rodger

2005

Chemistry A European J

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The binding of single-stranded DNAs and a neutral DNA analogue (peptide nucleic acid, PNA) to single-walled carbon nanotubes in solution phase has been probed by absorbance spectroscopy and linear dichroism. The nanotubes are solubilised by aqueous sodium dodecyl sulfate, in which the nucleic acids also dissolve. The linear dichroism (LD) of the nanotubes, when subtracted from that due to the nanotubes/nucleic acid samples, gives the LD of the bound nucleic acid. The binding of the single-stranded DNA to the single-walled nanotubes is quite different from that previously observed for double-stranded DNA. It is likely that the nucleic acid bases lie flat on the nanotube surface with the backbone wrapping round the nanotube at an oblique angle in the region of 45 degrees . The net effect is like beads on a string. The base orientation with the single-stranded PNA is inverted with respect to that of the single-stranded DNA, as shown by their oppositely signed LD signals.

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Section: Resultsmentioning

confidence: 95%

The Binding of Single‐Stranded DNA and PNA to Single‐Walled Carbon Nanotubes Probed by Flow Linear Dichroism

Rajendra

Rodger

2005

Chemistry A European J

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“…LD Measurements of f210 Fibrils under Flow-LD had been used to investigate the orientation of absorbing chromophores within elongated molecules (23)(24)(25)(26)(27)(28). Although chiral compounds generate CD signals, oriented compounds either intrinsically or during the measurements often exhibit LD signals.…”

Section: Resultsmentioning

confidence: 99%

“…where A is the absorption of the fibrils, S is the orientation factor that defines the efficiency of the fibril orientation (S ϭ 1 and 0 for perfect and random orientations, respectively), and ␣ is the angle that the transition moment makes with the orientation axis (23)(24)(25)(26)(27)(28). The reduced LD spectrum indicated a dominant peak at 198.5 nm with LD r ϭ Ϫ0.28 (Fig.…”

Section: Orientation Of Functional Groups In Fibrils-mentioning

confidence: 99%

Flow-induced Alignment of Amyloid Protofilaments Revealed by Linear Dichroism

Adachi

Yamaguchi

Yagi

et al. 2007

Journal of Biological Chemistry

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Amyloid fibrils underlying various serious amyloidoses including Alzheimer and prion diseases form characteristic deposits in which linear fibrils with an unbranched and rigid morphology associate laterally or radially, e.g. radial senile amyloid plaques of amyloid ␤. To clarify the formation of these high order amyloid deposits, studying the rheology is important. A 22-residue K3 peptide fragment of ␤ 2 -microglobulin, a protein responsible for dialysis-related amyloidosis, forms long and homogeneous protofilament-like fibrils in 20% (v/v) 2,2,2-trifluoroethanol and 10 mM HCl (pH ϳ2). Here, using circular dichroism and linear dichroism, we observed the flow-induced alignment of fibrils. Analysis of far-and near-UV linear dichroism spectra suggested that both the net -* transition moment of the backbone carbonyl group and L b transition moment of the Tyr 26 side chain are oriented in parallel to the fibril axis, revealing the structural details of amyloid protofilaments. Moreover, the intensities of flow-induced circular dichroism or linear dichroism signals depended critically on the length and type of fibrils, suggesting that they are useful for detecting and characterizing amyloid fibrils.Although the structural features of amyloid fibrils are of urgent importance (1-3), the high molecular weight and noncrystalline assembly preclude the use of conventional methods, such as x-ray crystallography or multidimensional NMR, to acquire high resolution structural images. Recently, it has been found that amyloid fibrils are, in general, amenable to the magic angle-spinning solid-state nuclear magnetic resonance methods, providing a general view of the atomic level structure in which ␤-strands are stacked in a parallel and registered manner (4 -6). A structure revealed by x-ray diffraction of a crystalline short amyloidogenic peptide was consistent with this view (7).More recently, the mechanical characterization of individual fibrils by atomic force microscopy revealed that the insulin fibrils have a strength and stiffness comparable with that of steel and silk, respectively (8). Among various structural features, although the long and rigid morphology of amyloid fibrils might be responsible for their biological consequences, rheological details remain unknown.␤ 2 -Microglobulin (␤2-m), 4 a typical immunoglobulin domain made of 99 residues, is a target of extensive study because of its clinical importance and suitable size for examining the relationship between protein folding and amyloid formation (9 -14). Dialysis-related amyloidosis is a common and serious complication among patients on long term hemodialysis, in which amyloid fibrils of ␤2-m deposit in the synovia of the carpal tunnel (9, 15). A 22-residue K3 peptide, corresponding to Ser 20 -Lys 41 of ␤2-m forms protofilament-like fibrils in various solvents including a low concentration of 2,2,2-trifluoroethanol (16 -19). Although the efficient fibrillation of intact ␤2-m requires seeding, K3 forms fibrils spontaneously. Furthermore, K3 formed two types of fibr...

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“…The correction method used for light scattering is based on the correction for the background turbidity of brain microtubules described by J. Nordh et al [20] and modified by R. Marrington [21]. It is based on the equation LD scattering (λ) = a λ − k .…”

Section: Methodsmentioning

confidence: 99%

The pH Dependence of Polymerization and Bundling by the Essential Bacterial Cytoskeltal Protein FtsZ

et al. 2011

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There is a growing body of evidence that bacterial cell division is an intricate coordinated process of comparable complexity to that seen in eukaryotic cells. The dynamic assembly of Escherichia coli FtsZ in the presence of GTP is fundamental to its activity. FtsZ polymerization is a very attractive target for novel antibiotics given its fundamental and universal function. In this study our aim was to understand further the GTP-dependent FtsZ polymerization mechanism and our main focus is on the pH dependence of its behaviour. A key feature of this work is the use of linear dichroism (LD) to follow the polymerization of FtsZ monomers into polymeric structures. LD is the differential absorption of light polarized parallel and perpendicular to an orientation direction (in this case that provided by shear flow). It thus readily distinguishes between FtsZ polymers and monomers. It also distinguishes FtsZ polymers and less well-defined aggregates, which light scattering methodologies do not. The polymerization of FtsZ over a range of pHs was studied by right-angled light scattering to probe mass of FtsZ structures, LD to probe real-time formation of linear polymeric fibres, a specially developed phosphate release assay to relate guanosine triphosphate (GTP) hydrolysis to polymer formation, and electron microscopy (EM) imaging of reaction products as a function of time and pH. We have found that lowering the pH from neutral to 6.5 does not change the nature of the FtsZ polymers in solution—it simply facilitates the polymerization so the fibres present are longer and more abundant. Conversely, lowering the pH to 6.0 has much the same effect as introducing divalent cations or the FtsZ-associated protein YgfE (a putative ZapA orthologue in E. coli)—it stablizes associations of protofilaments.

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A new method for fibrous protein analysis illustrated by application to tubulin microtubule polymerisation and depolymerisation

Cited by 34 publications

References 30 publications

The Binding of Single‐Stranded DNA and PNA to Single‐Walled Carbon Nanotubes Probed by Flow Linear Dichroism

The Binding of Single‐Stranded DNA and PNA to Single‐Walled Carbon Nanotubes Probed by Flow Linear Dichroism

Flow-induced Alignment of Amyloid Protofilaments Revealed by Linear Dichroism

The pH Dependence of Polymerization and Bundling by the Essential Bacterial Cytoskeltal Protein FtsZ

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