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Gerald Stubbs - One of the best experts on this subject based on the ideXlab platform.
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Fiber Diffraction data indicate a hollow core for the Alzheimer's aβ 3-fold symmetric fibril.
Journal of molecular biology, 2012Co-Authors: Michele Mcdonald, Amy Kendall, Wen Bian, Hayden Box, Robert Tycko, Gerald StubbsAbstract:Amyloid β protein (Aβ), the principal component of the extracellular plaques found in the brains of patients with Alzheimer's disease, forms fibrils well suited to structural study by X-ray Fiber Diffraction. Fiber Diffraction patterns from the 40-residue form Aβ(1–40) confirm a number of features of a 3-fold symmetric Aβ model from solid‐state NMR (ssNMR) but suggest that the fibrils have a hollow core not present in the original ssNMR models. Diffraction patterns calculated from a revised 3-fold hollow model with a more regular β-sheet structure are in much better agreement with the observed Diffraction data than patterns calculated from the original ssNMR model. Refinement of a hollow-core model against ssNMR data led to a revised ssNMR model, similar to the Fiber Diffraction model.
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Structure of hibiscus latent singapore virus by Fiber Diffraction: a nonconserved his122 contributes to coat protein stability.
Journal of molecular biology, 2010Co-Authors: Sunil Kumar Tewary, Amy Kendall, Wen Bian, Gerald Stubbs, Toshiro Oda, Sek-man Wong, Kunchithapadam SwaminathanAbstract:Abstract Hibiscus latent Singapore virus (HLSV) is a rigid rod-shaped plant virus and a new member of the Tobamovirus family. Unlike all other Tobamoviruses , the HLSV genome contains a unique poly(A) tract in its 3′ untranslated region. The virion is composed of a monomeric coat protein (CP) unit of 18 kDa, arranged as a right-handed helix around the virus axis. We have determined the structure of HLSV at 3.5 A by X-ray Fiber Diffraction and refined it to an R -factor of 0.096. While the overall structure of the HLSV CP resembles that of other Tobamoviruses , there are a few unique differences. There is a kink in the LR helix due to the presence of His122. Also, the adjacent Lys123 may further destabilize the helix by positive charge repulsion, making the kink more pronounced. The His122-Asp88 salt bridge provides significant stability to the loop adjacent to the RR helix. Carboxyl–carboxylate interactions that drive viral disassembly are also different in HLSV. The nucleotide recognition mechanisms for virus assembly between HLSV and ribgrass mosaic virus are similar, but different between tobacco mosaic virus and cucumber green mottle mosaic virus.
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Enclosed chambers for humidity control and sample containment in Fiber Diffraction
Journal of Applied Crystallography, 2008Co-Authors: Michele Mcdonald, Amy Kendall, Motomasa Tanaka, Jonathan S. Weissman, Gerald StubbsAbstract:A chamber and stretch frame for making Fibers for Diffraction is described. The chamber is made from a simple plastic cuvette with silicon nitride windows. It is suitable for maintaining constant humidity during Fiber drying and data collection, and allows stretching of the Fiber and exposure to magnetic fields during sample preparation. If necessary, it provides primary containment for toxic and infectious biological materials. The chamber has been used in Fiber Diffraction experiments with filamentous plant viruses and a yeast prion protein, and is shown to produce excellent orientation and to maintain hydration and order at the molecular level.
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Flexible filamentous virus structures from Fiber Diffraction
Powder Diffraction, 2008Co-Authors: Gerald Stubbs, Amy Kendall, Wen Bian, Ian Mccullough, Michele Mcdonald, Timothy M. Bowles, Sarah C. Baumgarten, Jian Shi, Phoebe L. Stewart, Esther BullittAbstract:Fiber Diffraction data have been obtained from Narcissus mosaic virus, a potexvirus from the family Flexiviridae, and soybean mosaic virus (SMV), a potyvirus from the family Potyviridae. Analysis of the data in conjunction with cryo-electron microscopy data allowed us to determine the symmetry of the viruses and to make reconstructions of SMV at 19 {angstrom} resolution and of another potexvirus, papaya mosaic virus, at 18 {angstrom} resolution. These data include the first well-ordered data ever obtained for the potyviruses and the best-ordered data from the potexviruses, and offer the promise of eventual high resolution structure determinations.
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WCEN: a computer program for initial processing of Fiber Diffraction patterns
Journal of Applied Crystallography, 2006Co-Authors: Wen Bian, Ian Mccullough, Hong Wang, Gerald StubbsAbstract:Processing of Fiber Diffraction patterns is generally more difficult than for single-crystal patterns, and requires different algorithms and software. The program WCEN has been developed to determine experimental and specimen parameters and to convert Diffraction data from detector to reciprocal space, and offers a variety of input and output formats, running under Mac OS X and Linux. The program is described and examples from oriented sols of filamentous plant viruses, illustrating different strategies for parameter determination and refinement, are given.
Rick P. Millane - One of the best experts on this subject based on the ideXlab platform.
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Modelling X-ray Diffraction From The Myosin Superlattice Of Vertebrate Muscle
Biophysical Journal, 2009Co-Authors: David H. Wojtas, Rick P. Millane, C.h. Yoon, John M. SquireAbstract:Muscular force is generated by molecular interactions between the contractile proteins actin and myosin. The myosin filaments in the sarcomere of vertebrate muscle pack on a triangular array into which the actin filaments are interdigitated. High resolution studies of the actin-myosin interactions are performed by x-ray Fiber Diffraction analysis of whole muscle Fibers. In most vertebrate muscles however, the myosin filaments pack in a so-called “superlattice” arrangement that involves a semi-random distribution of two filament rotations. The unknown effects of this disorder on Diffraction by muscle fibres have so far prevented a rigorous analysis of x-ray Fiber Diffraction patterns in terms of the structure of this complex system. We report a quantitative model of the disorder and its incorporation into calculations of x-ray Fiber Diffraction patterns from model structures. This allows rapid calculation of the Diffraction and does not involve numerical averaging over the disorder. Calculations show that the disorder modulates the Bragg reflections in Diffraction patterns and introduces diffuse Diffraction. The results of this analysis will allow the effects of the disorder to be included in muscle structure refinement programs, allowing more accurate structure determination from x-ray Fiber Diffraction data.
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CPRL: a program to plot the cylindrically projected reciprocal lattice for Fiber Diffraction patterns
Journal of Applied Crystallography, 1999Co-Authors: Victoria L. Finkenstadt, J. L. Van Der Plas, Rick P. MillaneAbstract:The positions of reflections on the Diffraction pattern from a polycrystalline Fiber are described by a cylindrical projection of the reciprocal lattice. The characteristics of the projection depend on the crystal system and the orientation of the Fiber axis relative to the unit-cell axes. The program CPRL plots the positions of the reflections on a Diffraction pattern from an oriented polycrystalline Fiber (the cylindrically projected reciprocal lattice), for a triclinic unit cell with any orientation of the Fiber axis. The results provided by the program facilitate interpretation of Fiber Diffraction patterns for complex systems.
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MODEL BIAS IN BAYESIAN IMAGE RECONSTRUCTION FROM X-RAY Fiber Diffraction DATA
Journal of the Optical Society of America A, 1999Co-Authors: Shyamsunder Baskaran, Rick P. MillaneAbstract:An image-reconstruction problem in x-ray Fiber Diffraction analysis (which is used to determine the atomic structure of biopolymers) is considered. The problem is to reconstruct an image (the electron density function) given data that are squared sums of the Fourier coefficients of the image as well as partial information (the model) on the image. A Bayesian estimation approach based on a prior for the missing part of the image is considered. Current (heuristic) approaches to this problem correspond to certain maximum a posteriori estimates. These estimates exhibit bias toward the model, and current methods to reduce the bias are based on scaling of the Fourier coefficients. A new procedure to remove bias, based on orthogonalization, is derived and shown by simulations to be superior to scaling. Bias and unbiasing are compared for the different maximum a posteriori estimates, for different amounts of missing information. These results are also compared with a new minimum mean-square-error estimate for this problem that has the form of weighted maximum a posteriori Fourier coefficients. The minimum mean-square-error estimate is free from bias and gives results superior to the unbiased maximum a posteriori estimates.
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Bayesian image reconstruction with a hyperellipsoidal posterior in x-ray Fiber Diffraction
Bayesian Inference for Inverse Problems, 1998Co-Authors: Shyamsunder Baskaran, Rick P. MillaneAbstract:The structure completion problem in x-ray Fiber Diffraction is addressed from a Bayesian perspective. The experimental data are sums of the squares of the amplitudes of particular sets of Fourier coefficients of the electron density. In addition, a part of the electron density. In addition, a part of the electron density is known. The image reconstruction problem is to estimate the missing part of the electron density. A Bayesian approach is taken in which the prior model for the image is based on the fact that it consists of atoms, i.e., the unknown electron density consists of separated sharp peaks. The posterior for the Fourier coefficients typically takes the form of an independent and identically distributed multivariate normal density restricted to the surface of a hypersphere. However, the electron density often exhibits symmetry, in which case, the Fourier coefficient components are not longer independent or identically distributed. A diagonalization process results in an independent multivariate normal probability density function, restricted to a hyperspherical surface. the analytical form for the mean of the posterior density function is derived. The mean can be expressed as a weighting function on the Fourier coefficients of the known part of the electron density. The weighting function for the hyperellipsoidal and hyperspherical cases are compared.
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bayesian estimation in an image restoration problem in x ray Fiber Diffraction
International Conference on Acoustics Speech and Signal Processing, 1998Co-Authors: Subramanian Baskaran, Rick P. MillaneAbstract:The restoration of an incomplete image from a known part and experimental data in the form of the Fourier amplitude squared sums is formulated as a Bayesian estimation problem. This problem is motivated by the structure completion problem in X-ray Fiber Diffraction analysis. An appropriate prior of uniformly distributed impulses is used. The Bayesian MMSE and MAP estimates are obtained. Simulations are used to compare the performance of the estimates. The results show that the MMSE estimate significantly outperforms the other estimates. The restored images exhibit some bias towards the known part of the image. This can be partly reduced by an unbiasing procedure.
Amy Kendall - One of the best experts on this subject based on the ideXlab platform.
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Fiber Diffraction data indicate a hollow core for the Alzheimer's aβ 3-fold symmetric fibril.
Journal of molecular biology, 2012Co-Authors: Michele Mcdonald, Amy Kendall, Wen Bian, Hayden Box, Robert Tycko, Gerald StubbsAbstract:Amyloid β protein (Aβ), the principal component of the extracellular plaques found in the brains of patients with Alzheimer's disease, forms fibrils well suited to structural study by X-ray Fiber Diffraction. Fiber Diffraction patterns from the 40-residue form Aβ(1–40) confirm a number of features of a 3-fold symmetric Aβ model from solid‐state NMR (ssNMR) but suggest that the fibrils have a hollow core not present in the original ssNMR models. Diffraction patterns calculated from a revised 3-fold hollow model with a more regular β-sheet structure are in much better agreement with the observed Diffraction data than patterns calculated from the original ssNMR model. Refinement of a hollow-core model against ssNMR data led to a revised ssNMR model, similar to the Fiber Diffraction model.
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Structure of hibiscus latent singapore virus by Fiber Diffraction: a nonconserved his122 contributes to coat protein stability.
Journal of molecular biology, 2010Co-Authors: Sunil Kumar Tewary, Amy Kendall, Wen Bian, Gerald Stubbs, Toshiro Oda, Sek-man Wong, Kunchithapadam SwaminathanAbstract:Abstract Hibiscus latent Singapore virus (HLSV) is a rigid rod-shaped plant virus and a new member of the Tobamovirus family. Unlike all other Tobamoviruses , the HLSV genome contains a unique poly(A) tract in its 3′ untranslated region. The virion is composed of a monomeric coat protein (CP) unit of 18 kDa, arranged as a right-handed helix around the virus axis. We have determined the structure of HLSV at 3.5 A by X-ray Fiber Diffraction and refined it to an R -factor of 0.096. While the overall structure of the HLSV CP resembles that of other Tobamoviruses , there are a few unique differences. There is a kink in the LR helix due to the presence of His122. Also, the adjacent Lys123 may further destabilize the helix by positive charge repulsion, making the kink more pronounced. The His122-Asp88 salt bridge provides significant stability to the loop adjacent to the RR helix. Carboxyl–carboxylate interactions that drive viral disassembly are also different in HLSV. The nucleotide recognition mechanisms for virus assembly between HLSV and ribgrass mosaic virus are similar, but different between tobacco mosaic virus and cucumber green mottle mosaic virus.
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X-Ray Fiber Diffraction Reveals Major Structural Differences Between Brain-Derived Prions and Recombinant Prion Protein Amyloid
Biophysical Journal, 2010Co-Authors: Holger Wille, Amy Kendall, Wen Bian, Michele Mcdonald, David W. Colby, Lillian Bloch, Julian Ollesch, Alexander L. Borovinskiy, Fred E. Cohen, Stanley B. PrusinerAbstract:X-ray Fiber Diffraction was used to study the structure of brain-derived prions and recombinant prion protein amyloid. Partially oriented, dried Fibers were prepared from brain-derived PrP 27-30 and recombinant PrP amyloid. Fiber Diffraction patterns were analyzed and used to interrogate models for the structure of the infectious prion.Fiber Diffraction patterns of recombinant PrP amyloid displayed characteristic, meridional reflections at ∼4.8 A and equatorial reflections at ∼10 A. These patterns were similar to those of other amyloids and are consistent with a basic cross-β architecture. In contrast, Diffraction patterns from brain-derived PrP 27-30 displayed meridional reflections at ∼9.6, ∼6.4, and ∼4.8 A, which correspond to the 2nd, 3rd, and 4th order of a ∼19.2-A repeating unit, suggesting that PrP 27-30 contains four β-strands in a cross-β architecture. Furthermore, PrP 27-30 lacked the typical, equatorial reflection at ∼10 A, but instead produced equatorial reflections characterizing the diameter of the amyloid Fiber and of individual protofilaments. Therefore, PrP 27-30 seems to have a structure consistent with a β-helix or β-solenoid, not unlike the model that was proposed earlier (Govaerts et al., 2004). This interpretation was also supported by extensive modeling, simulation of Diffraction, electron microscopy, and FTIR.In a previous study, recombinant PrP amyloid induced a transmissible prion disease in transgenic mice overexpressing PrP, and was thus termed a "synthetic prion" (Legname et al., 2004). Serially transmitted, synthetic prions were purified from mouse brains and analyzed by Fiber Diffraction. These brain-derived, synthetic prions showed the same structural characteristics as natural prion isolates and not those of its recombinant protein precursor. The relationship between structural differences and prion infectivity can be explained by several hypotheses. It remains to be determined which one, if any, is correct.
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natural and synthetic prion structure from x ray Fiber Diffraction
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: Holger Wille, Amy Kendall, Wen Bian, Michele Mcdonald, David W. Colby, Lillian Bloch, Julian Ollesch, Alexander L. Borovinskiy, Fred E. Cohen, Stanley B. PrusinerAbstract:A conformational isoform of the mammalian prion protein (PrP Sc ) is the sole component of the infectious pathogen that causes the prion diseases. We have obtained X-ray Fiber Diffraction patterns from infectious prions that show cross-β Diffraction: meridional intensity at 4.8 A resolution, indicating the presence of β strands running approximately at right angles to the filament axis and characteristic of amyloid structure. Some of the patterns also indicated the presence of a repeating unit along the Fiber axis, corresponding to four β-strands. We found that recombinant (rec) PrP amyloid differs substantially from highly infectious brain-derived prions, both in structure as demonstrated by the Diffraction data, and in heterogeneity as shown by electron microscopy. In addition to the strong 4.8 A meridional reflection, the recPrP amyloid Diffraction is characterized by strong equatorial intensity at approximately 10.5 A, absent from brain-derived prions, and indicating the presence of stacked β-sheets. Synthetic prions recovered from transgenic mice inoculated with recPrP amyloid displayed structural characteristics and homogeneity similar to those of naturally occurring prions. The relationship between the structural differences and prion infectivity is uncertain, but might be explained by any of several hypotheses: only a minority of recPrP amyloid possesses a replication-competent conformation, the majority of recPrP amyloid has to undergo a conformational maturation to acquire replication competency, or inhibitory forms of recPrP amyloid interfere with replication during the initial transmission.
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Enclosed chambers for humidity control and sample containment in Fiber Diffraction
Journal of Applied Crystallography, 2008Co-Authors: Michele Mcdonald, Amy Kendall, Motomasa Tanaka, Jonathan S. Weissman, Gerald StubbsAbstract:A chamber and stretch frame for making Fibers for Diffraction is described. The chamber is made from a simple plastic cuvette with silicon nitride windows. It is suitable for maintaining constant humidity during Fiber drying and data collection, and allows stretching of the Fiber and exposure to magnetic fields during sample preparation. If necessary, it provides primary containment for toxic and infectious biological materials. The chamber has been used in Fiber Diffraction experiments with filamentous plant viruses and a yeast prion protein, and is shown to produce excellent orientation and to maintain hydration and order at the molecular level.
David A. Middleton - One of the best experts on this subject based on the ideXlab platform.
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Cross-beta spine architecture of fibrils formed by the amyloidogenic segment NFGSVQFV of medin from solid-state NMR and X-ray Fiber Diffraction measurements.
Biochemistry, 2009Co-Authors: Jillian Madine, Louise C. Serpell, Alastair Copland, David A. MiddletonAbstract:Over 30 polypeptides are known to assemble into highly ordered fibrils associated with pathological disorders known collectively as amyloidoses. Structural studies of short model peptides are beginning to reveal trends in the types of molecular interactions that drive aggregation and stabilize the packing of s-sheet layers within fibrillar assemblies. This work investigates the molecular architecture of fibrils formed by the peptide AMed42-49 representing residues 42-49 of the 50 amino acid polypeptide medin associated with aortic medial amyloid, the most common form of senile localized amyloid. The peptide aggregates within 2 days to form bundles of microcrystalline-like needles displaying a high degree of order. Fibrils were prepared from peptides containing up to 23 13C labels, and the solid-state nuclear magnetic resonance (SSNMR) method rotational resonance (RR) was used to determine constraints on the distances between selective atomic sites within fibrils. The constraints are consistent with unbroken s-strands hydrogen bonded in a parallel in-register arrangement within s-sheets. Further RR measurements identify close (>6.5 A) contacts between residues F43 and V46 and between S45 and V46, which can only occur between s-sheet layers and which are consistent with two principal models of s-sheet arrangements. X-ray Fiber Diffraction from partially aligned fibrils revealed the classical amyloid Diffraction pattern, and comparison of patterns calculated from model coordinates with experimental data allowed determination of a consistent molecular model.
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structural insights into the polymorphism of amyloid like fibrils formed by region 20 29 of amylin revealed by solid state nmr and x ray Fiber Diffraction
Journal of the American Chemical Society, 2008Co-Authors: Jillian Madine, Louise C. Serpell, Edward R. Jack, Peter G. Stockley, Sheena E. Radford, David A. MiddletonAbstract:Many unrelated proteins and peptides can assemble into amyloid or amyloid-like nanostructures, all of which share the cross-beta motif of repeat arrays of beta-strands hydrogen-bonded along the fibril axis. Yet, paradoxically, structurally polymorphic fibrils may derive from the same initial polypeptide sequence. Here, solid-state nuclear magnetic resonance (SSNMR) analysis of amyloid-like fibrils of the peptide hIAPP 20-29, corresponding to the region S (20)NNFGAILSS (29) of the human islet amyloid polypeptide amylin, reveals that the peptide assembles into two amyloid-like forms, (1) and (2), which have distinct structures at the molecular level. Rotational resonance SSNMR measurements of (13)C dipolar couplings between backbone F23 and I26 of hIAPP 20-29 fibrils are consistent with form (1) having parallel beta-strands and form (2) having antiparallel strands within the beta-sheet layers of the protofilament units. Seeding hIAPP 20-29 with structurally homogeneous fibrils from a 30-residue amylin fragment (hIAPP 8-37) produces morphologically homogeneous fibrils with similar NMR properties to form (1). A model for the architecture of the seeded fibrils is presented, based on the analysis of X-ray Fiber Diffraction data, combined with an extensive range of SSNMR constraints including chemical shifts, torsional angles, and interatomic distances. The model features a cross-beta spine comprising two beta-sheets with an interface defined by residues F23, A25, and L27, which form a hydrophobic zipper. We suggest that the energies of formation for fibril form containing antiparallel and parallel beta-strands are similar when both configurations can be stabilized by a core of hydrophobic contacts, which has implications for the relationship between amino acid sequence and amyloid polymorphism in general.
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Structural Insights into the Polymorphism of Amyloid-Like Fibrils Formed by Region 20−29 of Amylin Revealed by Solid-State NMR and X-ray Fiber Diffraction
Journal of the American Chemical Society, 2008Co-Authors: Jillian Madine, Louise C. Serpell, Edward R. Jack, Peter G. Stockley, Sheena E. Radford, David A. MiddletonAbstract:Many unrelated proteins and peptides can assemble into amyloid or amyloid-like nanostructures, all of which share the cross-beta motif of repeat arrays of beta-strands hydrogen-bonded along the fibril axis. Yet, paradoxically, structurally polymorphic fibrils may derive from the same initial polypeptide sequence. Here, solid-state nuclear magnetic resonance (SSNMR) analysis of amyloid-like fibrils of the peptide hIAPP 20-29, corresponding to the region S (20)NNFGAILSS (29) of the human islet amyloid polypeptide amylin, reveals that the peptide assembles into two amyloid-like forms, (1) and (2), which have distinct structures at the molecular level. Rotational resonance SSNMR measurements of (13)C dipolar couplings between backbone F23 and I26 of hIAPP 20-29 fibrils are consistent with form (1) having parallel beta-strands and form (2) having antiparallel strands within the beta-sheet layers of the protofilament units. Seeding hIAPP 20-29 with structurally homogeneous fibrils from a 30-residue amylin fragment (hIAPP 8-37) produces morphologically homogeneous fibrils with similar NMR properties to form (1). A model for the architecture of the seeded fibrils is presented, based on the analysis of X-ray Fiber Diffraction data, combined with an extensive range of SSNMR constraints including chemical shifts, torsional angles, and interatomic distances. The model features a cross-beta spine comprising two beta-sheets with an interface defined by residues F23, A25, and L27, which form a hydrophobic zipper. We suggest that the energies of formation for fibril form containing antiparallel and parallel beta-strands are similar when both configurations can be stabilized by a core of hydrophobic contacts, which has implications for the relationship between amino acid sequence and amyloid polymorphism in general.
Katsuzo Wakabayashi - One of the best experts on this subject based on the ideXlab platform.
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X-ray Fiber Diffraction modeling of structural changes of the thin filament upon activation of live vertebrate skeletal muscles.
Biophysics (Nagoya-shi Japan), 2010Co-Authors: Tatsuhito Matsuo, Yasunobu Sugimoto, Yasunori Takezawa, Yutaka Ueno, Toshiro Oda, Katsuzo WakabayashiAbstract:In order to clarify the structural changes of the thin filaments related to the regulation mechanism in skeletal muscle contraction, the intensities of thin filament-based reflections in the X-ray Fiber Diffraction patterns from live frog skeletal muscles at non-filament overlap length were investigated in the relaxed state and upon activation. Modeling the structural changes of the whole thin filament due to Ca2+-activation was systematically performed using the crystallographic data of constituent molecules (actin, tropomyosin and troponin core domain) as starting points in order to determine the structural changes of the regulatory proteins and actin. The results showed that the globular core domain of troponin moved toward the filament axis by ∼6 A and rotated by ∼16° anticlockwise (viewed from the pointed end) around the filament axis by Ca2+-binding to troponin C, and that tropomyosin together with the tail of troponin T moved azimuthally toward the inner domains of actin by ∼12° and radially by ∼7 A from the relaxed position possibly to partially open the myosin binding region of actin. The domain structure of the actin molecule in F-actin we obtained for frog muscle thin filament was slightly different from that of the Holmes F-actin model in the relaxed state, and upon activation, all subdomains of actin moved in the direction to closing the nucleotide-binding pocket, making the actin molecule more compact. We suggest that the troponin movements and the structural changes within actin molecule upon activation are also crucial components of the regulation mechanism in addition to the steric blocking movement of tropomyosin.
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Quick Shear-Flow Alignment of Biological Filaments for X-ray Fiber Diffraction Facilitated by Methylcellulose
Biophysical journal, 2009Co-Authors: Takaaki Sugiyama, Katsuzo Wakabayashi, Yasunobu Sugimoto, Daisuke Miyashiro, Daisuke Takao, Hiroyuki Iwamoto, Shinji KamimuraAbstract:X-ray Fiber Diffraction is one of the most useful methods for examining the structural details of live biological filaments under physiological conditions. To investigate biologically active or labile materials, it is crucial to finish Fiber alignment within seconds before Diffraction analysis. However, the conventional methods, e.g., magnetic field alignment and low-speed centrifugations, are time-consuming and not very useful for such purposes. Here, we introduce a new alignment method using a rheometer with two parallel disks, which was applied to observe Fiber Diffractions of axonemes, tobacco mosaic tobamovirus, and microtubules. We found that Fibers were aligned within 5 s by giving high shear flow (1000–5000 s−1) to the medium and that methylcellulose contained in the medium (∼1%) was essential to the accomplishment of uniform orientation with a small angular deviation (
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structural changes of the regulatory proteins bound to the thin filaments in skeletal muscle contraction by x ray Fiber Diffraction
Biochemical and Biophysical Research Communications, 2008Co-Authors: Yasunobu Sugimoto, Yasunori Takezawa, Yutaka Ueno, Shiho Minakata, Tatsuhito Matsuo, Hidehiro Tanaka, Katsuzo WakabayashiAbstract:Abstract In order to clarify the structural changes related to the regulation mechanism in skeletal muscle contraction, the intensity changes of thin filament-based reflections were investigated by X-ray Fiber Diffraction. The time course and extent of intensity changes of the first to third order troponin (TN)-associated meridional reflections with a basic repeat of 38.4 nm were different for each of these reflections. The intensity of the first and second thin filament layer lines changed in a reciprocal manner both during initial activation and during the force generation process. The axial spacings of the TN-meridional reflections decreased by ∼0.1% upon activation relative to the relaxing state and increased by ∼0.24% in the force generation state, in line with that of the 2.7-nm reflection. Ca 2+ -binding to TN triggered the shortening and a change in the helical symmetry of the thin filaments. Modeling of the structural changes using the intensities of the thin filament-based reflections suggested that the conformation of the globular core domain of TN altered upon activation, undergoing additional conformational changes at the tension plateau. The tail domain of TN moved together with tropomyosin during contraction. The results indicate that the structural changes of regulatory proteins bound to the actin filaments occur in two steps, the first in response to the Ca 2+ -binding and the second induced by actomyosin interaction.
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structural alterations of thin actin filaments in muscle contraction by synchrotron x ray Fiber Diffraction
Advances in Experimental Medicine and Biology, 2007Co-Authors: Katsuzo Wakabayashi, Yasunobu Sugimoto, Yasunori Takezawa, Yutaka Ueno, Shiho Minakata, Kanji Oshima, Tatsuhito Matsuo, Takakazu KobayashiAbstract:Strong evidence has been accumulated that the conformational changes of the thin actin filaments are occurring and playing an important role in the entire process of muscle contraction. The conformational changes and the mechanical properties of the thin actin filaments we have found by X-ray Fiber Diffraction on skeletal muscle contraction are explored. Recent studies on the conformational changes of regulatory proteins bound to actin filaments upon activation and in the force generation process are also described. Finally, the roles of structural alterations and dynamics of the actin filaments are discussed in conjunction with the regulation mechanism and the force generation mechanism.