Journal of Structural Biology

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  • Holoenzyme structures of endothelial nitric oxide synthase – An allosteric role for calmodulin in pivoting the FMN domain for electron transfer
    [Oct 2014]

    Publication date: October 2014
    Source:Journal of Structural Biology, Volume 188, Issue 1

    Author(s): Niels Volkmann , Pavel Martásek , Linda J. Roman , Xiao-Ping Xu , Christopher Page , Mark Swift , Dorit Hanein , Bettie Sue Masters

    While the three-dimensional structures of heme- and flavin-binding domains of the NOS isoforms have been determined, the structures of the holoenzymes remained elusive. Application of electron cryo-microscopy and structural modeling of the bovine endothelial nitric oxide synthase (eNOS) holoenzyme produced detailed models of the intact holoenzyme in the presence and absence of Ca2+/calmodulin (CaM). These models accommodate the cross-electron transfer from the reductase in one monomer to the heme in the opposite monomer. The heme domain acts as the anchoring dimeric structure for the entire enzyme molecule, while the FMN domain is activated by CaM to move flexibly to bridge the distance between the reductase and oxygenase domains. Our results indicate that the key regulatory role of CaM involves the stabilization of structural intermediates and precise positioning of the pivot for the FMN domain tethered shuttling motion to accommodate efficient and rapid electron transfer in the homodimer of eNOS.





    Categories: Journal Articles
  • Nanoscale three-dimensional imaging of the human myocyte
    [Oct 2014]

    Publication date: October 2014
    Source:Journal of Structural Biology, Volume 188, Issue 1

    Author(s): Matthew S. Sulkin , Fei Yang , Katherine M. Holzem , Brandon Van Leer , Cliff Bugge , Jacob I. Laughner , Karen Green , Igor R. Efimov

    The ventricular human myocyte is spatially organized for optimal ATP and Ca2+ delivery to sarcomeric myosin and ionic pumps during every excitation–contraction cycle. Comprehension of three-dimensional geometry of the tightly packed ultrastructure has been derived from discontinuous two-dimensional images, but has never been precisely reconstructed or analyzed in human myocardium. Using a focused ion beam scanning electron microscope, we created nanoscale resolution serial images to quantify the three-dimensional ultrastructure of a human left ventricular myocyte. Transverse tubules (t-tubule), lipid droplets, A-bands, and mitochondria occupy 1.8, 1.9, 10.8, and 27.9% of the myocyte volume, respectively. The complex t-tubule system has a small tortuosity (1.04±0.01), and is composed of long transverse segments with diameters of 317±24nm and short branches. Our data indicates that lipid droplets located well beneath the sarcolemma are proximal to t-tubules, where 59% (13 of 22) of lipid droplet centroids are within 0.50μm of a t-tubule. This spatial association could have an important implication in the development and treatment of heart failure because it connects two independently known pathophysiological alterations, a substrate switch from fatty acids to glucose and t-tubular derangement.





    Categories: Journal Articles
  • Visualisation by high resolution synchrotron X-ray phase contrast micro-tomography of gas films on submerged superhydrophobic leaves
    [Oct 2014]

    Publication date: October 2014
    Source:Journal of Structural Biology, Volume 188, Issue 1

    Author(s): Torsten Lauridsen , Kyriaki Glavina , Timothy David Colmer , Anders Winkel , Sarah Irvine , Kim Lefmann , Robert Feidenhans’l , Ole Pedersen

    Floods can completely submerge terrestrial plants but some wetland species can sustain O2 and CO2 exchange with the environment via gas films forming on superhydrophobic leaf surfaces. We used high resolution synchrotron X-ray phase contrast micro-tomography in a novel approach to visualise gas films on submerged leaves of common cordgrass (Spartina anglica). 3D tomograms enabled a hitherto unmatched level of detail regarding the micro-topography of leaf gas films. Gas films formed only on the superhydrophobic adaxial leaf side (water droplet contact angle, Φ =162°) but not on the abaxial side (Φ =135°). The adaxial side of the leaves of common cordgrass is plicate with a longitudinal system of parallel grooves and ridges and the vast majority of the gas film volume was found in large ∼180μm deep elongated triangular volumes in the grooves and these volumes were connected to each neighbouring groove via a fine network of gas tubules (∼1.7μm diameter) across the ridges. In addition to the gas film retained on the leaf exterior, the X-ray phase contrast micro-tomography also successfully distinguished gas spaces internally in the leaf tissues, and the tissue porosity (gas volume per unit tissue volume) ranged from 6.3% to 20.3% in tip and base leaf segments, respectively. We conclude that X-ray phase contrast micro-tomography is a powerful tool to obtain quantitative data of exterior gas features on biological samples because of the significant difference in electron density between air, biological tissues and water.





    Categories: Journal Articles
  • Robust and low cost uniform 15N-labeling of proteins expressed in Drosophila S2 cells and Spodoptera frugiperda Sf9 cells for NMR applications
    [Oct 2014]

    Publication date: October 2014
    Source:Journal of Structural Biology, Volume 188, Issue 1

    Author(s): Annalisa Meola , Célia Deville , Scott A. Jeffers , Pablo Guardado-Calvo , Ieva Vasiliauskaite , Christina Sizun , Christine Girard-Blanc , Christian Malosse , Carine van Heijenoort , Julia Chamot-Rooke , Thomas Krey , Eric Guittet , Stéphane Pêtres , Félix A. Rey , François Bontems

    Nuclear magnetic resonance spectroscopy is a powerful tool to study structural and functional properties of proteins, provided that they can be enriched in stable isotopes such as 15N, 13C and 2H. This is usually easy and inexpensive when the proteins are expressed in Escherichia coli, but many eukaryotic (human in particular) proteins cannot be produced this way. An alternative is to express them in insect cells. Labeled insect cell growth media are commercially available but at prohibitive prices, limiting the NMR studies to only a subset of biologically important proteins. Non-commercial solutions from academic institutions have been proposed, but none of them is really satisfying. We have developed a 15N-labeling procedure based on the use of a commercial medium depleted of all amino acids and supplemented with a 15N-labeled yeast autolysate for a total cost about five times lower than that of the currently available solutions. We have applied our procedure to the production of a non-polymerizable mutant of actin in Sf9 cells and of fragments of eukaryotic and viral membrane fusion proteins in S2 cells, which typically cannot be produced in E. coli, with production yields comparable to those obtained with standard commercial media. Our results support, in particular, the putative limits of a self-folding domain within a viral glycoprotein of unknown structure.





    Categories: Journal Articles
  • Dynamics of bovine opsin bound to G-protein fragments
    [Oct 2014]

    Publication date: October 2014
    Source:Journal of Structural Biology, Volume 188, Issue 1

    Author(s): Minoru Sugihara , Makiko Suwa , Ana-Nicoleta Bondar

    G protein-coupled receptors (GPCRs) are a large class of membrane proteins that mediate communication of the cell with the outer environment. Upon activation by an agonist, GPCRs undergo large-scale conformational changes that enable binding of the G protein to the receptor. A key open question concerns the mechanism of the long-distance coupling between the agonist-binding site and the cytoplasmic site where G protein binds. Here we address this question by exploring the molecular dynamics of bovine opsin bound to three different fragments of G-proteins. We find that an extended network of hydrogen bonds connects the agonist retinal binding site to the G protein binding site via conserved amino acid residues. The dynamics of the hydrogen-bonding network inside opsin couples to interactions at the G protein binding site.





    Categories: Journal Articles
  • 2D and 3D crystallization of a bacterial homologue of human vitamin C membrane transport proteins
    [Oct 2014]

    Publication date: October 2014
    Source:Journal of Structural Biology, Volume 188, Issue 1

    Author(s): Jean-Marc Jeckelmann , Daniel Harder , Zöhre Ucurum , Dimitrios Fotiadis

    Most organisms are able to synthesize vitamin C whereas humans are not. In order to contribute to the elucidation of the molecular working mechanism of vitamin C transport through biological membranes, we cloned, overexpressed, purified, functionally characterized, and 2D- and 3D-crystallized a bacterial protein (UraDp) with 29% of amino acid sequence identity to the human sodium-dependent vitamin C transporter 1 (SVCT1). Ligand-binding experiments by scintillation proximity assay revealed that uracil is a substrate preferably bound to UraDp. For structural analysis, we report on the production of tubular 2D crystals and present a first projection structure of UraDp from negatively stained tubes. On the other hand the successful growth of UraDp 3D crystals and their crystallographic analysis is described. These 3D crystals, which diffract X-rays to 4.2Å resolution, pave the way towards the high-resolution crystal structure of a bacterial homologue with high amino acid sequence identity to human SVCT1.





    Categories: Journal Articles
  • Changes in tissue directionality reflect differences in myelin content after demyelination in mice spinal cords
    [Oct 2014]

    Publication date: Available online 1 October 2014
    Source:Journal of Structural Biology

    Author(s): Mohammad K. Ansari , Heather Y.F. Yong , Luanne Metz , V. Wee Yong , Yunyan Zhang

    Changes in myelin integrity are key manifestations of many neurological diseases including multiple sclerosis but precise measurement of myelin in vivo is challenging. The goal of this study was to evaluate myelin content in histological images obtained from a lysolecithin mouse model of demyelination, using a new quantitative method named structure tensor analysis. Injury was targeted at the dorsal column of mice spinal cords. We obtained 16 histological images stained with luxol fast blue for myelin from 9 mice: 9 images from lesion epicenter and 7 from a distant area 500-μm away from the epicenter. In each image, we categorized 3 tissue types: healthy, completely demyelinated, and partially demyelinated. Structure tensor analysis was applied to quantify the coherency (anisotropy), energy (trace of dominant directions), and angular entropy (degree of disorder) of each tissue. We found that completely demyelinated lesions had significantly lower coherency and energy but higher angular entropy than partially demyelinated and healthy tissues at both the epicenter and distant areas of the injury. In addition, the coherency of healthy tissue was greater than partially demyelinated tissue at each site. Within tissue category, we did not find differences in any measure between spinal cord locations. Our findings suggest that greater myelin integrity is associated with better tissue anisotropy, independent of injury location. Structure tensor analysis may serve as a new tool for quantitative measurement of myelin content in white matter, and this may help understand disease mechanisms and development in MS and other demyelinating disorders.





    Categories: Journal Articles
  • Near-atomic resolution reconstructions using a mid-range electron microscope operated at 200kV
    [Oct 2014]

    Publication date: Available online 30 September 2014
    Source:Journal of Structural Biology

    Author(s): Melody G. Campbell , Bradley M. Kearney , Anchi Cheng , Clinton S. Potter , John E. Johnson , Bridget Carragher , David Veesler

    A new era has begun for single particle cryo-electron microscopy (cryoEM) which can now compete with X-ray crystallography for determination of protein structures. The development of direct detectors constitutes a revolution that has led to a wave of near-atomic resolution cryoEM reconstructions. However, regardless of the sample studied, virtually all high-resolution reconstructions reported to date have been achieved using high-end microscopes. We demonstrate that the new generation of direct detectors coupled to a widely used mid-range electron microscope also enables obtaining cryoEM maps of sufficient quality for de novo modeling of protein structures of different sizes and symmetries. We provide an outline of the strategy used to achieve a 3.7Å resolution reconstruction of Nudaurelia capensis ω virus and a 4.2Å resolution reconstruction of the Thermoplasma acidophilum T20S proteasome.





    Categories: Journal Articles
  • Designability landscape reveals sequence features that define axial helix rotation in four-helical homo-oligomeric antiparallel coiled-coil structures
    [Oct 2014]

    Publication date: Available online 30 September 2014
    Source:Journal of Structural Biology

    Author(s): Krzysztof Szczepaniak , Grzegorz Lach , Janusz M. Bujnicki , Stanislaw Dunin-Horkawicz

    Coiled coils are widespread protein domains comprising α-helices wound around each other in a regular fashion. Owing to their regularity, coiled-coil structures can be fully described by parametric equations. This in turn makes them an excellent model for studying sequence–structure relationships in proteins. Here, we used computational design to identify sequence features that determine the degree of helix axial rotation in four-helical homo-oligomeric antiparallel coiled coils. We designed 135,000 artificial sequences for a repertoire of backbone models representing all theoretically possible axial rotation states. Analysis of the designed sequences revealed features that precisely define the rotation of the helices. Based on these features we implemented a bioinformatic tool, which given a coiled-coil sequence, predicts the rotation of the helices in its structure. Moreover, we showed that another structural parameter, helix axial shift, is coupled to helix axial rotation and that dependence between these two parameters narrows the number of possible axial rotation states.





    Categories: Journal Articles
  • Revisiting the NMR solution structure of the Cel48S type-I dockerin module from Clostridium thermocellum reveals a cohesin-primed conformation
    [Oct 2014]

    Publication date: Available online 28 September 2014
    Source:Journal of Structural Biology

    Author(s): Chao Chen , Zhenling Cui , Yan Xiao , Qiu Cui , Steven P. Smith , Raphael Lamed , Edward A. Bayer , Yingang Feng

    Dockerin modules of the cellulosomal enzyme subunits play an important role in the assembly of the cellulosome by binding tenaciously to cohesin modules of the scaffoldin subunit. A previously reported NMR-derived solution structure of the type-I dockerin module from Cel48S of Clostridium thermocellum, which utilized two-dimensional homonuclear 1H-1H NOESY and three-dimensional 15N-edited NOESY distance restraints, displayed substantial conformational differences from subsequent structures of dockerin modules in complex with their cognate cohesin modules, raising the question whether the source of the observed differences resulted from cohesin-induced structural rearrangements. Here, we determined the solution structure of the Cel48S type-I dockerin based on 15N- and 13C-edited NOESY-derived distance restraints. The structure adopted a fold similar to X-ray crystal structures of dockerin modules in complex with their cohesin partners. A unique cis-peptide bond between Leu-65 and Pro-66 in the Cel48S type-I dockerin module was also identified in the present structure. Our structural analysis of the Cel48S type-I dockerin module indicates that it does not undergo appreciable cohesin-induced structural alterations but rather assumes an inherent calcium-dependent cohesin-primed conformation.





    Categories: Journal Articles
  • Hybrid Electron Microscopy Normal Mode Analysis graphical interface and protocol
    [Oct 2014]

    Publication date: Available online 27 September 2014
    Source:Journal of Structural Biology

    Author(s): Carlos Oscar S. Sorzano , José Miguel de la Rosa-Trevín , Florence Tama , Slavica Jonić

    This article presents an integral graphical interface to the Hybrid Electron Microscopy Normal Mode Analysis (HEMNMA) approach that was developed for capturing continuous motions of large macromolecular complexes from single-particle EM images. HEMNMA was shown to be a good approach to analyze multiple conformations of a macromolecular complex but it could not be widely used in the EM field due to a lack of an integral interface. In particular, its use required switching among different software sources as well as selecting modes for image analysis was difficult without the graphical interface. The graphical interface was thus developed to simplify the practical use of HEMNMA. It is implemented in the open-source software package Xmipp 3.1 (http://xmipp.cnb.csic.es) and only a small part of it relies on MATLAB that is accessible through the main interface. Such integration provides the user with an easy way to perform the analysis of macromolecular dynamics and forms a direct connection to the single-particle reconstruction process. A step-by-step HEMNMA protocol with the graphical interface is given in full details in Supplementary material. The graphical interface will be useful to experimentalists who are interested in studies of continuous conformational changes of macromolecular complexes beyond the modeling of continuous heterogeneity in single particle reconstruction.





    Categories: Journal Articles
  • Comparative structure of vertebrate sperm chromatin
    [Oct 2014]

    Publication date: Available online 27 September 2014
    Source:Journal of Structural Biology

    Author(s): Juan Ausió , Rodrigo González-Romero , Christopher L. Woodcock

    A consistent feature of sperm nuclei is its exceptionally compact state in comparison with somatic nuclei. Here, we have examined the structural organization of sperm chromatin from representatives of three vertebrate lineages, bony fish (Danio rerio), birds (Gallus gallus domesticus) and mammals (Mus musculus) using light and transmission electron microscopy (TEM). Although the three sperm nuclei are all highly compact, they differ in morphology and in the complement of compaction-inducing proteins. Whereas zebrafish sperm retain somatic histones and a nucleosomal organization, in the rooster and mouse, histones are largely replaced by small, arginine-rich protamines. In contrast to the mouse, the rooster protamine contains no cysteine residues and lacks the potential stabilizing effects of S–S bonds. Protamine driven chromatin compaction results in a stable, highly condensed chromatin, markedly different from the somatic nucleosome-based beads-on-a-string architecture, but its structure remains poorly understood. When prepared gently for whole mount TEM, the rooster and mouse sperm chromatin reveal striking rod-like units 40–50nm in width. Also present in the mouse, which has very flattened sperm nuclei, but not rooster, where nuclei take the form of elongated cylinders, are toroidal shaped structures, with an external diameter of about 90nm. In contrast, similarly prepared zebrafish sperm exhibit nucleosomal chromatin. We also examined the early stages in the binding of salmine (the salmon protamine) to defined sequence DNA. These images suggest an initial side-by-side binding of linear DNA–protamine complexes leading to the nucleation of thin, flexible rods with the potential to bend, allowing the ends to come into contact and fuse to form toroidal structures. We discuss the relationship between these in vitro observations and the rods and toroids seen in nuclei, and suggest an explanation for the apparent absence of these structures in TEM images of fully condensed sperm nuclei.





    Categories: Journal Articles
  • Structural studies suggest a peptidoglycan hydrolase function for the Mycobacterium tuberculosis Tat-secreted protein Rv2525c
    [Oct 2014]

    Publication date: Available online 24 September 2014
    Source:Journal of Structural Biology

    Author(s): Marco Bellinzoni , Ahmed Haouz , Isabelle Miras , Sophie Magnet , Gwénaëlle André-Leroux , Raju Mukherjee , William Shepard , Stewart T. Cole , Pedro M. Alzari

    Among the few proteins shown to be secreted by the Tat system in Mycobacterium tuberculosis, Rv2525c is of particular interest, since its gene is conserved in the minimal genome of Mycobacterium leprae. Previous evidence linked this protein to cell wall metabolism and sensitivity to β-lactams. We describe here the crystal structure of Rv2525c that shows a TIM barrel-like fold characteristic of glycoside hydrolases of the GH25 family, which includes prokaryotic and phage-encoded peptidoglycan hydrolases. Structural comparison with other members of this family combined with substrate docking suggest that, although the ‘neighbouring group’ catalytic mechanism proposed for this family still appears as the most plausible, the identity of residues involved in catalysis in GH25 hydrolases might need to be revised.





    Categories: Journal Articles
  • Cover 2 - Editorial Board
    [Oct 2014]

    Publication date: September 2014
    Source:Journal of Structural Biology, Volume 187, Issue 3









    Categories: Journal Articles
  • Table of Contents / barcode
    [Oct 2014]

    Publication date: September 2014
    Source:Journal of Structural Biology, Volume 187, Issue 3









    Categories: Journal Articles
  • Delineating the reaction mechanism of reductase domains of Nonribosomal Peptide Synthetases from mycobacteria
    [Oct 2014]

    Publication date: September 2014
    Source:Journal of Structural Biology, Volume 187, Issue 3

    Author(s): Asfarul S. Haque , Ketan D. Patel , Mandar V. Deshmukh , Arush Chhabra , Rajesh S. Gokhale , Rajan Sankaranarayanan

    Substrate binding to enzymes often follows a precise order where catalysis is accomplished through programmed conformational changes. Short-chain dehydrogenase/reductase (SDR) enzymes follow sequential order ‘bi–bi’ reaction kinetics. The mechanistic study of a SDR homolog, reductase (R) domain, from multifunctional enzymes, e.g. Nonribosomal Peptide Synthetases (NRPSs) and Polyketide Synthases (PKSs) has revealed that it reductively releases 4′-phosphopantetheinyl arm-tethered peptidyl product. We report that the R-domains of NRPSs from Mycobacterium tuberculosis (RNRP) and Mycobacterium smegmatis (RGPL) do not strictly adhere to the obligatory mode of catalysis performed by SDRs, but instead can carry out reductive catalysis of substrate following random bi–bi reaction mechanism as deciphered by NMR and SAXS studies. The crucial conformational change associated with NADPH binding necessary to achieve catalytically competent conformation is also delineated by SAXS studies. Using ITC, we have demonstrated that mutation of catalytic tyrosine to phenylalanine in R-domains results in 3–4-fold decrease in affinity for NADPH and attribute this phenomenon to loss of the noncovalent cation–π interactions present between the tyrosine and nicotinamide ring. We propose that the adaptation to an alternative theme of bi–bi catalytic mechanism enables the R-domains to process the substrates transferred by upstream domains and maintain assembly-line enzymology.





    Categories: Journal Articles
  • Crystal structures of Ophiostoma piceae sterol esterase: Structural insights into activation mechanism and product release
    [Oct 2014]

    Publication date: September 2014
    Source:Journal of Structural Biology, Volume 187, Issue 3

    Author(s): Javier Gutiérrez-Fernández , María Eugenia Vaquero , Alicia Prieto , Jorge Barriuso , María Jesús Martínez , Juan A. Hermoso

    Sterol esterases are able to efficiently hydrolyze both sterol esters and triglycerides and to carry out synthesis reactions in the presence of organic solvents. Their high versatility makes them excellent candidates for biotechnological purposes. Sterol esterase from fungus Ophiostoma piceae (OPE) belongs to the family abH03.01 of the Candida rugosa lipase-like proteins. Crystal structures of OPE were solved in this study for the closed and open conformations. Enzyme activation involves a large displacement of the conserved lid, structural rearrangements of loop α16–α17, and formation of a dimer with a large opening. Three PEG molecules are placed in the active site, mimicking chains of the triglyceride substrate, demonstrating the position of the oxyanion hole and the three pockets that accommodate the sn-1, sn-2 and sn-3 fatty acids chains. One of them is an internal tunnel, connecting the active center with the outer surface of the enzyme 30Å far from the catalytic Ser220. Based on our structural and biochemical results we propose a mechanism by which a great variety of different substrates can be hydrolyzed in OPE paving the way for the construction of new variants to improve the catalytic properties of these enzymes and their biotechnological applications.





    Categories: Journal Articles
  • Crystal structure of the full-length ATPase GspE from the Vibrio vulnificus type II secretion system in complex with the cytoplasmic domain of GspL
    [Oct 2014]

    Publication date: September 2014
    Source:Journal of Structural Biology, Volume 187, Issue 3

    Author(s): Connie Lu , Konstantin V. Korotkov , Wim G.J. Hol

    The type II secretion system (T2SS) is present in many Gram-negative bacteria and is responsible for secreting a large number of folded proteins, including major virulence factors, across the outer membrane. The T2SS consists of 11–15 different proteins most of which are present in multiple copies in the assembled secretion machinery. The ATPase GspE, essential for the functioning of the T2SS, contains three domains (N1E, N2E and CTE) of which the N1E domain is associated with the cytoplasmic domain of the inner membrane protein GspL. Here we describe and analyze the structure of the GspE•cyto-GspL complex from Vibrio vulnificus in the presence of an ATP analog, AMPPNP. There are three such ∼83kDa complexes per asymmetric unit with essentially the same structure. The N2E and CTE domains of a single V. vulnificus GspE subunit adopt a mutual orientation that has not been seen before in any of the previous GspE structures, neither in structures of related ATPases from other secretion systems. This underlines the tremendous conformational flexibility of the T2SS secretion ATPase. Cyto-GspL interacts not only with the N1E domain, but also with the CTE domain and is even in contact with AMPPNP. Moreover, the cyto-GspL domains engage in two types of mutual interactions, resulting in two essentially identical, but crystallographically independent, “cyto-GspL rods” that run throughout the crystal. Very similar rods are present in previous crystals of cyto-GspL and of the N1E•cyto-GspL complex. This arrangement, now seen four times in three entirely different crystal forms, involves contacts between highly conserved residues suggesting a role in the biogenesis or the secretion mechanism or both of the T2SS.





    Categories: Journal Articles
  • The structural basis of differential inhibition of human calpain by indole and phenyl α-mercaptoacrylic acids
    [Oct 2014]

    Publication date: September 2014
    Source:Journal of Structural Biology, Volume 187, Issue 3

    Author(s): Sarah E. Adams , Pierre J. Rizkallah , David J. Miller , Emma J. Robinson , Maurice B. Hallett , Rudolf K. Allemann

    Excessive activity of neutrophils has been linked to many pathological conditions, including rheumatoid arthritis, cancer and Alzheimer’s disease. Calpain-I is a Ca2+-dependent protease that plays a key role in the extravasation of neutrophils from the blood stream prior to causing damage within affected tissues. Inhibition of calpain-I with small molecule mercaptoacrylic acid derivatives slows the cell spreading process of live neutrophils and so these compounds represent promising drug leads. Here we present the 2.05 and 2.03Å co-crystal X-ray structures of the pentaEF hand region, PEF(S), from human calpain with (Z)-3-(4-chlorophenyl)-2-mercaptoacrylic acid and (Z)-3-(5-bromoindol-3-yl)-2-mercaptoacrylic acid. In both structures, the α-mercaptoacrylic acid derivatives bind between two α-helices in a hydrophobic pocket that is also exploited by a leucine residue of the endogenous regulatory calpain inhibitor calpastatin. Hydrophobic interactions between the aromatic rings of both inhibitors and the aliphatic residues of the pocket are integral for tight binding. In the case of (Z)-3-(5-bromoindol-3-yl)-2-mercaptoacrylic acid, hydrogen bonds form between the mercaptoacrylic acid substituent lying outside the pocket and the protein and the carboxylate group is coplanar with the aromatic ring system. Multiple conformations of (Z)-3-(5-bromoindol-3-yl)-2-mercaptoacrylic acid were found within the pocket. The increased potency of (Z)-3-(5-bromoindol-3-yl)-2-mercaptoacrylic acid relative to (Z)-3-(4-chlorophenyl)-2-mercaptoacrylic acid may be a consequence of the indole group binding more deeply in the hydrophobic pocket of PEF(S) than the phenyl ring.





    Categories: Journal Articles
  • Quantification of aortic and cutaneous elastin and collagen morphology in Marfan syndrome by multiphoton microscopy
    [Oct 2014]

    Publication date: September 2014
    Source:Journal of Structural Biology, Volume 187, Issue 3

    Author(s): Jason Z. Cui , Arash Y. Tehrani , Kimberly A. Jett , Pascal Bernatchez , Cornelis van Breemen , Mitra Esfandiarei

    In a mouse model of Marfan syndrome, conventional Verhoeff-Van Gieson staining displays severe fragmentation, disorganization and loss of the aortic elastic fiber integrity. However, this method involves chemical fixatives and staining, which may alter the native morphology of elastin and collagen. Thus far, quantitative analysis of fiber damage in aorta and skin in Marfan syndrome has not yet been explored. In this study, we have used an advanced noninvasive and label-free imaging technique, multiphoton microscopy to quantify fiber fragmentation, disorganization, and total volumetric density of aortic and cutaneous elastin and collagen in a mouse model of Marfan syndrome. Aorta and skin samples were harvested from Marfan and control mice aged 3-, 6- and 9-month. Elastin and collagen were identified based on two-photon excitation fluorescence and second-harmonic-generation signals, respectively, without exogenous label. Measurement of fiber length indicated significant fragmentation in Marfan vs. control. Fast Fourier transform algorithm analysis demonstrated markedly lower fiber organization in Marfan mice. Significantly reduced volumetric density of elastin and collagen and thinner skin dermis were observed in Marfan mice. Cutaneous content of elastic fibers and thickness of dermis in 3-month Marfan resembled those in the oldest control mice. Our findings of early signs of fiber degradation and thinning of skin dermis support the potential development of a novel non-invasive approach for early diagnosis of Marfan syndrome.





    Categories: Journal Articles