Journal of Molecular Biology

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Vinculin: An unfolding tale

Sun, 11/29/2015 - 23:48
Publication date: Available online 12 November 2015
Source:Journal of Molecular Biology

Author(s): Charles Sindelar, Andrew Huehn







Categories: Journal Articles

Novel Features of DAG-Activated PKC Isozymes Reveal a Conserved 3-D Architecture

Sun, 11/29/2015 - 23:48
Publication date: Available online 12 November 2015
Source:Journal of Molecular Biology

Author(s): Iva Lučić, Linda Truebestein, Thomas A. Leonard

Diacylglycerol (DAG) activates the eight conventional and novel isozymes of protein kinase C (PKC) by binding to their C1 domains. The crystal structure of PKCβII in a partially activated conformation showed how the C1B domain regulates activity by clamping a helix in the C-terminal AGC extension of the kinase domain. Here we show that the global three-dimensional shape of the conventional and novel PKCs is conserved despite differences in the order of the domains in their primary sequences. The membrane translocation phenotypes of mutants in the C1B clamp are consistent across all DAG-activated PKCs, demonstrating conservation of this regulatory interface. We now identify a novel interface that sequesters the C1A domain in PKCβII in a membrane-inaccessible state and we generalize this to all DAG-activated PKCs. In the conventional PKCs, we identify a novel element of their C2 domains that additionally contributes to the stability of the inactive conformation. We demonstrate that the interdomain linkers play important roles in permitting and stabilizing this state. We propose a multi-step activation mechanism in which the sequential and cooperative binding of the regulatory domains to the membrane is coupled to allosteric activation of the kinase domain by DAG and that acquisition of full catalytic activity requires DAG binding to the C1B domain. In light of the conservation of shape and intramolecular architecture, we propose that this mechanism is common to all DAG-activated PKCs.
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Synthetic Ecology of Microbes: Mathematical Models and Applications

Sun, 11/29/2015 - 23:48
Publication date: Available online 11 November 2015
Source:Journal of Molecular Biology

Author(s): Ali R. Zomorrodi, Daniel Segrè

As the indispensable role of natural microbial communities in many aspects of life on Earth is uncovered, the bottom-up engineering of synthetic microbial consortia with novel functions is becoming an attractive alternative to engineering single-species systems. Here, we summarize recent work on synthetic microbial communities with a particular emphasis on open challenges and opportunities in environmental sustainability and human health. We next provide a critical overview of mathematical approaches, ranging from phenomenological to mechanistic, to decipher the principles that govern the function, dynamics and evolution of microbial ecosystems. Finally, we present our outlook on key aspects of microbial ecosystems and synthetic ecology that require further developments, including the need for more efficient computational algorithms, a better integration of empirical methods and model-driven analysis, the importance of improving gene function annotation, and the value of a standardized library of well-characterized organisms to be used as building blocks of synthetic communities.
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Structural Stability and Local Dynamics in Disease-Causing Mutants of Human Apolipoprotein A-I: What Makes the Protein Amyloidogenic?

Sun, 11/29/2015 - 23:48
Publication date: Available online 10 November 2015
Source:Journal of Molecular Biology

Author(s): Madhurima Das, Christopher J. Wilson, Xiaohu Mei, Thomas Wales, John R. Engen, Olga Gursky

ApoA-I, the major protein of plasma high-density lipoprotein, removes cellular cholesterol and protects against atherosclerosis. ApoA-I mutations can cause familial amyloidosis, a life-threatening disease wherein N-terminal protein fragments form fibrils in vital organs. To unveil the protein misfolding mechanism and to understand why some mutations cause amyloidosis while others do not, we analyzed the structure, stability and lipid-binding properties of naturally occurring mutants of full-length human apoA-I causing either amyloidosis (G26R, W50R, F71Y, L170P) or aberrant lipid metabolism (L159R). Global and local protein conformation and dynamics in solution were assessed by circular dichroism, fluorescence, and hydrogen-deuterium exchange mass spectrometry. All mutants showed increased deuteration in residues 14-22, supporting our hypothesis that decreased protection of this major amyloid “hot spot” can trigger protein misfolding. In addition, L159R showed local helical unfolding near the mutation site, consistent with cleavage of this mutant in plasma to generate the labile 1-159 fragment. Together, the results suggest that reduced protection of the major amyloid “hot spot”, combined with structural integrity of the native helix-bundle conformation, shift the balance from protein clearance to β-aggregation. A delicate balance between the overall structural integrity of a globular protein and the local destabilization of its amyloidogenic segments may be a fundamental determinant of this and other amyloid diseases. Furthermore, mutation-induced conformational changes observed in the helix bundle, which comprises N-terminal 75% of apoA-I, and its flexible C-terminal tail suggest the propagation of structural perturbations to distant sites via an unexpected template-induced ensemble-based mechanism, challenging the classical structure-based view.
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Partial dedifferentiation of murine radial glia type neural stem cells by Brn2 and c-Myc yields early neuroepithelial progenitors

Sun, 11/29/2015 - 23:48
Publication date: Available online 10 November 2015
Source:Journal of Molecular Biology

Author(s): Raffaela Bung, Philipp Wörsdörfer, Marc Christian Thier, Kathrin Vogt, Martina Gebhardt, Frank Edenhofer

Direct cell conversion developed into an important paradigm for generating cells with enhanced differentiation capability. We combined a transcription factor-based cell fate conversion strategy with the use of pharmacological compounds to derive early neuroepithelial progenitor cells from developmentally more restricted radial glia type neural stem cells (RG-NSCs). By combining the small molecules CHIR99021, Tranylcypromine, SB431542 and valproic acid with viral transduction of the transcription factors c-Myc and the POU domain transcription factor Brn2 we dedifferentiated RG-NSCs into an early neuroepithelial progenitor cell state within 6days. RT-PCR analyses showed a rapid down-regulation of the radial glia markers Olig2 and Vimentin during conversion, whereas the neuroepithelial markers Dach1 and Sox1 were fastly up-regulated. Furthermore, a switch from N- to E-Cadherin indicates a mesenchymal-to-epithelial transition. The differentiation of cells converted by Brn-2/c-Myc yielded smooth muscle actin- and Peripherin-positive cells in addition to the neuronal marker TUJ1 and cells that are positive for the glial marker GFAP. This differentiation potential suggests that the applied reprogramming strategy induced an early neuroepithelial cell population, which might resemble cells of the neural border or even more primitive neuroepithelial cells.
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Crystal structure analysis of wild type and fast hydrolyzing mutant of EhRabX3, a tandem Ras superfamily GTPase from Entamoeba histolytica

Sun, 11/29/2015 - 23:48
Publication date: Available online 10 November 2015
Source:Journal of Molecular Biology

Author(s): Vijay kumar Srivastava, Mintu Chandra, Yumiko Saito-Nakano, Tomoyoshi Nozaki, Sunando Datta

The enteric protozoan parasite, Entamoeba histolytica, is the causative agent of amoebic dysentery, liver abscess and colitis in human. Vesicular trafficking plays a key role in the survival and virulence of the protozoan and is regulated by various Rab GTPases. EhRabX3 is a catalytically inefficient amoebic Rab protein, which is unique among the eukaryotic Ras superfamily by virtue of its tandem domain organization. Here, we report the crystal structures of GDP-bound fast hydrolyzing mutant (V71A/K73Q) and GTP-bound wild type EhRabX3 at 3.1 and 2.8 Å resolutions, respectively. Though both G-domains possess “P-loop containing nucleoside triphosphate hydrolases fold”, only the NTD binds to guanine nucleotide. The relative orientation of the NTD and CTD is stabilized by numerous inter-domain interactions. Compared to other Ras superfamily members, both the GTPase domains displayed large deviation in switch II, perhaps due to non-conservative substitutions in this region. As a result, entire switch II is restructured and moved away from the nucleotide binding pocket, providing a rationale for the diminished GTPase activity of EhRabX3. The N-terminal GTPase domain possesses unusually large number of cysteine residues. X-ray crystal structure of the fast hydrolyzing mutant of EhRabX3 revealed that C39 and C163 formed an intra-molecular disulfide bond. Subsequent mutational and biochemical studies suggest that C39 and C163 are critical for maintaining the structural integrity and function of EhRabX3. Structure-guided functional investigation of cysteine mutants could provide the physiological implications of the disulfide bond and allow us to design potential inhibitors for the better treatment of intestinal amebiasis.
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Meiotic Clade AAA ATPases: Protein Polymer Disassembly Machines

Sun, 11/29/2015 - 23:48
Publication date: Available online 10 November 2015
Source:Journal of Molecular Biology

Author(s): Nicole Monroe, Christopher P. Hill

Meiotic clade AAA ATPases, which were initially grouped on the basis of phylogenetic classification of their AAA ATPase cassette, include four relatively well characterized family members, Vps4, spastin, katanin, and fidgetin. These enzymes all function to disassemble specific polymeric protein structures, with Vps4 disassembling the ESCRT-III polymers that are central to the many membrane-remodeling activities of the ESCRT pathway, and spastin, katanin p60 and fidgetin affecting multiple aspects of cellular dynamics by severing microtubules. They share a common domain architecture that features an N-terminal MIT domain followed by a single AAA ATPase cassette. Meiotic clade AAA ATPases function as hexamers that can cycle between the active assembly and inactive monomers/dimers in a regulated process, and appear to disassemble their polymeric substrates by translocating subunits through the central pore of their hexameric ring. Recent studies with Vps4 have shown that nucleotide-induced asymmetry is a requirement for substrate binding to the pore loops, and that recruitment to the protein lattice via MIT domains also relieves auto-inhibition and primes the AAA ATPase cassettes for substrate binding. The most striking, unifying feature of meiotic clade AAA ATPases may be their MIT domain, which is a module that is found in a wide variety of proteins that localize to ESCRT-III polymers. Spastin also displays an adjacent microtubule-binding sequence, and the presence of both ESCRT-III and microtubule binding elements may underlie the recent findings that the ESCRT-III disassembly function of Vps4 and the microtubule-severing function of spastin, and potentially katanin and fidgetin, are highly coordinated.
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A Substrate Mimic Allows High-Throughput Assay of the FabA Protein and Consequently the Identification of a Novel Inhibitor of Pseudomonas aeruginosa FabA

Sun, 11/29/2015 - 23:48
Publication date: Available online 10 November 2015
Source:Journal of Molecular Biology

Author(s): Lucile Moynié, Anthony G. Hope, Kara Finzel, Jason Schmidberger, Stuart M. Leckie, Gunter Schneider, Michael D. Burkart, Andrew D. Smith, David W. Gray, James H. Naismith

Eukaryotes and prokaryotes possess fatty acid synthase (FAS) biosynthetic pathways that comprise iterative chain elongation, reduction, and dehydration reactions. The bacterial FASII pathway differs significantly from human FAS pathways and is a long-standing target for antibiotic development against Gram-negative bacteria due to differences from the human FAS, and several existing antibacterial agents are known to inhibit FASII enzymes. N-Acetylcysteamine (NAC) fatty acid thioesters have been used as mimics of the natural acyl carrier protein pathway intermediates to assay FASII enzymes, and we now report an assay of FabV from Pseudomonas aeruginosa using (E)-2-decenoyl-NAC. In addition, we have converted an existing UV absorbance assay for FabA, the bifunctional dehydration/epimerization enzyme and key target in the FASII pathway, into a high-throughput enzyme coupled fluorescence assay that has been employed to screen a library of diverse small molecules. With this approach, N-(4-chlorobenzyl)-3-(2-furyl)-1H-1,2,4-triazol-5-amine (N42FTA) was found to competitively inhibit (pIC50 =5.7±0.2) the processing of 3-hydroxydecanoyl-NAC by P. aeruginosa FabA. N42FTA was shown to be potent in blocking crosslinking of Escherichia coli acyl carrier protein and FabA, a direct mimic of the biological process. The co-complex structure of N42FTA with P. aeruginosa FabA protein rationalises affinity and suggests future design opportunities. Employing NAC fatty acid mimics to develop further high-throughput assays for individual enzymes in the FASII pathway should aid in the discovery of new antimicrobials.
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Efficient Genome Manipulation by Variants of Site-Specific Recombinases R and TD

Sun, 11/29/2015 - 23:48
Publication date: Available online 7 November 2015
Source:Journal of Molecular Biology

Author(s): Eugenia Voziyanova, Rachelle P. Anderson, Riddhi Shah, Feng Li, Yuri Voziyanov

Genome engineering benefits from the availability of DNA modifying enzymes that have different target specificities and have optimized performance in different cell types. This variety of site-specific enzymes can be used to develop complex genome engineering applications at multiple loci. Although eight yeast site-specific tyrosine recombinases are known, only Flp is actively used in genome engineering. To expand the pool of the yeast site-specific tyrosine recombinases capable of mediating genome manipulations in mammalian cells, we engineered and analyzed variants of two tyrosine recombinases: R and TD. The activity of the evolved variants, unlike the activity of the native R and TD recombinases, is suitable for genome engineering in Escherichia coli and mammalian cells. Unexpectedly, we found that R recombinase benefits from the shortening of its C-terminus. We also found that the activity of wild-type R can be modulated by its non-consensus “head” sequence but this modulation became not apparent in the evolved R variants. The engineered recombinase variants were found to be active in all recombination reactions tested: excision, integration, and dual recombinase-mediated cassette exchange. The analysis of the latter reaction catalyzed by the R/TD recombinase pair shows that the condition supporting the most efficient replacement reaction favors efficient TD-mediated integration reaction while favoring efficient R-mediated integration and deletion reactions.
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Editorial Board

Sun, 11/29/2015 - 23:48
Publication date: 6 November 2015
Source:Journal of Molecular Biology, Volume 427, Issue 22









Categories: Journal Articles

Editorial Board

Sun, 11/01/2015 - 22:48
Publication date: 6 November 2015
Source:Journal of Molecular Biology, Volume 427, Issue 22









Categories: Journal Articles

Contest List

Sun, 11/01/2015 - 22:48
Publication date: 6 November 2015
Source:Journal of Molecular Biology, Volume 427, Issue 22









Categories: Journal Articles

Navigating toward an Understanding of the Role of Regulator of Calcineurin in Thermotaxis

Sun, 11/01/2015 - 22:48
Publication date: 6 November 2015
Source:Journal of Molecular Biology, Volume 427, Issue 22

Author(s): Tami J Kingsbury







Categories: Journal Articles

Regulator of Calcineurin (RCAN-1) Regulates Thermotaxis Behavior in Caenorhabditis elegans

Sun, 11/01/2015 - 22:48
Publication date: 6 November 2015
Source:Journal of Molecular Biology, Volume 427, Issue 22

Author(s): Weixun Li, Harold W. Bell, Joohong Ahnn, Sun-Kyung Lee

Regulator of calcineurin (RCAN) is a calcineurin-interacting protein that inhibits calcineurin phosphatase when overexpressed, often upregulated under neuropathological conditions with impaired learning and memory processes, such as Down syndrome or Alzheimer's disease. Thermotactic behavior in the nematode Caenorhabditis elegans is a form of memory in which calcineurin signaling plays a pivotal role in the thermosensation of AFD neurons. In this study, we found that rcan-1 deletion mutants exhibited cryophilic behavior dependent on tax-6, which was rescued by expressing rcan-1 in AFD neurons. Interaction between RCAN-1 and TAX-6 requires the conserved PxIxIT motif of RCAN-1, without which thermotactic behavior could not be fully rescued. In addition, the loss of crh-1/CREB suppressed the thermotaxis phenotypes of rcan-1 and tax-6 mutants, indicating that crh-1 is crucial in thermotaxis memory in these mutants. Taken together, our results suggest that rcan-1 is an inhibitory regulator of tax-6 and that it acts in the formation of thermosensory behavioral memory in C. elegans.
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Gene Regulation Gets in Tune: How Riboswitch Tertiary-Structure Networks Adapt to Meet the Needs of Their Transcription Units

Sun, 11/01/2015 - 22:48
Publication date: 6 November 2015
Source:Journal of Molecular Biology, Volume 427, Issue 22

Author(s): Debapratim Dutta, Joseph E. Wedekind







Categories: Journal Articles

A Highly Coupled Network of Tertiary Interactions in the SAM-I Riboswitch and Their Role in Regulatory Tuning

Sun, 11/01/2015 - 22:48
Publication date: 6 November 2015
Source:Journal of Molecular Biology, Volume 427, Issue 22

Author(s): Christopher Wostenberg, Pablo Ceres, Jacob T. Polaski, Robert T. Batey

RNA folding in vivo is significantly influenced by transcription, which is not necessarily recapitulated by Mg2+-induced folding of the corresponding full-length RNA in vitro. Riboswitches that regulate gene expression at the transcriptional level are an ideal system for investigating this aspect of RNA folding as ligand-dependent termination is obligatorily co-transcriptional, providing a clear readout of the folding outcome. The folding of representative members of the SAM-I family of riboswitches has been extensively analyzed using approaches focusing almost exclusively upon Mg2+ and/or S-adenosylmethionine (SAM)-induced folding of full-length transcripts of the ligand binding domain. To relate these findings to co-transcriptional regulatory activity, we have investigated a set of structure-guided mutations of conserved tertiary architectural elements of the ligand binding domain using an in vitro single-turnover transcriptional termination assay, complemented with phylogenetic analysis and isothermal titration calorimetry data. This analysis revealed a conserved internal loop adjacent to the SAM binding site that significantly affects ligand binding and regulatory activity. Conversely, most single point mutations throughout key conserved features in peripheral tertiary architecture supporting the SAM binding pocket have relatively little impact on riboswitch activity. Instead, a secondary structural element in the peripheral subdomain appears to be the key determinant in observed differences in regulatory properties across the SAM-I family. These data reveal a highly coupled network of tertiary interactions that promote high-fidelity co-transcriptional folding of the riboswitch but are only indirectly linked to regulatory tuning.
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Acidic Residues in the Hfq Chaperone Increase the Selectivity of sRNA Binding and Annealing

Sun, 11/01/2015 - 22:48
Publication date: 6 November 2015
Source:Journal of Molecular Biology, Volume 427, Issue 22

Author(s): Subrata Panja, Andrew Santiago-Frangos, Daniel J. Schu, Susan Gottesman, Sarah A. Woodson

Hfq facilitates gene regulation by small non-coding RNAs (sRNAs), thereby affecting bacterial attributes such as biofilm formation and virulence. Escherichia coli Hfq recognizes specific U-rich and AAN motifs in sRNAs and target mRNAs, after which an arginine patch on the rim promotes base pairing between their complementary sequences. In the cell, Hfq must discriminate between many similar RNAs. Here, we report that acidic amino acids lining the sRNA binding channel between the inner pore and rim of the Hfq hexamer contribute to the selectivity of Hfq's chaperone activity. RNase footprinting, in vitro binding and stopped-flow fluorescence annealing assays showed that alanine substitution of D9, E18 or E37 strengthened RNA interactions with the rim of Hfq and increased annealing of non-specific or U-tailed RNA oligomers. Although the mutants were less able than wild-type Hfq to anneal sRNAs with wild-type rpoS mRNA, the D9A mutation bypassed recruitment of Hfq to an (AAN)4 motif in rpoS, both in vitro and in vivo. These results suggest that acidic residues normally modulate access of RNAs to the arginine patch. We propose that this selectivity limits indiscriminate target selection by E. coli Hfq and enforces binding modes that favor genuine sRNA and mRNA pairs.
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Mechanistic Analysis of Activation of the Innate Immune Sensor PKR by Bacterial RNA

Sun, 11/01/2015 - 22:48
Publication date: 6 November 2015
Source:Journal of Molecular Biology, Volume 427, Issue 22

Author(s): Chelsea M. Hull, Philip C. Bevilacqua

The protein kinase PKR (protein kinase R) is a sensor in innate immunity. PKR autophosphorylates in the presence of double-stranded RNA enabling it to phosphorylate its substrate, eIF2α (eukaryotic initiation factor 2α), halting cellular translation. Classical activators of PKR are long viral double-stranded RNAs, but recently, PKR has been found to be activated by bacterial RNA. However, the features of bacterial RNA that activate PKR are unknown. We studied the Bacillus subtilis trp 5′-UTR (untranslated region), which is an indirect riboswitch with secondary and tertiary RNA structures that regulate gene function. Additionally, the trp 5′-UTR binds a protein, TRAP (tryptophan RNA-binding attenuation protein), which recognizes l-tryptophan. We present the first evidence that multiple structural features in this RNA, which are typical of bacterial RNAs, activate PKR in TRAP-free and TRAP/l-Trp-bound forms. Segments from the 5′-UTR, including the terminator 5′-stem–loop and Shine–Dalgarno blocking hairpins, demonstrated 5′-triphosphate and flanking RNA tail dependence on PKR activation. Disruption of long-distance tertiary interactions in the 5′-UTR led to partial loss in activation, consistent with highly base-paired regions in bacterial RNA activating PKR. One physiological change a bacterial RNA would face in a human cell is a decrease in the concentration of free magnesium. Upon lowering the magnesium concentration to human physiological conditions of 0.5mM, the trp 5′-UTR continued to activate PKR potently. Moreover, total RNA from Escherichia coli, depleted of rRNA, also activated PKR under these ionic conditions. This study demonstrates that PKR can signal the presence of bacterial RNAs under physiological ionic conditions and offers a potential explanation for the apparent absence of riboswitches in the human genome.
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Mutations in RNA Polymerase Bridge Helix and Switch Regions Affect Active-Site Networks and Transcript-Assisted Hydrolysis

Sun, 11/01/2015 - 22:48
Publication date: 6 November 2015
Source:Journal of Molecular Biology, Volume 427, Issue 22

Author(s): Nan Zhang, Jorrit Schäfer, Amit Sharma, Lucy Rayner, Xiaodong Zhang, Roman Tuma, Peter Stockley, Martin Buck

In bacterial RNA polymerase (RNAP), the bridge helix and switch regions form an intricate network with the catalytic active centre and the main channel. These interactions are important for catalysis, hydrolysis and clamp domain movement. By targeting conserved residues in Escherichia coli RNAP, we are able to show that functions of these regions are differentially required during σ70-dependent and the contrasting σ54-dependent transcription activations and thus potentially underlie the key mechanistic differences between the two transcription paradigms. We further demonstrate that the transcription factor DksA directly regulates σ54-dependent activation both positively and negatively. This finding is consistent with the observed impacts of DksA on σ70-dependent promoters. DksA does not seem to significantly affect RNAP binding to a pre-melted promoter DNA but affects extensively activity at the stage of initial RNA synthesis on σ54-regulated promoters. Strikingly, removal of the σ54 Region I is sufficient to invert the action of DksA (from stimulation to inhibition or vice versa) at two test promoters. The RNAP mutants we generated also show a strong propensity to backtrack. These mutants increase the rate of transcript-hydrolysis cleavage to a level comparable to that seen in the Thermus aquaticus RNAP even in the absence of a non-complementary nucleotide. These novel phenotypes imply an important function of the bridge helix and switch regions as an anti-backtracking ratchet and an RNA hydrolysis regulator.
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Three-Dimensional Structure of Vertebrate Muscle Z-Band: The Small-Square Lattice Z-Band in Rat Cardiac Muscle

Sun, 11/01/2015 - 22:48
Publication date: 6 November 2015
Source:Journal of Molecular Biology, Volume 427, Issue 22

Author(s): Thomas Burgoyne, Edward P. Morris, Pradeep K. Luther

The Z-band in vertebrate striated muscle crosslinks actin filaments of opposite polarity from adjoining sarcomeres and transmits tension along myofibrils during muscular contraction. It is also the location of a number of proteins involved in signalling and myofibrillogenesis; mutations in these proteins lead to myopathies. Understanding the high-resolution structure of the Z-band will help us understand its role in muscle contraction and the role of these proteins in the function of muscle. The appearance of the Z-band in transverse-section electron micrographs typically resembles a small-square lattice or a basketweave appearance. In longitudinal sections, the Z-band width varies more with muscle type than species: slow skeletal and cardiac muscles have wider Z-bands than fast skeletal muscles. As the Z-band is periodic, Fourier methods have previously been used for three-dimensional structural analysis. To cope with variations in the periodic structure of the Z-band, we have used subtomogram averaging of tomograms of rat cardiac muscle in which subtomograms are extracted and compared and similar ones are averaged. We show that the Z-band comprises four to six layers of links, presumably α-actinin, linking antiparallel overlapping ends of the actin filaments from the adjoining sarcomeres. The reconstruction shows that the terminal 5–7nm of the actin filaments within the Z-band is devoid of any α-actinin links and is likely to be the location of capping protein CapZ.
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Categories: Journal Articles