Home About us Contact
|
Home About us Contact | ||
|
|
||
Structural Role (structural + role)
Selected AbstractsDifferential expression and localization of neuronal intermediate filament proteins within newly developing neurites in dissociated cultures of Xenopus laevis embryonic spinal cordCYTOSKELETON, Issue 1 2001Jayanthi Undamatla Abstract The molecular subunit composition of neurofilaments (NFs) progressively changes during axon development. In developing Xenopus laevis spinal cord, peripherin emerges at the earliest stages of neurite outgrowth. NF-M and XNIF (an ,-internexin-like protein) appear later, as axons continue to elongate, and NF-L is expressed after axons contact muscle. Because NFs are the most abundant component of the vertebrate axonal cytoskeleton, we must understand why these changes occur before we can fully comprehend how the cytoskeleton regulates axon growth and morphology. Knowing where these proteins are localized within developing neurites and how their expression changes with cell contact is essential for this understanding. Thus, we examined by immunofluorescence the expression and localization of these NF subunits within dissociated cultures of newly differentiating spinal cord neurons. In young neurites, peripherin was most abundant in distal neuritic segments, especially near branch points and extending into the central domain of the growth cone. In contrast, XNIF and NF-M were usually either absent from very young neurites or exhibited a proximal to distal gradient of decreasing intensity. In older neurites, XNIF and NF-M expression increased, whereas that of peripherin declined. All three of these proteins became more evenly distributed along the neurites, with some branches staining more intensely than others. At 24 h, NF-L appeared, and in 48-h cultures, its expression, along with that of NF-M, was greater in neurites contacting muscle cells, arguing that the upregulation of these two subunits is dependent on contact with target cells. Moreover, this contact had no effect on XNIF or peripherin expression. Our findings are consistent with a model in which peripherin plays an important structural role in growth cones, XNIF and NF-M help consolidate the intermediate filament cytoskeleton beginning in the proximal neurite, and increased levels of NF-L and NF-M help further solidify the cytoskeleton of axons that successfully reach their targets. Cell Motil. Cytoskeleton 49:16,32, 2001. © 2001 Wiley-Liss, Inc. [source] R120G ,B-crystallin promotes the unfolding of reduced ,-lactalbumin and is inherently unstableFEBS JOURNAL, Issue 3 2005Teresa M. Treweek ,-Crystallin is the principal lens protein which, in addition to its structural role, also acts as a molecular chaperone, to prevent aggregation and precipitation of other lens proteins. One of its two subunits, ,B-crystallin, is also expressed in many nonlenticular tissues, and a natural missense mutation, R120G, has been associated with cataract and desmin-related myopathy, a disorder of skeletal muscles [Vicart P, Caron A, Guicheney P, Li Z, Prevost MC, Faure A, Chateau D, Chapon F, Tome F, Dupret JM, Paulin D & Fardeau M (1998) Nat Genet20, 92,95]. In the present study, real-time 1H-NMR spectroscopy showed that the ability of R120G ,B-crystallin to stabilize the partially folded, molten globule state of ,-lactalbumin was significantly reduced in comparison with wild-type ,B-crystallin. The mutant showed enhanced interaction with, and promoted unfolding of, reduced ,-lactalbumin, but showed limited chaperone activity for other target proteins. Using NMR spectroscopy, gel electrophoresis, and MS, we observed that, unlike the wild-type protein, R120G ,B-crystallin is intrinsically unstable in solution, with unfolding of the protein over time leading to aggregation and progressive truncation from the C-terminus. Light scattering, MS, and size-exclusion chromatography data indicated that R120G ,B-crystallin exists as a larger oligomer than wild-type ,B-crystallin, and its size increases with time. It is likely that removal of the positive charge from R120 of ,B-crystallin causes partial unfolding, increased exposure of hydrophobic regions, and enhances its susceptibility to proteolysis, thus reducing its solubility and promoting its aggregation and complexation with other proteins. These characteristics may explain the involvement of R120G ,B-crystallin with human disease states. [source] Cover Picture: Structural Modifications to Polystyrene via Self-Assembling Molecules (Adv. Funct.ADVANCED FUNCTIONAL MATERIALS, Issue 3 2005Mater. Abstract The cover shows tensile failure of a sample of pure polystyrene (left), and a polystyrene sample with greater impact strength containing 1% by weight of dispersed nanoribbons (right), as reported in work by Stupp and co-workers on p.,487. The nanoribbons are formed by self-assembly of molecules known as dendron rodcoils (DRCs) in styrene monomer, resulting in the formation of a gel. This gel can then be polymerized thermally. We have previously reported that small quantities of self-assembling molecules known as dendron rodcoils (DRCs) can be used as supramolecular additives to modify the properties of polystyrene (PS). These molecules spontaneously assemble into supramolecular nanoribbons that can be incorporated into bulk PS in such a way that the orientation of the polymer is significantly enhanced when mechanically drawn above the glass-transition temperature. In the current study, we more closely evaluate the structural role of the DRC nanoribbons in PS by investigating the mechanical properties and deformation microstructures of polymers modified by self-assembly. In comparision to PS homopolymer, PS containing small amounts (,,1.0,wt.-%) of self-assembling DRC molecules exhibit greater Charpy impact strengths in double-notch four-point bending and significantly greater elongations to failure in uniaxial tension at 250,% prestrain. Although the DRC-modified polymer shows significantly smaller elongations to failure at 1000,% prestrain, both low- and high-prestrain specimens maintain tensile strengths that are comparable to those of the homopolymer. The improved toughness and ductility of DRC-modified PS appears to be related to the increased stress whitening and craze density that was observed near fracture surfaces. However, the mechanism by which the self-assembling DRC molecules toughen PS is different from that of conventional additives. These molecules assemble into supramolecular nanoribbons that enhance polymer orientation, which in turn modifies crazing patterns and improves impact strength and ductility. [source] The infectivity of transmissible spongiform encephalopathy agent at low doses: the importance of phospholipidJOURNAL OF APPLIED MICROBIOLOGY, Issue 2 2006P. Gale Abstract The issue of whether the mechanism of infection is independent or co-operative for low doses of transmissible spongiform encephalopathy (TSE) agent is critical for risk assessment. The susceptibility (and hence ID50) of individuals with the same prion protein (PrP) genotype may vary considerably with a small proportion being very susceptible. Assuming independent action, the incubation period (IP) would continue to increase until the dose is below the ID50 of the most susceptible individuals in the experiment, at which point it would become constant. This may explain the observed increase in IP with decreasing dose below the apparent ID50 in experiments with untreated TSE agent. In contrast, IPs for autoclaved or NaOH-treated TSE agent increase greatly at doses WRN, the protein deficient in Werner syndrome, plays a critical structural role in optimizing DNA repairAGING CELL, Issue 4 2003Lishan Chen Summary Werner syndrome (WS) predisposes patients to cancer and premature aging, owing to mutations in WRN. The WRN protein is a RECQ-like helicase and is thought to participate in DNA double-strand break (DSB) repair by non-homologous end joining (NHEJ) or homologous recombination (HR). It has been previously shown that non-homologous DNA ends develop extensive deletions during repair in WS cells, and that this WS phenotype was complemented by wild-type (wt) WRN. WRN possesses both 3, , 5, exonuclease and 3, , 5, helicase activities. To determine the relative contributions of each of these distinct enzymatic activities to DSB repair, we examined NHEJ and HR in WS cells (WRN,/,) complemented with either wtWRN, exonuclease-defective WRN (E,), helicase-defective WRN (H,) or exonuclease/helicase-defective WRN (E,H,). The single E, and H, mutants each partially complemented the NHEJ abnormality of WRN,/, cells. Strikingly, the E,H, double mutant complemented the WS deficiency nearly as efficiently as did wtWRN. Similarly, the double mutant complemented the moderate HR deficiency of WS cells nearly as well as did wtWRN, whereas the E, and H, single mutants increased HR to levels higher than those restored by either E,H, or wtWRN. These results suggest that balanced exonuclease and helicase activities of WRN are required for optimal HR. Moreover, WRN appears to play a structural role, independent of its enzymatic activities, in optimizing HR and efficient NHEJ repair. Another human RECQ helicase, BLM, suppressed HR but had little or no effect on NHEJ, suggesting that mammalian RECQ helicases have distinct functions that can finely regulate recombination events. [source] Synthesis of heterotrimeric collagen peptides containing the ,1,1 integrin recognition site of collagen type IVJOURNAL OF PEPTIDE SCIENCE, Issue 5 2002Barbara Saccá Abstract Collagen type IV provides a biomechanically stable scaffold into which the other constituents of basement membranes are incorporated, but it also plays an important role in cell adhesion. This occurs with collagen type IV mainly via the ,1,1 integrin, and the proposed epitope involved in this type of collagen/integrin interaction corresponds to a non-sequential R/Xaa/D motif, where the arginine and aspartate residues are provided by the ,2 and ,1 chains of the collagen molecule, respectively. Since the stagger of the three , chains in native collagen type IV is still unknown and different alignments of the chains lead to different spatial epitopes, two heterotrimeric collagen peptides containing the natural 457,469 sequences of the cell adhesion site were synthesized in which the single chains were assembled via disulfide bonds into the two most plausible ,1,2,1, and ,2,1,1, registers. The differentiated triple-helical stabilities of the two heterotrimers suggest a significant structural role of the chain register in collagen, although the binding to ,1,1 integrin is apparently less affected as indicated by preliminary experiments. Copyright © 2002 European Peptide Society and John Wiley & Sons, Ltd. [source] Hg2+ Reacts with Different Components of the NADPH: Protochlorophyllide Oxidoreductase MacrodomainsPLANT BIOLOGY, Issue 3 2004K. Solymosi Abstract: The molecular background of Hg2+ -induced inhibition of protochlorophyllide (Pchlide) photoreduction was investigated in homogenates of dark-grown wheat leaves. Our earlier work showed that 15 min incubation with 10 -2 M Hg2+ completely inhibits the activity of NADPH: Pchlide oxidoreductase. Detailed analysis of spectra recorded at 10 K indicated the appearance of emission bands at 638 and 650 nm, which are characteristic for NADP+ -Pchlide complexes. Fluorescence emission spectra recorded with different excitation wavelengths, fluorescence lifetime measurements and the analysis of acetone extractions revealed that Hg2+ can also react directly with Pchlide, resulting in protopheophorbide formation. At 10 -3 M Hg2+, the phototransformation was complete but the blue shift of the chlorophyllide emission band speeded up remarkably. This indicates oxidation of the NADPH molecules that have a structural role in keeping together the etioplast inner membrane components. We suggest a complex model for the Hg2+ effect: depending on concentration it can react with any components of the NADPH: Pchlide oxidoreductase macrodomains. [source] Structures and molecular-dynamics studies of three active-site mutants of bovine pancreatic phospholipase A2ACTA CRYSTALLOGRAPHICA SECTION D, Issue 10 2008Shankar Prasad Kanaujia Phospholipase A2 hydrolyzes phospholipids at the sn -2 position to cleave the fatty-acid ester bond of l -glycerophospholipids. The catalytic dyad (Asp99 and His48) along with a nucleophilic water molecule is responsible for enzyme hydrolysis. Furthermore, the residue Asp49 in the calcium-binding loop is essential for controlling the binding of the calcium ion and the catalytic action of phospholipase A2. To elucidate the structural role of His48 and Asp49, the crystal structures of three active-site single mutants H48N, D49N and D49K have been determined at 1.9,Å resolution. Although the catalytically important calcium ion is present in the H48N mutant, the crystal structure shows that proton transfer is not possible from the catalytic water to the mutated residue. In the case of the Asp49 mutants, no calcium ion was found in the active site. However, the tertiary structures of the three active-site mutants are similar to that of the trigonal recombinant enzyme. Molecular-dynamics simulation studies provide a good explanation for the crystallographic results. [source] Structural asymmetry and intersubunit communication in muscle creatine kinaseACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2007Jeffrey F. Ohren The structure of a transition-state analog complex of a highly soluble mutant (R134K) of rabbit muscle creatine kinase (rmCK) has been determined to 1.65,Å resolution in order to elucidate the structural changes that are required to support and regulate catalysis. Significant structural asymmetry is seen within the functional homodimer of rmCK, with one monomer found in a closed conformation with the active site occupied by the transition-state analog components creatine, MgADP and nitrate. The other monomer has the two loops that control access to the active site in an open conformation and only MgADP is bound. The N-terminal region of each monomer makes a substantial contribution to the dimer interface; however, the conformation of this region is dramatically different in each subunit. Based on this structural evidence, two mutational modifications of rmCK were conducted in order to better understand the role of the amino-terminus in controlling creatine kinase activity. The deletion of the first 15 residues of rmCK and a single point mutant (P20G) both disrupt subunit cohesion, causing the dissociation of the functional homodimer into monomers with reduced catalytic activity. This study provides support for a structural role for the amino-terminus in subunit association and a mechanistic role in active-site communication and catalytic regulation. [source] A poke in the eye: Inhibiting HIV-1 protease through its flap-recognition pocketBIOPOLYMERS, Issue 8 2008Kelly L. Damm Abstract A novel mechanism of inhibiting HIV-1 protease (HIVp) is presented. Using computational solvent mapping to identify complementary interactions and the Multiple Protein Structure method to incorporate protein flexibility, we generated a receptor-based pharmacophore model of the flexible flap region of the semiopen, apo state of HIVp. Complementary interactions were consistently observed at the base of the flap, only within a cleft with a specific structural role. In the closed, bound state of HIVp, each flap tip docks against the opposite monomer, occupying this cleft. This flap-recognition site is filled by the protein and cannot be identified using traditional approaches based on bound, closed structures. Virtual screening and dynamics simulations show how small molecules can be identified to complement this cleft. Subsequent experimental testing confirms inhibitory activity of this new class of inhibitor. This may be the first new inhibitor class for HIVp since dimerization inhibitors were introduced 17 years ago. © 2008 Wiley Periodicals, Inc. Biopolymers 89: 643,652, 2008. This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com [source] A possible molecular mechanism of hanatoxin binding-modified gating in voltage-gated K+ -channelsJOURNAL OF MOLECULAR RECOGNITION, Issue 6 2003Kuo-Long Lou Abstract While S4 is known as the voltage sensor in voltage-gated potassium channels, the carboxyl terminus of S3 (S3C) is of particular interest concerning the site for gating modifier toxins like hanatoxin. The thus derived helical secondary structural arrangement for S3C, as well as its surrounding environment, has since been intensively and vigorously debated. Our previous structural analysis based on molecular simulation has provided sufficient information to describe reasonable docking conformation and further experimental designs (Lou et al., 2002. J. Mol. Recognit. 15: 175,179). However, if one only relies on such information, more advanced structure,functional interpretations for the roles S3C may play in the modification of gating behavior upon toxin binding will remain unknown. In order to have better understanding of the molecular details regarding this issue, we have performed the docking simulation with the S3C sequence from the hanatoxin-insensitive K+ -channel, shaker, and analyzed the conformational changes resulting from such docking. Compared with other functional data from previous studies with respect to the proximity of the S3,S4 linker region, we suggested a significant movement of drk1 S3C, but not shaker S3C, in the direction presumably towards S4, which was comprehended as a possible factor interfering with S4 translocation during drk1 gating in the presence of toxin. In combination with the discussions for structural roles of the length of the S3,S4 linker, a possible molecular mechanism to illustrate the hanatoxin binding-modified gating is proposed. Copyright © 2003 John Wiley & Sons, Ltd. [source] The identification of conserved interactions within the SH3 domain by alignment of sequences and structuresPROTEIN SCIENCE, Issue 11 2000Stefan M. Larson Abstract The SH3 domain, comprised of approximately 60 residues, is found within a wide variety of proteins, and is a mediator of protein,protein interactions. Due to the large number of SH3 domain sequences and structures in the databases, this domain provides one of the best available systems for the examination of sequence and structural conservation within a protein family. In this study, a large and diverse alignment of SH3 domain sequences was constructed, and the pattern of conservation within this alignment was compared to conserved structural features, as deduced from analysis of eighteen different SH3 domain structures. Seventeen SH3 domain structures solved in the presence of bound peptide were also examined to identify positions that are consistently most important in mediating the peptide-binding function of this domain. Although residues at the two most conserved positions in the alignment are directly involved in peptide binding, residues at most other conserved positions play structural roles, such as stabilizing turns or comprising the hydrophobic core. Surprisingly, several highly conserved side-chain to main-chain hydrogen bonds were observed in the functionally crucial RT-Src loop between residues with little direct involvement in peptide binding. These hydrogen bonds may be important for maintaining this region in the precise conformation necessary for specific peptide recognition. In addition, a previously unrecognized yet highly conserved ,-bulge was identified in the second ,-strand of the domain, which appears to provide a necessary kink in this strand, allowing it to hydrogen bond to both sheets comprising the fold. [source] Alternative roles for Cdk5 in learning and synaptic plasticityBIOTECHNOLOGY JOURNAL, Issue 8 2007Ammar H. Hawasli Abstract Protein kinases mediate the intracellular signal transduction pathways controlling synaptic plasticity in the central nervous system. While the majority of protein kinases achieve this function via the phosphorylation of synaptic substrates, some kinases may contribute through alternative mechanisms in addition to enzymatic activity. There is growing evidence that protein kinases may often play structural roles in plasticity as well. Cyclin-dependent kinase 5 (Cdk5) has been implicated in learning and synaptic plasticity. Initial scrutiny focused on its enzymatic activity using pharmacological inhibitors and genetic modifications of Cdk5 cofactors. Quite recently Cdk5 has been shown to govern learning and plasticity via regulation of glutamate receptor degradation, a function that may not dependent on phosphorylation of downstream effectors. From these new studies, two roles emerge for Cdk5 in plasticity: one in which it controls structural plasticity via phosphorylation of synaptic substrates, and a second where it regulates functional plasticity via protein-protein interactions. [source] Coxiella burnetii inhabits a cholesterol-rich vacuole and influences cellular cholesterol metabolismCELLULAR MICROBIOLOGY, Issue 3 2006Dale Howe Summary Coxiella burnetii directs the synthesis of a large parasitophorous vacuole (PV) required for replication. While some lysosomal characteristics of the PV have been described, the origin and composition of the PV membrane is largely undefined. Cholesterol is an essential component of mammalian cell membranes where it plays important regulatory and structural roles. Here we investigated the role of host cholesterol in biogenesis and maintenance of the C. burnetii PV in Vero cells. The C. burnetii PV membrane stained with filipin and was positive for the lipid raft protein flotillin-1, suggesting PV membranes are enriched in cholesterol and contain lipid raft microdomains. C. burnetii infection increased host cell cholesterol content by 1.75-fold with a coincident upregulation of host genes involved in cholesterol metabolism. Treatment with U18666A, lovastatin, or 25-hydroxycholesterol, pharmacological agents that inhibit cholesterol uptake and/or biosynthesis, altered PV morphology and partially inhibited C. burnetii replication. Complete inhibition of C. burnetii PV development and replication was observed when infected cells were treated with imipramine or ketoconazole, inhibitors of cholesterol uptake and biosynthesis respectively. We conclude that C. burnetii infection perturbs host cell cholesterol metabolism and that free access to host cholesterol stores is required for optimal C. burnetii replication. [source]
| | ||||||||||||