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Articles 1 - 9 of 9

Full-Text Articles in Biochemistry, Biophysics, and Structural Biology

Planning Combinatorial Disulfide Cross-Links For Protein Fold Determination, Fei Xiong, Alan M Friedman, Chris Bailey-Kellogg Nov 2011

Planning Combinatorial Disulfide Cross-Links For Protein Fold Determination, Fei Xiong, Alan M Friedman, Chris Bailey-Kellogg

Open Dartmouth: Faculty Open Access Scholarship

Fold recognition techniques take advantage of the limited number of overall structural organizations, and have become increasingly effective at identifying the fold of a given target sequence. However, in the absence of sufficient sequence identity, it remains difficult for fold recognition methods to always select the correct model. While a native-like model is often among a pool of highly ranked models, it is not necessarily the highest-ranked one, and the model rankings depend sensitively on the scoring function used. Structure elucidation methods can then be employed to decide among the models based on relatively rapid biochemical/biophysical experiments.


A Lipid-Anchored Snare Supports Membrane Fusion, Hao Xu, Michael Zick, William T. Wickner, Youngsoo Jun Oct 2011

A Lipid-Anchored Snare Supports Membrane Fusion, Hao Xu, Michael Zick, William T. Wickner, Youngsoo Jun

Open Dartmouth: Faculty Open Access Scholarship

Intracellular membrane fusion requires R-SNAREs and Q-SNAREs to assemble into a four-helical parallel coiled-coil, with their hydrophobic anchors spanning the two apposed membranes. Based on the fusion properties of chemically defined SNARE- proteoliposomes, it has been proposed that the assembly of this helical bundle transduces force through the entire bilayer via the transmembrane SNARE anchor domains to drive fusion. However, an R-SNARE, Nyv1p, with a genetically engineered lipid anchor that spans half of the bilayer suffices for the fusion of isolated vacuoles, although this organelle has other R-SNAREs. To demonstrate unequivocally the fusion activity of lipid-anchored Nyv1p, we reconstituted proteoliposomes ...


Splice Variant–Specific Cellular Function Of The Formin Inf2 In Maintenance Of Golgi Architecture, Vinay Ramabhadran, Farida Korobova, Gilbert J. Rahme, Henry N. Higgs Oct 2011

Splice Variant–Specific Cellular Function Of The Formin Inf2 In Maintenance Of Golgi Architecture, Vinay Ramabhadran, Farida Korobova, Gilbert J. Rahme, Henry N. Higgs

Open Dartmouth: Faculty Open Access Scholarship

INF2 is a unique formin that can both polymerize and depolymerize actin filaments. Mutations in INF2 cause the kidney disease focal and segmental glomerulosclerosis. INF2 can be expressed as two C-terminal splice variants: CAAX and non-CAAX. The CAAX isoform contains a C-terminal prenyl group and is tightly bound to endoplasmic reticulum (ER). The localization pattern and cellular function of the non-CAAX isoform have not been studied. Here we find that the two isoforms are expressed in a cell type-dependent manner, with CAAX predominant in 3T3 fibroblasts and non-CAAX predominant in U2OS, HeLa, and Jurkat cells. Although INF2-CAAX is ER localized ...


Differential Interactions Of The Formins Inf2, Mdia1, And Mdia2 With Microtubules, Jeremie Gaillard, Bvinay Ramabhadran, Emmanuelle Neumanne, Pinar Gurel, Laurent Blanchoin, Marylin Vantard, Henry N. Higgs Sep 2011

Differential Interactions Of The Formins Inf2, Mdia1, And Mdia2 With Microtubules, Jeremie Gaillard, Bvinay Ramabhadran, Emmanuelle Neumanne, Pinar Gurel, Laurent Blanchoin, Marylin Vantard, Henry N. Higgs

Open Dartmouth: Faculty Open Access Scholarship

A number of cellular processes use both microtubules and actin filaments, but the molecular machinery linking these two cytoskeletal elements remains to be elucidated in detail. Formins are actin-binding proteins that have multiple effects on actin dynamics, and one formin, mDia2, has been shown to bind and stabilize microtubules through its formin homology 2 (FH2) domain. Here we show that three formins, INF2, mDia1, and mDia2, display important differences in their interactions with microtubules and actin. Constructs containing FH1, FH2, and C-terminal domains of all three formins bind microtubules with high affinity (K(d) < 100 nM). However, only mDia2 binds microtubules at 1:1 stoichiometry, with INF2 and mDia1 showing saturating binding at approximately 1:3 (formin dimer:tubulin dimer). INF2-FH1FH2C is a potent microtubule-bundling protein, an effect that results in a large reduction in catastrophe rate. In contrast, neither mDia1 nor mDia2 is a potent microtubule bundler. The C-termini of mDia2 and INF2 have different functions in microtubule interaction, with mDia2's C-terminus required for high-affinity binding and INF2's C-terminus required for bundling. mDia2's C-terminus directly binds microtubules with submicromolar affinity. These formins also differ in their abilities to bind actin and microtubules simultaneously. Microtubules strongly inhibit actin polymerization by mDia2, whereas they moderately inhibit mDia1 and have no effect on INF2. Conversely, actin monomers inhibit microtubule binding/bundling by INF2 but do not affect mDia1 or mDia2. These differences in interactions with microtubules and actin suggest differential function in cellular processes requiring both cytoskeletal elements.


The Pcdp1 Complex Coordinates The Activity Of Dynein Isoforms To Produce Wild-Type Ciliary Motility, Christen G. Dipetrillo, Elizabeth F. Smith Sep 2011

The Pcdp1 Complex Coordinates The Activity Of Dynein Isoforms To Produce Wild-Type Ciliary Motility, Christen G. Dipetrillo, Elizabeth F. Smith

Open Dartmouth: Faculty Open Access Scholarship

Generating the complex waveforms characteristic of beating cilia requires the coordinated activity of multiple dynein isoforms anchored to the axoneme. We previously identified a complex associated with the C1d projection of the central apparatus that includes primary ciliary dyskinesia protein 1 (Pcdp1). Reduced expression of complex members results in severe motility defects, indicating that C1d is essential for wild-type ciliary beating. To define a mechanism for Pcdp1/C1d regulation of motility, we took a functional and structural approach combined with mutants lacking C1d and distinct subsets of dynein arms. Unlike mutants completely lacking the central apparatus, dynein-driven microtubule sliding velocities ...


The Filament-Forming Protein Pil1 Assembles Linear Eisosomes In Fission Yeast, Ruth Kabeche, Suzanne Baldissard, John Hammond, Louisa Howard, James B. Moseley Aug 2011

The Filament-Forming Protein Pil1 Assembles Linear Eisosomes In Fission Yeast, Ruth Kabeche, Suzanne Baldissard, John Hammond, Louisa Howard, James B. Moseley

Open Dartmouth: Faculty Open Access Scholarship

The cortical cytoskeleton mediates a range of cellular activities such as endocytosis, cell motility, and the maintenance of cell rigidity. Traditional polymers, including actin, microtubules, and septins, contribute to the cortical cytoskeleton, but additional filament systems may also exist. In yeast cells, cortical structures called eisosomes generate specialized domains termed MCCs to cluster specific proteins at sites of membrane invaginations. Here we show that the core eisosome protein Pil1 forms linear cortical filaments in fission yeast cells and that purified Pil1 assembles into filaments in vitro. In cells, Pil1 cortical filaments are excluded from regions of cell growth and are ...


Mutant Alcohol Dehydrogenase Leads To Improved Ethanol Tolerance In Clostridium Thermocellum, Steven D. Brown, Adam M. Guss, Tatiana V. Karpinets, Jerry M. Parks Aug 2011

Mutant Alcohol Dehydrogenase Leads To Improved Ethanol Tolerance In Clostridium Thermocellum, Steven D. Brown, Adam M. Guss, Tatiana V. Karpinets, Jerry M. Parks

Open Dartmouth: Faculty Open Access Scholarship

Clostridium thermocellum is a thermophilic, obligately anaerobic, Gram-positive bacterium that is a candidate microorganism for converting cellulosic biomass into ethanol through consolidated bioprocessing. Ethanol intolerance is an important metric in terms of process economics, and tolerance has often been described as a complex and likely multigenic trait for which complex gene interactions come into play. Here, we resequence the genome of an ethanol-tolerant mutant, show that the tolerant phenotype is primarily due to a mutated bifunctional acetaldehyde-CoA/alcohol dehydrogenase gene (adhE), hypothesize based on structural analysis that cofactor specificity may be affected, and confirm this hypothesis using enzyme assays. Biochemical ...


Serum- And Glucocorticoid-Induced Kinase 3 In Recycling Endosomes Mediates Acute Activation Of Na+/H+ Exchanger Nhe3 By Glucocorticoids, Peijian He, Sei-Jung Lee, Songbai Lin, Ursula Seidler, Florian Lang, Geza Fejes-Toth, Aniko Naray-Fejes-Toth, C. Chris Yun Aug 2011

Serum- And Glucocorticoid-Induced Kinase 3 In Recycling Endosomes Mediates Acute Activation Of Na+/H+ Exchanger Nhe3 By Glucocorticoids, Peijian He, Sei-Jung Lee, Songbai Lin, Ursula Seidler, Florian Lang, Geza Fejes-Toth, Aniko Naray-Fejes-Toth, C. Chris Yun

Open Dartmouth: Faculty Open Access Scholarship

Na(+)/H(+) exchanger 3 (NHE3) is the major Na(+) transporter in the intestine. Serum- and glucocorticoid-induced kinase (SGK) 1 interacts with NHE regulatory factor 2 (NHERF2) and mediates activation of NHE3 by dexamethasone (Dex) in cultured epithelial cells. In this study, we compared short-term regulation of NHE3 by Dex in SGK1-null and NHERF2-null mice. In comparison to wild-type mice, loss of SGK1 or NHERF2 significantly attenuated regulation of NHE3 by Dex but did not completely obliterate the effect. We show that transfection of SGK2 or SGK3 in PS120 cells resulted in robust activation of NHE3 by Dex. However, unlike SGK1 ...


The Csc Is Required For Complete Radial Spoke Assembly And Wild-Type Ciliary Motility, Erin E. Dymek, Thomas Heuser, Daniela Nicastro, Elizabeth F. Smith May 2011

The Csc Is Required For Complete Radial Spoke Assembly And Wild-Type Ciliary Motility, Erin E. Dymek, Thomas Heuser, Daniela Nicastro, Elizabeth F. Smith

Open Dartmouth: Faculty Open Access Scholarship

The ubiquitous calcium binding protein, calmodulin (CaM), plays a major role in regulating the motility of all eukaryotic cilia and flagella. We previously identified a CaM and Spoke associated Complex (CSC) and provided evidence that this complex mediates regulatory signals between the radial spokes and dynein arms. We have now used an artificial microRNA (amiRNA) approach to reduce expression of two CSC subunits in Chlamydomonas. For all amiRNA mutants, the entire CSC is lacking or severely reduced in flagella. Structural studies of mutant axonemes revealed that assembly of radial spoke 2 is defective. Furthermore, analysis of both flagellar beating and ...