Other Biochemistry, Biophysics, and Structural Biology Commons^™

Identification And Characterization Of Oxalate Oxidoreductase, A Novel Thiamine Pyrophosphate-Dependent 2-Oxoacid Oxidoreductase That Enables Anaerobic Growth On Oxalate, Elizabeth Pierce, Donald F. Becker, Stephen W. Ragsdale

Department of Biochemistry: Faculty Publications

Moorella thermoacetica is an anaerobic acetogen, a class of bacteria that is found in the soil, the animal gastrointestinal tract, and the rumen. This organism engages the Wood-Ljungdahl pathway of anaerobic CO₂ fixation for heterotrophic or autotrophic growth. This paper describes a novel enzyme, oxalate oxidoreductase (OOR), that enables M. thermoacetica to grow on oxalate, which is produced in soil and is a common component of kidney stones. Exposure to oxalate leads to the induction of three proteins that are subunits of OOR, which oxidizes oxalate coupled to the production of two electrons and CO₂ or bicarbonate. Like …

Structure Of The Proline Utilization A Proline Dehydrogenase Domain Inactivated By N-Propargylglycine Provides Insight Into Conformational Changes Induced By Substrate Binding And Flavin Reduction, Dhiraj Srivastava, Weidong Zhu, William H. Johnson Jr., Christian P. Whitman, Donald F. Becker, John J. Tanner

Department of Biochemistry: Faculty Publications

Proline utilization A (PutA) from Escherichia coli is a flavoprotein that has mutually exclusive roles as a transcriptional repressor of the put regulon and a membrane-associated enzyme that catalyzes the oxidation of proline to glutamate. Previous studies have shown that the binding of proline in the proline dehydrogenase (PRODH) active site and subsequent reduction of the FAD trigger global conformational changes that enhance PutA-membrane affinity. These events cause PutA to switch from its repressor to enzymatic role, but the mechanism by which this signal is propagated from the active site to the distal membrane-binding domain is largely unknown. Here, it …

Identification And Characterization Of Small Compound Inhibitors Of Human Fatp2, Angel Sandoval, Aalap Chokshi, Elliot D. Jesch, Paul N. Black, Concetta C. Dirusso

Department of Biochemistry: Faculty Publications

Fatty acid transport proteins (FATPs) are bifunctional proteins, which transport long chain fatty acids into cells and activate very long chain fatty acids by esterification with coenzyme A. In an effort to understand the linkage between cellular fatty acid transport and the pathology associated with excessive accumulation of exogenous fatty acids, we targeted FATP-mediated fatty acid transport in a high throughput screen of more than 100,000 small diverse chemical compounds in yeast expressing human FATP2 (hsFATP2). Compounds were selected for their ability to depress the transport of the fluorescent long chain fatty acid analogue, C₁-BODIPY-C₁₂. Among …

Quantitative Nuclear Proteomics Identifies Mtor Regulation Of Dna Damage Response, Sricharan Bandhakavi, Young-Mi Kim, Seung-Hyun Ro, Hongwei Xie, Getiria Onsongo, Chang-Bong Jun, Do-Hyung Kim, Timothy J. Griffin

Department of Biochemistry: Faculty Publications

Cellular nutritional and energy status regulates a wide range of nuclear processes important for cell growth, survival, and metabolic homeostasis. Mammalian target of rapamycin (mTOR) plays a key role in the cellular responses to nutrients. However, the nuclear processes governed by mTOR have not been clearly defined. Using isobaric peptide tagging coupled with linear ion trap mass spectrometry, we performed quantitative proteomics analysis to identify nuclear processes in human cells under control of mTOR. Within 3 h of inhibiting mTOR with rapamycin in HeLa cells, we observed downregulation of nuclear abundance of many proteins involved in translation and RNA modification. …

Methanogenesis By Methanosarcina Acetivorans Involves Two Structurally And Functionally Distinct Classes Of Heterodisulfide Reductase, Nicole R. Buan, William W. Metcalf

Department of Biochemistry: Faculty Publications

Biochemical studies have revealed two distinct classes of Coenzyme B-Coenzyme M heterodisulfide (CoB-S-S-CoM) reductase (Hdr), a key enzyme required for anaerobic respiration in methaneproducing archaea. A cytoplasmic HdrABC enzyme complex is found in most methanogens, whereas a membrane-bound HdrED complex is found exclusively in members of the order Methanosarcinales. Unexpectedly, genomic data indicate that multiple copies of both Hdr classes are found in all sequenced Methanosarcinales genomes. The Methanosarcina acetivorans hdrED1 operon is constitutively expressed and required for viability under all growth conditions examined, consistent with HdrED being the primary Hdr. HdrABC appears to be specifically involved in methylotrophic …

Analysis Of Leigh Syndrome Mutations In The Yeast Surf1 Homolog Reveals A New Member Of The Cytochrome Oxidase Assembly Factor Family, Megan Bestwick, Mi-Young Jeong, Oleh Khalimonchuk, Hyung Kim, Dennis R. Winge

Department of Biochemistry: Faculty Publications

Three missense SURF1 mutations identified in patients with Leigh syndrome (LS) were evaluated in the yeast homolog Shy1 protein. Introduction of two of the Leigh mutations, F²⁴⁹T and Y³⁴⁴D, in Shy1 failed to significantly attenuate the function of Shy1 in cytochrome c oxidase (CcO) biogenesis as seen with the human mutations. In contrast, a G¹³⁷E substitution in Shy1 results in a nonfunctional protein conferring a CcO deficiency. The G¹³⁷E Shy1 mutant phenocopied shy1 Δ cells in impaired Cox1 hemylation and low mitochondrial copper. A genetic screen for allele-specific suppressors of the G137E …

The Role Of Coa2 In Hemylation Of Yeast Cox1 Revealed By Its Genetic Interaction With Cox10, Megan Bestwick, Oleh Khalimonchuk, Fabien Pierrel, Dennis R. Winge

Department of Biochemistry: Faculty Publications

Saccharomyces cerevisiae cells lacking the cytochrome c oxidase (CcO) assembly factor Coa2 are impaired in Cox1 maturation and exhibit a rapid degradation of newly synthesized Cox1. The respiratory deficiency of coa2 Δ cells is suppressed either by the presence of a mutant allele of the Cox10 farnesyl transferase involved in heme a biosynthesis or through impaired proteolysis by the disruption of the mitochondrial Oma1 protease. Cox10 with an N196K substitution functions as a robust gain-of-function suppressor of the respiratory deficiency of coa2 Δ cells but lacks suppressor activity for two other CcO assembly mutant strains, the coa1 Δ and shy1 …

Mzm1 Influences A Labile Pool Of Mitochondrial Zinc Important For Respiratory Function, Aaron Atkinson, Oleh Khalimonchuk, Pamela Smith, Hana Sabic, David Eide, Dennis R. Winge

Department of Biochemistry: Faculty Publications

Zinc is essential for function of mitochondria as a cofactor for

several matrix zinc metalloproteins. We demonstrate that a

labile cationic zinc component of low molecular mass exists in

the yeast mitochondrial matrix. This zinc pool is homeostatically

regulated in response to the cellular zinc status. This pool

of zinc is functionally important because matrix targeting of a

cytosolic zinc-binding protein reduces the level of labile zinc

and interferes with mitochondrial respiratory function. We

identified a series of proteins that modulate the matrix zinc

pool, one of which is a novel conserved mitochondrial protein

designated Mzm1. Mutant mzm1∆ cells …

Evolution Of New Enzymatic Function By Structural Modulation Of Cysteine Reactivity In Pseudomonas Fluorescens Isocyanide Hydratase, Mahadevan Lakshminarasimhan, Peter Madzelan, Ruth Nan, Nicole Marie Milkovic, Mark A. Wilson

Department of Biochemistry: Faculty Publications

Isocyanide (formerly isonitrile) hydratase (EC 4.2.1.103) is an enzyme of the DJ-1 superfamily that hydrates isocyanides to yield the corresponding N-formamide. In order to understand the structural basis for isocyanide hydratase (ICH) catalysis, we determined the crystal structures of wild-type and several site-directed mutants of Pseudomonas fluorescens ICH at resolutions ranging from 1.0 to 1.9 Å. We also developed a simple UV-visible spectrophotometric assay for ICH activity using 2-naphthyl isocyanide as a substrate. ICH contains a highly conserved cysteine residue (Cys101) that is required for catalysis and interacts with Asp17, Thr102, and an ordered water molecule in the active …

Functional Hybrid Rubisco Enzymes With Plant Small Subunits And Algal Large Subunits Engineered Rbcs Cdna For Expression In Chlamydomonas, Todor Genkov, Moritz Meyer, Howard Griffiths, Robert J. Spreitzer

Department of Biochemistry: Faculty Publications

There has been much interest in the chloroplast-encoded large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) as a target for engineering an increase in net CO₂ fixation in photosynthesis. Improvements in the enzyme would lead to an increase in the production of food, fiber, and renewable energy. Although the large subunit contains the active site, a family of rbcS nuclear genes encodes the Rubisco small subunits, which can also influence the carboxylation catalytic efficiency and CO₂/O₂ specificity of the enzyme. To further define the role of the small subunit in Rubisco function, small subunits from spinach, Arabidopsis, …

Regulation Of Sealing Ring Formation By L-Plastin And Cortactin In Osteoclasts, Tao Ma, Kavitha Sadashivalah, Nandakumar Madayiputhiya, Meenakshi A. Chellaia

Department of Biochemistry: Faculty Publications

The aim of this study is to identify the exact mechanism(s) by which cytoskeletal structures are modulated during bone resorption. In this study, we have shown the possible role of different actin-binding and signaling proteins in the regulation of sealing ring formation. Our analyses have demonstrated a significant increase in cortactin and a corresponding decrease in L-plastin protein levels in osteoclasts subjected to bone resorption for 18 h in the presence of RANKL, M-CSF, and native bone particles. Time-dependent changes in the localization of L-plastin (in actin aggregates) and cortactin (in the sealing ring) suggest that these proteins may be …

Formation Of The Redox Cofactor Centers During Cox1 Maturation In Yeast Cytochrome Oxidase, Oleh Khalimonchuk, Megan Bestwick, Brigitte Meunier, Talina C. Watts, Dennis R. Winge

Department of Biochemistry: Faculty Publications

The biogenesis of cytochrome c oxidase initiates with synthesis and maturation of the mitochondrionencoded Cox1 subunit prior to the addition of other subunits. Cox1 contains redox cofactors, including the low-spin heme a center and the heterobimetallic heme a₃:Cu_B center. We sought to identify the step in the maturation of Cox1 in which the redox cofactor centers are assembled. Newly synthesized Cox1 is incorporated within one early assembly intermediate containing Mss51 in Saccharomyces cerevisiae. Subsequent Cox1 maturation involves the progression to downstream assembly intermediates involving Coa1 and Shy1. We show that the two heme a cofactor sites …

Targeting The Fatty Acid Transport Proteins (Fatp) To Understand The Mechanisms Linking Fatty Acid Transport To Metabolism, Paul N. Black, Angel Sandoval, Elsa Arias-Barrau, Concetta C. Dirusso

Department of Biochemistry: Faculty Publications

One principal process driving fatty acid transport is vectorial acylation, where fatty acids traverse the membrane concomitant with activation to CoA thioesters. Current evidence is consistent with the proposal that specific fatty acid transport (FATP) isoforms alone or in concert with specific long chain acyl CoA synthetase (Acsl) isoforms function to drive this energy-dependent process. Understanding the details of vectorial acylation is of particular importance as disturbances in lipid metabolism many times leads to elevated levels of circulating free fatty acids, which in turn increases fatty acid internalization and ectopic accumulation of triglycerides. This is associated with changes in fatty …

Crystal Structure Of Acivicin-Inhibited Γ-Glutamyltranspeptidase Reveals Critical Roles For Its C-Terminus In Autoprocessing And Catalysis, Kristin Williams, Sierra Cullati, Aaron Sand, Ekaterina I. Biterova, Joseph J. Barycki

Department of Biochemistry: Faculty Publications

Helicobacter pylori γ-glutamyltranspeptidase (HpGT) is a general γ-glutamyl hydrolase and a

demonstrated virulence factor. The enzyme confers a growth advantage to the bacterium, providing essential amino acid precursors by initiating the degradation of extracellular glutathione and glutamine. HpGT is a member of the N-terminal nucleophile (Ntn) hydrolase superfamily and undergoes autoprocessing to generate the active form of the enzyme. Acivicin is a widely used γ-glutamyltranspeptidase inhibitor that covalently modifies the enzyme, but its precise mechanism of action remains unclear. The time-dependent inactivation of HpGT exhibits a hyperbolic dependence on acivicin concentration with kmax = 0.033 ± 0.006 sec^{−1 …}

Hyaluronidase Activity Of Human Hyal1 Requires Active Site Acidic And Tyrosine Residues, Ling Zhang, Alamelu G. Bharadwaj, Andrew Casper, Joel Barkley, Joseph J. Barycki, Melanie A. Simpson

Department of Biochemistry: Faculty Publications

Hyaluronidases are a family of endolytic glycoside hydrolases

that cleave the β1–4 linkage between N-acetylglucosamine and

glucuronic acid in hyaluronan polymers via a substrate-assisted

mechanism. In humans, turnover of hyaluronan by this enzyme

family is critical for normal extracellular matrix remodeling.

However, elevated expression of the Hyal1 isozyme accelerates

tumor growth and metastatic progression. In this study, we used

structural information, site-directed mutagenesis, and steady

state enzyme kinetics to probe molecular determinants of

human Hyal1 function. Mutagenesis of active site residues

Glu¹³¹ and Tyr²⁴⁷ to Gln and Phe, respectively, eliminated activity

at all hyaluronan concentrations (to 125 …

Mechanistic Details Of Glutathione Biosynthesis Revealed By Crystal Structures Of Saccharomyces Cerevisiae Glutamate Cysteine Ligase, Ekaterina I. Biterova, Joseph J. Barycki

Department of Biochemistry: Faculty Publications

Glutathione is a thiol-disulfide exchange peptide critical for

buffering oxidative or chemical stress, and an essential cofactor

in several biosynthesis and detoxification pathways. The ratelimiting

step in its de novo biosynthesis is catalyzed by glutamate

cysteine ligase, a broadly expressed enzyme for which limited

structural information is available in higher eukaryotic species.

Structural data are critical to the understanding of clinical glutathione

deficiency, as well as rational design of enzyme modulators

that could impact human disease progression. Here, we

have determined the structures of Saccharomyces cerevisiae glutamate

cysteine ligase (ScGCL) in the presence of glutamate and

MgCl2 (2.1 Å; R …

A Role For The Atp7a Copper-Transporting Atpase In Macrophage Bactericidal Activity, Carine White, Jaekwon Lee, Taiho Kambe, Kevin Fritsche, Michael J. Petris

Department of Biochemistry: Faculty Publications

Copper is an essential micronutrient that is necessary for healthy immune function. This requirement is underscored by an increased susceptibility to bacterial infection in copper-deficient animals; however, a molecular understanding of its importance in immune defense is unknown. In this study, we investigated the effect of proinflammatory agents on copper homeostasis in RAW264.7 macrophages. Interferon-γ was found to increase expression of the high affinity copper importer, CTR1, and stimulate copper uptake. This was accompanied by copper-stimulated trafficking of the ATP7A copper exporter from the Golgi to vesicles that partially overlapped with phagosomal compartments. Silencing of ATP7A expression attenuated bacterial killing, …

Copper Transport Activity Of Yeast Ctr1 Is Down-Regulated Via Its C Terminus In Response To Excess Copper, Xiaobin Wu, Devis Sinani, Heejeong Kim, Jaekwon Lee

Department of Biochemistry: Faculty Publications

Copper is an essential yet toxic trace element. The Ctr1 family of proteins plays a critical role for copper uptake in eukaryotes. However, the mechanisms of action of Ctr1 are largely unknown. Our previous data demonstrated that copper transport induces conformational changes in the cytosolic C terminus of the yeast Saccharomyces cerevisiae Ctr1. To define the physiological significance of this molecular event and gain better insights into the mechanism of Ctr1-mediated copper uptake, we have characterized the functional roles of the Ctr1 C terminus.ACtr1 mutant lacking the entire C-terminal cytosolic tail is functional in high affinity copper uptake; however, yeast …

Ulk-Atg13-Fip200 Complexes Mediate Mtor Signaling To The Autophagy Machinery, Chang Hwa Jung, Chang Bong Jun, Seung-Hyun Ro, Young-Mi Kim, Neil Michael Otto, Jing Cao, Mondira Kundu, Do-Hyung Kim

Department of Biochemistry: Faculty Publications

Autophagy, the starvation-induced degradation of bulky cytosolic components, is up-regulated in mammalian cells when nutrient supplies are limited. Although mammalian target of rapamycin (mTOR) is known as the key regulator of autophagy induction, the mechanism by which mTOR regulates autophagy has remained elusive. Here, we identify that mTOR phosphorylates a mammalian homologue of Atg13 and the mammalian Atg1 homologues ULK1 and ULK2. The mammalian Atg13 binds both ULK1 and ULK2 and mediates the interaction of the ULK proteins with FIP200. The binding of Atg13 stabilizes and activates ULK and facilitates the phosphorylation of FIP200 by ULK, whereas knockdown of Atg13 …

Editorial: Dietary Lipid Absorption, Concetta Dirusso, Paul N. Black

Department of Biochemistry: Faculty Publications

The composition of dietary fat influences tissue fatty acid composition, which in turn impacts cellular function through a number of different processes. This includes changes in signaling, lipid metabolism, and transcriptional activities that normally function to maintain intracellular fatty acid homeostasis. The consumption of high levels of dietary fat in excess of caloric expenditure is linked with obesity and the disruption of normal homeostatic mechanisms governing lipid metabolism. Data from the Centers for Disease Control and Prevention show that obesity (defined as a BMI≥30) represents a considerable health concern in the United States. Of particular note is that for adult …

Contribution Of Impaired Myocardial Insulin Signaling To Mitochondrial Dysfunction And Oxidative Stress In The Heart, Sihem Boudina, Heiko Bugger, Sandra Sena, Brian T. O'Neill, Vlad G. Zaha, Olesya Ilkun, Jordan J. Wright, Pradip K. Mazumber, Eric Palfreyman, Timothy J. Tidwell, Heather Theobald, Oleh Khalimonchuk, Benjamin Wayment, Xiaoming Sheng, Kenneth J. Rodnick, Ryan Centini, Dong Chen, Sheldon E. Litwin, Bart E. Weimer, E. Dale Abel

Department of Biochemistry: Faculty Publications

Background—Diabetes-associated cardiac dysfunction is associated with mitochondrial dysfunction and oxidative stress, which may contribute to LV dysfunction. The contribution of altered myocardial insulin action, independently of associated changes in systemic metabolism is incompletely understood. The present study tested the hypothesis that perinatal loss of insulin signaling in the heart impairs mitochondrial function.

Methods and Results—In 8-week-old mice with cardiomyocyte deletion of insulin receptors (CIRKO), inotropic reserves were reduced and mitochondria manifested respiratory defects for pyruvate that was associated with proportionate reductions in catalytic subunits of pyruvate dehydrogenase. Progressive age-dependent defects in oxygen consumption and ATP synthesis with the substrates glutamate …

Proteomic Analysis Of Endodontic Infections By Liquid Chromatography–Tandem Mass Spectrometry, R. Nandakumar, Nandakumar Madayiputhiya, A. F. Fouad

Department of Biochemistry: Faculty Publications

Introduction—Endodontic infections are very prevalent and have a polymicrobial etiology characterized by complex interrelationships between endodontic microorganisms and the host defenses. Proteomic analysis of endodontic infections can provide global insights into the invasion, pathogenicity mechanisms, and multifactorial interactions existing between root canal bacteria and the host in the initiation and progression of apical periodontitis. The purpose of this study was to apply proteomic techniques such as liquid chromatography–tandem mass spectrometry (LC–MS/ MS) for the identification of proteins of bacterial origin present in endodontic infections.

Methods—Endodontic specimens were aseptically obtained from seven patients with root canal infections. Protein mixtures were subjected …

Modulation Of The Heme Electronic Structure And Cystathionine Β-Synthase Activity By Second Coordination Sphere Ligands: The Role Of Heme Ligand Switching In Redox Regulation, Sangita Singh, Peter Madzelan, Jay Stasser, Colin L. Weeks, Donald F. Becker, Thomas G. Spiro, James Penner-Hahn, Ruma Banerjee

Department of Biochemistry: Faculty Publications

In humans, cystathionine β-synthase (CBS) is a hemeprotein, which catalyzes a pyridoxal phosphate (PLP)-dependent condensation reaction. Changes in the heme environment are communicated to the active site, which is ~20 Å away. In this study, we have examined the role of H67 and R266, which are in the second coordination sphere of the heme ligands, H65 and C52 respectively, in modulating the heme's electronic properties and in transmitting information between the heme and active sites. While the H67A mutation is comparable to wild-type CBS, interesting differences are revealed by mutations at the R266 site. The pathogenic mutant, R266K, is moderately …

Heme Regulation Of Human Cystathionine Β-Synthase Activity: Insights From Fluorescence And Raman Spectroscopy, Colin L. Weeks, Sangita Singh, Peter Madzelan, Ruma Banerjee, Thomas G. Spiro

Department of Biochemistry: Faculty Publications

Cystathionine β-synthase (CBS) plays a central role in cysteine metabolism, and malfunction of the enzyme leads to homocystinuria, a devastating metabolic disease. CBS contains a pyridoxal 5′- phosphate (PLP) cofactor which catalyzes the synthesis of cystathionine from homocysteine and serine. Mammalian forms of the enzyme also contain a heme group, which is not involved in catalysis. It may, however, play a regulatory role, since the enzyme is inhibited when CO or NO are bound to the heme. We have investigated the mechanism of this inhibition using fluorescence and resonance Raman spectroscopies. CO binding is found to induce a tautomeric shift …

Glucose Promotes Stress Resistance In The Fungal Pathogen Candida Albicans, Alexandra Rodaki, Iryna M. Bohovych, Brice Enjalbert, Tim Young, Frank C. Odds, Neil A.R. Gow, Alistair J.P. Brown

Department of Biochemistry: Faculty Publications

Metabolic adaptation, and in particular the modulation of carbon assimilatory pathways during disease progression, is thought to contribute to the pathogenicity of Candida albicans. Therefore, we have examined the global impact of glucose upon the C. albicans transcriptome, testing the sensitivity of this pathogen to wide-ranging glucose levels (0.01, 0.1, and 1.0%). We show that, like Saccharomyces cerevisiae, C. albicans is exquisitely sensitive to glucose, regulating central metabolic genes even in response to 0.01% glucose. This indicates that glucose concentrations in the bloodstream (approximate range 0.05–0.1%) have a significant impact upon C. albicans gene regulation. However, in contrast …

Sdh5, A Gene Required For Flavination Of Succinate Dehydrogenase, Is Mutated In Paraganglioma, Huai-Xiang Hao, Oleh Khalimonchuk, Margit Schraders, Noah Dephoure, Jean-Pierre Bayley, Henricus Kunst, Peter Devilee, Cor W.R.J. Cremers, Joshua D. Schiffman, Brandon G. Bentz, Steven P. Gygi, Dennis R. Winge, Hannie Kremer, Jared Rutter

Department of Biochemistry: Faculty Publications

Mammalian mitochondria contain about 1100 proteins, nearly 300 of which are uncharacterized. Given the well-established role of mitochondrial defects in human disease, functional characterization of these proteins may shed new light on disease mechanisms. Starting with yeast as a model system, we investigated an uncharacterized but highly conserved mitochondrial protein (named here Sdh5). Both yeast and human Sdh5 interact with the catalytic subunit of the succinate dehydrogenase (SDH) complex, a component of both the electron transport chain and the tricarboxylic acid cycle. Sdh5 is required for SDH-dependent respiration and for Sdh1 flavination (incorporation of the flavin adenine dinucleotide cofactor). Germline …

Chemchains: A Platform For Simulation And Analysis Of Biochemical Networks Aimed To Laboratory Scientists, Tomáš Helikar, Jim A. Rogers

Department of Biochemistry: Faculty Publications

Background: New mathematical models of complex biological structures and computer simulation software allow modelers to simulate and analyze biochemical systems in silico and form mathematical predictions. Due to this potential predictive ability, the use of these models and software has the possibility to compliment laboratory investigations and help refine, or even develop, new hypotheses. However, the existing mathematical modeling techniques and simulation tools are often difficult to use by laboratory biologists without training in high-level mathematics, limiting their use to trained modelers.

Results: We have developed a Boolean network-based simulation and analysis software tool, ChemChains, which combines the advantages of …

Emergent Decision-Making In Biological Signal Transduction Networks, Tomáš Helikar, John Konvalina, Jack Heidel, Jim A. Rogers

Department of Biochemistry: Faculty Publications

The complexity of biochemical intracellular signal transduction networks has led to speculation that the high degree of interconnectivity that exists in these networks transforms them into an information processing network. To test this hypothesis directly, a large scale model was created with the logical mechanism of each node described completely to allow simulation and dynamical analysis. Exposing the network to tens of thousands of random combinations of inputs and analyzing the combined dynamics of multiple outputs revealed a robust system capable of clustering widely varying input combinations into equivalence classes of biologically relevant cellular responses. This capability was nontrivial in …

Mapping The Functional Interaction Of Sco1 And Cox2 In Cytochrome Oxidase Biogenesis, Kevin Rigby, Paul A. Cobine, Oleh Khalimonchuk, Dennis R. Winge

Department of Biochemistry: Faculty Publications

Sco1 is implicated in the copper metallation of the Cu_A site in

Cox2 of cytochrome oxidase. The structure of Sco1 in the metallated

and apo-conformers revealed structural dynamics primarily

in an exposed region designated loop 8. The structural

dynamics of loop 8 in Sco1 suggests it may be an interface for

interactions with Cox17, the Cu(I) donor and/or Cox2. A series

of conserved residues in the sequence motif ²¹⁷KKYRVYF²²³ on

the leading edge of this loop are shown presently to be important

for yeast Sco1 function. Cells harboring Y219D, R220D, V221D,

and Y222D mutant Sco1 proteins failed …

Function And Redox State Of Mitochondrial Localized Cysteine-Rich Proteins Important In The Assembly Of Cytochrome C Oxidase, Oleh Khalimonchuk, Dennis R. Winge

Department of Biochemistry: Faculty Publications

The cytochrome c oxidase (CcO) complex of the mitochondrial respiratory chain exists within the mitochondrial inner membrane (IM). The biogenesis of the complex is a multi-faceted process requiring multiple assembly factors that function on both faces of the IM. Formation of the two copper centers of CcO occurs within the intermembrane space (IMS) and is dependent on assembly factors with critical cysteinyl thiolates. Two classes of assembly factors exist, one group being soluble IMS proteins and the second class being proteins tethered to the IM. A common motif in the soluble assembly factors is a duplicated Cx₉C sequence …