Research and Publications - Department of Chemistry & Biochemistry

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Synthesis and Characterization of Multifunctional Chitosan- MnFe2O4 Nanoparticles for Magnetic Hyperthermia and Drug Delivery
(MDPI, 2010) Kim, Dong-Hyun; Nikles, David E.; Brazel, Christopher S.; University of Alabama Tuscaloosa
Multifunctional nanoparticles composed of MnFe2O4 were encapsulated in chitosan for investigation of system to combine magnetically-triggered drug delivery and localized hyperthermia for cancer treatment with the previously published capacity of MnFe2O4 to be used as an efficient MRI contrast agent for cancer diagnosis. This paper focuses on the synthesis and characterization of magnetic MnFe2O4 nanoparticles, their dispersion in water and their incorporation in chitosan, which serves as a drug carrier. The surface of the MnFe2O4 nanoparticles was modified with meso-2,3-di-mercaptosuccinic acid (DMSA) to develop stable aqueous dispersions. The nanoparticles were coated with chitosan, and the magnetic properties, heat generation and hydrodynamic size of chitosan-coated MnFe2O4 were evaluated for various linker concentrations and in a range of pH conditions.
Catecholamines up integrates dopamine synthesis and synaptic trafficking
(Wiley, 2011) Wang, Zhe; Ferdousy, Faiza; Lawal, Hakeem; Huang, Zhinong; Daigle, J. Gavin; Izevbaye, Iyare; Doherty, Olugbenga; Thomas, Jerrad; Stathakis, Dean G.; O'Donnell, Janis M.; University of Alabama Tuscaloosa; University of Mississippi Medical Center; University of Mississippi; University of Virginia
The highly reactive nature of dopamine renders dopaminergic neurons vulnerable to oxidative damage. We recently demonstrated that loss-of-function mutations in the Drosophila gene Catecholamines up (Catsup) elevate dopamine pools but, paradoxically, also confer resistance to paraquat, an herbicide that induces oxidative stress-mediated toxicity in dopaminergic neurons. We now report a novel association of the membrane protein, Catsup, with GTP cyclohydrolase rate-limiting enzyme for tetrahydrobiopterin (BH4) biosynthesis and tyrosine hydroxylase, rate-limiting enzyme for dopamine biosynthesis, which requires BH4 as a cofactor. Loss-of-function Catsup mutations cause dominant hyperactivation of both enzymes. Elevated dopamine levels in Catsup mutants coincide with several distinct characteristics, including hypermobility, minimal basal levels of 3,4-dihydroxy-phenylacetic acid, an oxidative metabolite of dopamine, and resistance to the vesicular monoamine transporter inhibitor, reserpine, suggesting that excess dopamine is synaptically active and that Catsup functions in the regulation of synaptic vesicle loading and release of dopamine. We conclude that Catsup regulates and links the dopamine synthesis and transport networks.
Inhibitors of LRRK2 kinase attenuate neurodegeneration and Parkinson-like phenotypes in Caenorhabditis elegans and Drosophila Parkinson's disease models
(Oxford University Press, 2011) Liu, Zhaohui; Hamamichi, Shusei; Lee, Byoung Dae; Yang, Dejun; Ray, Arpita; Caldwell, Guy A.; Caldwell, Kim A.; Dawson, Ted M.; Smith, Wanli W.; Dawson, Valina L.; University of Alabama Tuscaloosa; University of Maryland Baltimore; Johns Hopkins University; University of Alabama Birmingham
Mutations in leucine-rich repeat kinase 2 (LRRK2) have been identified as a genetic cause of familial Parkinson's disease (PD) and have also been found in the more common sporadic form of PD, thus positioning LRRK2 as important in the pathogenesis of PD. Biochemical studies of the disease-causing mutants of LRRK2 implicates an enhancement of kinase activity as the basis of neuronal toxicity and thus possibly the pathogenesis of PD due to LRRK2 mutations. Previously, a chemical library screen identified inhibitors of LRRK2 kinase activity. Here, two of these inhibitors, GW5074 and sorafenib, are shown to protect against G2019S LRRK2-induced neurodegeneration in vivo in Caenorhabditis elegans and in Drosophila. These findings indicate that increased kinase activity of LRRK2 is neurotoxic and that inhibition of LRRK2 activity can have a disease-modifying effect. This suggests that inhibition of LRRK2 holds promise as a treatment for PD.
Polymer Micelles with Crystalline Cores for Thermally Triggered Release
(American Chemical Society, 2012) Glover, Amanda L.; Nikles, Sarah M.; Nikles, Jacqueline A.; Brazel, Christopher S.; Nikles, David E.; University of Alabama Tuscaloosa; University of Alabama Birmingham
Interest in the use of poly(ethylene glycol)-b-polycaprolactone diblock copolymers in a targeted, magnetically triggered drug delivery system has led to this study of the phase behavior of the polycaprolactone core. Four different diblock copolymers were prepared by the ring-opening polymerization of caprolactone from the alcohol terminus of poly(ethylene glycol) monomethylether, M-n approximate to 2000. The critical micelle concentration depended on the degree of polymerization for the polycaprolactone block and was in the range of 2.9 to 41 mg/L. Differential scanning calorimetry curves for polymer solutions with a-concentration above the critical micelle concentration showed a melting endotherm in the range of 40 to 45 degrees C, indicating the polycaprolactone core was semicrystalline. Pyrene was entrapped in the micelle core without interfering with the ability of the polycaprolactone to crystallize. When the polymer solution was heated above the melting point of the micelle core, the pyrene was free to leave the core. Temperature-dependent measurements of the critical micelle concentration and temperature-dependent dynamic light scattering showed that the micelles remain intact at temperatures above the melting point of the polycaprolactone core.
Nrf2b, Novel Zebrafish Paralog of Oxidant-responsive Transcription Factor NF-E2-related Factor 2 (NRF2)
(American Society of Biochemistry and Molecular Biology, 2012) Timme-Laragy, Alicia R.; Karchner, Sibel I.; Franks, Diana G.; Jenny, Matthew J.; Harbeitner, Rachel C.; Goldstone, Jared V.; McArthur, Andrew G.; Hahn, Mark E.; Woods Hole Oceanographic Institution; University of Alabama Tuscaloosa
NF-E2-related factor 2 (NRF2; also called NFE2L2) and related NRF family members regulate antioxidant defenses by activating gene expression via antioxidant response elements (AREs), but their roles in embryonic development are not well understood. We report here that zebrafish (Danio rerio), an important developmental model species, possesses six nrf genes, including duplicated nrf1 and nrf2 genes. We cloned a novel zebrafish nrf2 paralog, nrf2b. The predicted Nrf2b protein sequence shares several domains with the original Nrf2 (now Nrf2a) but lacks the Neh4 transactivation domain. Zebrafish-human comparisons demonstrate conserved synteny involving nrf2 and hox genes, indicating that nrf2a and nrf2b are co-orthologs of human NRF2. nrf2a and nrf2b displayed distinct patterns of expression during embryonic development; nrf2b was more highly expressed at all stages. Embryos in which Nrf2a expression had been knocked down with morpholino oligo-nucleotides were more sensitive to tert-butylhydroperoxide but not tert-butylhydroquinone, whereas knockdown of Nrf2b did not affect sensitivity of embryos to either chemical. Gene expression profiling by microarray identified a specific role for Nrf2b as a negative regulator of several genes, including p53, cyclin G1, and heme oxygenase 1, in embryos. Nrf2a and Nrf2b exhibited different mechanisms of cross-talk with the Ahr2 signaling pathway. Together, these results demonstrate distinct roles for nrf2a and nrf2b, consistent with sub-function partitioning, and identify a novel negative regulatory role for Nrf2b during development. The identification of zebrafish nrf2 co-orthologs will facilitate new understanding of the multiple roles of NRF2 in protecting vertebrate embryos from oxidative damage.
Purification of full-length VP22 from cells infected with HSV-1: A two-pronged approach for the solubilization and purification of viral proteins for use in biochemical studies
(Elsevier, 2012) Dewberry, Ebony J.; Dunkerley, Eric; Duffy, Carol; University of Alabama Tuscaloosa
VP22, encoded by the U(L)49 gene, is one of the most abundant proteins of the herpes simplex virus type 1 (HSV-1) tegument and has been shown to be important for virus replication and spread. However, the exact role(s) played by VP22 in the HSV-1 replication cycle have yet to be delineated. The lack of a procedure to purify full-length VP22 has limited molecular studies on VP22 function. A procedure was developed for the purification of soluble, full-length VP22 from cells infected with HSV-1. A recombinant virus encoding His-tagged VP22 was generated and found to express VP22 at levels comparable to the wild type virus upon infection of Vero cells. By experimenting with a wide variety of cell lysis buffer conditions, several buffers that promote the solubility of full-length VP22 were identified. Buffers that gave the highest levels of solubility were then used in immobilized metal ion affinity chromatography experiments to identify conditions that provided the greatest level of VP22 binding and recovery from cobalt and nickel affinity resins. Using this strategy soluble, full-length VP22 was purified from cells infected with HSV-1. (C) 2012 Elsevier B.V. All rights reserved.
A new species of leopard frog (Anura: Ranidae) from the urban northeastern US
(Elsevier, 2012) Newman, Catherine E.; Feinberg, Jeremy A.; Rissler, Leslie J.; Burger, Joanna; Shaffer, H. Bradley; University of California Davis; Rutgers State University New Brunswick; University of Alabama Tuscaloosa
Past confusion about leopard frog (genus Rana) species composition in the Tri-State area of the US that includes New York (NY), New Jersey (NJ), and Connecticut (CT) has hindered conservation and management efforts, especially where populations are declining or imperiled. We use nuclear and mitochondrial genetic data to clarify the identification and distribution of leopard frog species in this region. We focus on four problematic frog populations of uncertain species affiliation in northern NJ, southeastern mainland NY, and Staten Island to test the following hypotheses: (1) they are conspecific with Rana sphenocephala or R. pipiens, (2) they are hybrids between R. sphenocephala and R. pipiens, or (3) they represent one or more previously undescribed cryptic taxa. Bayesian phylogenetic and cluster analyses revealed that the four unknown populations collectively form a novel genetic lineage, which represents a previously undescribed cryptic leopard frog species, Rana sp. nov. Statistical support for R. sp. nov. was strong in both the Bayesian (pp = 1.0) and maximum-likelihood (bootstrap = 99) phylogenetic analyses as well as the Structure cluster analyses. While our data support recognition of R. sp. nov. as a novel species, we recommend further study including fine-scaled sampling and ecological, behavioral, call, and morphological analyses before it is formally described. (C) 2012 Elsevier Inc. All rights reserved.
Myristoylation Exerts Direct and Allosteric Effects on G alpha Conformation and Dynamics in Solution
(American Chemical Society, 2012) Preininger, Anita M.; Kaya, Ali I.; Gilbert, James A., III; Busenlehner, Laura S.; Armstrong, Richard N.; Hamm, Heidi E.; Vanderbilt University; University of Alabama Tuscaloosa
Coupling of heterotrimeric G proteins to activated G protein-coupled receptors results in nucleotide exchange on the G alpha subunit, which in turn decreases its affinity for both G beta gamma and activated receptors. N-Terminal myristoylation of G alpha subunits aids in membrane localization of inactive G proteins. Despite the presence of the covalently attached myristoyl group, G alpha proteins are highly soluble after GTP binding. This study investigated factors facilitating the solubility of the activated, myristoylated protein. In doing so, we also identified myristoylation-dependent differences in regions of G alpha known to play important roles in interactions with receptors, effectors, and nucleotide binding. Amide hydrogen deuterium exchange and site-directed fluorescence of activated proteins revealed a solvent-protected amino terminus that was enhanced by myristoylation. Furthermore, fluorescence quenching confirmed that the myristoylated amino terminus is in proximity to the Switch H region in the activated protein. Myristoylation also stabilized the interaction between the guanine ring and the base of the alpha 5 helix that contacts the bound nucleotide. The allosteric effects of myristoylation on protein structure, function, and localization indicate that the myristoylated terminus of G alpha(i) functions as a myristoyl switch, with implications for myristoylation in the stabilization of nucleotide binding and in the spatial regulation of G protein signaling.
Biodegradable Orthopedic Magnesium-Calcium (MgCa) Alloys, Processing, and Corrosion Performance
(MDPI, 2012) Salahshoor, Meisam; Guo, Yuebin; University of Alabama Tuscaloosa
Magnesium-Calcium (Mg-Ca) alloy has received considerable attention as an emerging biodegradable implant material in orthopedic fixation applications. The biodegradable Mg-Ca alloys avoid stress shielding and secondary surgery inherent with permanent metallic implant materials. They also provide sufficient mechanical strength in load carrying applications as opposed to biopolymers. However, the key issue facing a biodegradable Mg-Ca implant is the fast corrosion in the human body environment. The ability to adjust degradation rate of Mg-Ca alloys is critical for the successful development of biodegradable orthopedic implants. This paper focuses on the functions and requirements of bone implants and critical issues of current implant biomaterials. Microstructures and mechanical properties of Mg-Ca alloys, and the unique properties of novel magnesium-calcium implant materials have been reviewed. Various manufacturing techniques to process Mg-Ca based alloys have been analyzed regarding their impacts on implant performance. Corrosion performance of Mg-Ca alloys processed by different manufacturing techniques was compared. In addition, the societal and economical impacts of developing biodegradable orthopedic implants have been emphasized.
Abdominal segment reduction Development and evolution of a deeply fixed trait
(Landes Biocience, 2012) Yoder, John H.; University of Alabama Tuscaloosa
When a new student first begins to push flies, an immediate skill that must be learned is sorting the sexes. In Drosophila melanogaster several sexually dimorphic characters can be used to readily distinguish males from females including abdominal pigmentation, male sex combs and genital morphology. Another, often-overlooked, sexual dimorphism is adult abdominal segment number. Externally, adult Drosophila males possess one fewer abdominal segment than females; the terminal pregenital segment apparently either absent or fused with the next-most anterior segment. Beyond known roles for the homeotic protein Abdominal-B (Abd-B) and the sex-determining transcription factor Doublesex (Dsx) as key regulators of this trait, surprisingly little is known about either the morphogenetic processes or the downstream genetics responsible for patterning these events. We have explored both and found that rapid epithelial reorganization during pupation eliminates a nascent terminal male segment. We found this Abd-B-dependent process results from sex-and segment-specific regulation of diverse developmental targets including the wingless gene and surprisingly, dsx itself.(1,2) Here, I review our observations and discuss this trait as a model to explore both dynamics of epithelial morphogenesis as well as the evolution of developmental mechanisms.
Identification of novel ATP13A2 interactors and their role in alpha-synuclein misfolding and toxicity
(Oxford University Press, 2012) Usenovic, Marija; Knight, Adam L.; Ray, Arpita; Wong, Victoria; Brown, Kevin R.; Caldwell, Guy A.; Caldwell, Kim A.; Stagljar, Igor; Krainc, Dimitri; Harvard University; Massachusetts General Hospital; University of Alabama Tuscaloosa; University of Toronto; University of Alabama Birmingham
Lysosomes are responsible for degradation and recycling of bulky cell material, including accumulated misfolded proteins and dysfunctional organelles. Increasing evidence implicates lysosomal dysfunction in several neurodegenerative disorders, including Parkinson's disease and related synucleinopathies, which are characterized by the accumulation of alpha-synuclein (alpha-syn) in Lewy bodies. Studies of lysosomal proteins linked to neurodegenerative disorders present an opportunity to uncover specific molecular mechanisms and pathways that contribute to neurodegeneration. Loss-of-function mutations in a lysosomal protein, ATP13A2 (PARK9), cause Kufor-Rakeb syndrome that is characterized by early-onset parkinsonism, pyramidal degeneration and dementia. While loss of ATP13A2 function plays a role in alpha-syn misfolding and toxicity, the normal function of ATP13A2 in the brain remains largely unknown. Here, we performed a screen to identify ATP13A2 interacting partners, as a first step toward elucidating its function. Utilizing a split-ubiquitin membrane yeast two-hybrid system that was developed to identify interacting partners of full-length integral membrane proteins, we identified 43 novel interactors that primarily implicate ATP13A2 in cellular processes such as endoplasmic reticulum (ER) translocation, ER-to-Golgi trafficking and vesicular transport and fusion. We showed that a subset of these interactors modified alpha-syn aggregation and alpha-syn-mediated degeneration of dopaminergic neurons in Caenorhabditis elegans, further suggesting that ATP13A2 and alpha-syn are functionally linked in neurodegeneration. These results implicate ATP13A2 in vesicular trafficking and provide a platform for further studies of ATP13A2 in neurodegeneration.
1,2,3-Triazole-Heme Interactions in Cytochrome P450: Functionally Competent Triazole-Water-Heme Complexes
(American Chemical Society, 2012) Conner, Kip P.; Vennam, Preethi; Woods, Caleb M.; Krzyaniak, Matthew D.; Bowman, Michael K.; Atkins, William M.; University of Washington; University of Washington Seattle; University of Alabama Tuscaloosa
In comparison to imidazole (IMZ) and 1,2,4-triazole (1,2,4-TRZ), the isosteric 1,2,3-triazole (1,2,3-TRZ) is unrepresented among cytochrome P450 (CYP) inhibitors. This is surprising because 1,2,3-TRZs are easily obtained via "click" chemistry. To understand this underrepresentation of 1,2,3-TRZs among CYP inhibitors, thermodynamic and density functional theory computational studies were performed with unsubstituted IMZ, 1,2,4-TRZ, and 1,2,3-TRZ. The results indicate that the lower affinity of 1,2,3-TRZ for the heme iron includes a large unfavorable entropy term likely originating in solvent-1,2,3-TRZ interactions; the difference is not solely due to differences in the enthalpy of heme-ligand interactions. In addition, the 1,2,3-TRZ fragment was incorporated into a well-established CYP3A4 substrate and mechanism-based inactivator, 17-alpha-ethynylestradiol (17EE), via click chemistry. This derivative, 17-click, yielded optical spectra consistent with low-spin ferric heme iron (type II) in contrast to 17EE, which yields a high-spin complex (type I). Furthermore, the rate of CYP3A4-mediated metabolism of 17-click was comparable to that of 17EE, with a different regioselectivity. Surprisingly, continuous-wave electron paramagnetic resonance (EPR) and HYSCORE EPR spectroscopy indicate that 17-click does not displace water from the sixth axial ligand position of CYP3A4 as expected for a type II ligand. We propose a binding model in which 17-click pendant 1,2,3-TRZ hydrogen bonds with the sixth axial water ligand. The results demonstrate the potential for 1,2,3-TRZ to form metabolically labile water-bridged low-spin heme complexes, consistent with recent evidence that nitrogenous type II ligands of CYPs can be efficiently metabolized. The specific case of [CYP3A4 center dot 17-click] highlights the risk of interpreting CYP-ligand complex structure on the basis of optical spectra.
Different 8-Hydroxyquinolines Protect Models of TDP-43 Protein, alpha-Synuclein, and Polyglutamine Proteotoxicity through Distinct Mechanisms
(American Society of Biochemistry and Molecular Biology, 2012) Tardiff, Daniel F.; Tucci, Michelle L.; Caldwell, Kim A.; Caldwell, Guy A.; Lindquist, Susan; Massachusetts Institute of Technology (MIT); Whitehead Institute; University of Alabama Tuscaloosa; Howard Hughes Medical Institute
No current therapies target the underlying cellular pathologies of age-related neurodegenerative diseases. Model organisms provide a platform for discovering compounds that protect against the toxic, misfolded proteins that initiate these diseases. One such protein, TDP-43, is implicated in multiple neurodegenerative diseases, including amyotrophic lateral sclerosis and frontotemporal lobar degeneration. In yeast, TDP-43 expression is toxic, and genetic modifiers first discovered in yeast have proven to modulate TDP-43 toxicity in both neurons and humans. Here, we describe a phenotypic screen for small molecules that reverse TDP-43 toxicity in yeast. One group of hit compounds was 8-hydroxyquinolines (8-OHQ), a class of clinically relevant bioactive metal chelators related to clioquinol. Surprisingly, in otherwise wild-type yeast cells, different 8-OHQs had selectivity for rescuing the distinct toxicities caused by the expression of TDP-43, alpha-synuclein, or polyglutamine proteins. In fact, each 8-OHQ synergized with the other, clearly establishing that they function in different ways. Comparative growth and molecular analyses also revealed that 8-OHQs have distinct metal chelation and ionophore activities. The diverse bioactivity of 8-OHQs indicates that altering different aspects of metal homeostasis and/or metalloprotein activity elicits distinct protective mechanisms against several neurotoxic proteins. Indeed, phase II clinical trials of an 8-OHQ has produced encouraging results in modifying Alzheimer disease. Our unbiased identification of 8-OHQs in a yeast TDP-43 toxicity model suggests that tailoring 8-OHQ activity to a particular neurodegenerative disease may be a viable therapeutic strategy.
Variational approach for nonpolar solvation analysis
(American Institute of Physics, 2012) Chen, Zhan; Zhao, Shan; Chun, Jaehun; Thomas, Dennis G.; Baker, Nathan A.; Bates, Peter W.; Wei, G. W.; Michigan State University; University of Alabama Tuscaloosa; United States Department of Energy (DOE); Pacific Northwest National Laboratory
Solvation analysis is one of the most important tasks in chemical and biological modeling. Implicit solvent models are some of the most popular approaches. However, commonly used implicit solvent models rely on unphysical definitions of solvent-solute boundaries. Based on differential geometry, the present work defines the solvent-solute boundary via the variation of the nonpolar solvation free energy. The solvation free energy functional of the system is constructed based on a continuum description of the solvent and the discrete description of the solute, which are dynamically coupled by the solvent-solute boundaries via van der Waals interactions. The first variation of the energy functional gives rise to the governing Laplace-Beltrami equation. The present model predictions of the nonpolar solvation energies are in an excellent agreement with experimental data, which supports the validity of the proposed nonpolar solvation model. (C) 2012 American Institute of Physics. [http://dx.doi.org/10.1063/1.4745084]
Evaluation of Chewing and Swallowing Sensors for Monitoring Ingestive Behavior
(American Scientific, 2013) Fontana, Juan M.; Sazonov, Edward S.; University of Alabama Tuscaloosa
Monitoring Ingestive Behavior (MIB) of individuals is of special importance to identify and treat eating patterns associated with obesity and eating disorders. Current methods for MIB require subjects reporting every meal consumed, which is burdensome and tend to increase the reporting bias over time. This study presents an evaluation of the burden imposed by two wearable sensors for MIB during unrestricted food intake: a strain sensor to detect chewing events and a throat microphone to detect swallowing sounds. A total of 30 healthy subjects with various levels of adiposity participated in experiments involving the consumption of four meals in four different visits. A questionnaire was handled to subjects at the end of the last visit to evaluate the sensors burden in terms of the comfort levels experienced. Results showed that sensors presented high comfort levels as subjects indicated that the way they ate their meal was not considerably affected by the presence of the sensors. A statistical analysis showed that chewing sensor presented significantly higher comfort levels than the swallowing sensor. The outcomes of this study confirmed the suitability of the chewing and swallowing sensors for MIB and highlighted important aspects of comfort that should be addressed to obtain acceptable and less burdensome wearable sensors for MIB.
A Caged, Destabilized, Free Radical Intermediate in the Q-Cycle
(Wiley-VCH, 2013) Vennam, Preethi R.; Fisher, Nicholas; Krzyaniak, Matthew D.; Kramer, David M.; Bowman, Michael K.; University of Alabama Tuscaloosa; Michigan State University; United States Department of Energy (DOE)
The Rieske/cytochrome b complexes, also known as cytochrome bc complexes, catalyze a unique oxidant-induced reduction reaction at their quinol oxidase (Q(o)) sites, in which substrate hydroquinone reduces two distinct electron transfer chains, one through a series of high-potential electron carriers, the second through low-potential cytochrome b. This reaction is a critical step in energy storage by the Q-cycle. The semiquinone intermediate in this reaction can reduce O-2 to produce deleterious superoxide. It is yet unknown how the enzyme controls this reaction, though numerous models have been proposed. In previous work, we trapped a Q-cycle semiquinone anion intermediate, termed SQ(o), in bacterial cytochrome bc(1) by rapid freeze-quenching. In this work, we apply pulsed-EPR techniques to determine the location and properties of SQ(o) in the mitochondrial complex. In contrast to semiquinone inter-mediates in other enzymes, SQ(o) is not thermodynamically stabilized, and can even be destabilized with respect to solution. It is trapped in Q(o) at a site that is distinct from previously described inhibitor-binding sites, yet sufficiently close to cytochrome b(L) to allow rapid electron transfer. The binding site and EPR analyses show that SQ(o) is not stabilized by hydrogen bonds to proteins. The formation of SQ(o) involves "stripping" of both substrate -OH protons during the initial oxidation step, as well as conformational changes of the semiquinone and Q(o) proteins. The resulting charged radical is kinetically trapped, rather than thermodynamically stabilized (as in most enzymatic semiquinone species), conserving redox energy to drive electron transfer to cytochrome b(L) while minimizing certain Q-cycle bypass reactions, including oxidation of prereduced cytochrome b and reduction of O-2.
Escherichia coli SufE Sulfur Transfer Protein Modulates the SufS Cysteine Desulfurase through Allosteric Conformational Dynamics
(American Society of Biochemistry and Molecular Biology, 2013) Singh, Harsimran; Dai, Yuyuan; Outten, F. Wayne; Busenlehner, Laura S.; University of Alabama Tuscaloosa; University of South Carolina Columbia
Background: SufS cysteine desulfurase mobilizes sulfur for stress-responsive iron-sulfur cluster biogenesis in bacteria. Results: Interaction with the sulfur transfer protein SufE triggers conformational changes in the SufS active site. Conclusion: SufE participates in allosteric regulation of SufS activity in addition to being a sulfur acceptor. Significance: New insight into sulfur mobilization and transfer during iron-sulfur cluster metallocofactor assembly is provided. Fe-S clusters are critical metallocofactors required for cell function. Fe-S cluster biogenesis is carried out by assembly machinery consisting of multiple proteins. Fe-S cluster biogenesis proteins work together to mobilize sulfide and iron, form the nascent cluster, traffic the cluster to target metalloproteins, and regulate the assembly machinery in response to cellular Fe-S cluster demand. A complex series of protein-protein interactions is required for the assembly machinery to function properly. Despite considerable progress in obtaining static three-dimensional structures of the assembly proteins, little is known about transient protein-protein interactions during cluster assembly or the role of protein dynamics in the cluster assembly process. The Escherichia coli cysteine desulfurase SufS (EC 2.8.1.7) and its accessory protein SufE work together to mobilize persulfide from l-cysteine, which is then donated to the SufB Fe-S cluster scaffold. Here we use amide hydrogen/deuterium exchange mass spectrometry (HDX-MS) to characterize SufS-SufE interactions and protein dynamics in solution. HDX-MS analysis shows that SufE binds near the SufS active site to accept persulfide from Cys-364. Furthermore, SufE binding initiates allosteric changes in other parts of the SufS structure that likely affect SufS catalysis and alter SufS monomer-monomer interactions. SufE enhances the initial l-cysteine substrate binding to SufS and formation of the external aldimine with pyridoxal phosphate required for early steps in SufS catalysis. Together, these results provide a new picture of the SufS-SufE sulfur transferase pathway and suggest a more active role for SufE in promoting the SufS cysteine desulfurase reaction for Fe-S cluster assembly.
Long-Term Exposure to [Cr3O(O2CCH2CH3)(6)(H2O)(3)](+) in Wistar Rats Fed Normal or High-Fat Diets Does Not Alter Glucose Metabolism
(Humana Press, 2013) Herring, Betty J.; Logsdon, Amanda L.; Lockard, Jarrett E.; Miller, Brittany M.; Kim, Hanna; Calderon, Eric A.; Vincent, John B.; Bailey, Melissa M.; University of Alabama Tuscaloosa
The essentiality of chromium(III) has been the subject of much debate, particularly in healthy subjects. Chromium(III)-containing supplements are widely used for body mass loss, building of lean muscle mass, and improving glucose and lipid metabolism. [Cr3O(O2CCH2CH3)(6)(H2O)(3)](+), Cr3, is one of the most-studied chromium nutritional supplements. The current study evaluates the effects of long-term (15 months) supplementation with Cr3 on body mass and glucose metabolism in Wistar rats on traditional and cafeteria-style (high fat, high carbohydrate) diets. Male Wistar rats were randomly assigned to one of four treatment groups: (1) control diet (milled Harlan Teklad LM-485 rodent diet), (2) control diet + 1 mg Cr3/kg body mass/day, (3) a cafeteria-style (CAF) diet (high fat, high carbohydrate), or (4) CAF diet + 1 mg Cr3/kg/day. Cr3 supplementation had no effect on fasting blood glucose levels or blood glucose levels in response to glucose and insulin challenges. Rats consuming the CAF + Cr3 diet tended to have a significantly higher body mass than rats consuming the CAF diet, but necropsy results showed no difference in visceral fat or body wall thickness between groups. These data suggest that long-term Cr3 supplementation does not significantly affect body mass in rats consuming a normal diet or glucose levels or metabolism in rats consuming either diet.
Skew projection of echo-detected EPR spectra for increased sensitivity and resolution
(Elsevier, 2013) Bowman, Michael K.; Krzyaniak, Matthew D.; Cruce, Alex A.; Weber, Ralph T.; University of Alabama Tuscaloosa; Bruker Corporation; Bruker BioSpin GmbH
The measurement of EPR spectra during pulsed EPR experiments is commonly accomplished by recording the integral of the electron spin echo as the applied magnetic field is stepped through the spectrum. This approach to echo-detected EPR spectral measurement (ED-EPR) limits sensitivity and spectral resolution and can cause gross distortions in the resulting spectra because some of the information present in the electron spin echo is discarded in such measurements. However, Fourier transformation of echo shapes measured at a series of magnetic field values followed by skew projection onto either a magnetic field or resonance frequency axis can increase both spectral resolution and sensitivity without the need to trade one against the other. Examples of skew-projected spectra with single crystals, glasses and powders show resolution improvements as large as a factor of seven with sensitivity increases of as much as a factor of five. (C) 2013 Elsevier Inc. All rights reserved.
Site-Specific Variability in the Chemical Diversity of the Antarctic Red Alga Plocamium cartilagineum
(MDPI, 2013) Young, Ryan M.; von Salm, Jacqueline L.; Amsler, Margaret O.; Lopez-Bautista, Juan; Amsler, Charles D.; McClintock, James B.; Baker, Bill J.; University of South Florida; University of Alabama Birmingham; University of Alabama Tuscaloosa
Plocamium cartilagineum is a common red alga on the benthos of Antarctica and can be a dominant understory species along the western Antarctic Peninsula. Algae from this region have been studied chemically, and like "P. cartilagineum" from other worldwide locations where it is common, it is rich in halogenated monoterpenes, some of which have been implicated as feeding deterrents toward sympatric algal predators. Secondary metabolites are highly variable in this alga, both qualitatively and quantitatively, leading us to probe individual plants to track the possible link of variability to genetic or other factors. Using cox1 and rbcL gene sequencing, we find that the Antarctic alga divides into two closely related phylogroups, but not species, each of which is further divided into one of five chemogroups. The chemogroups themselves, defined on the basis of Bray-Curtis similarity profiling of GC/QqQ chromatographic analyses, are largely site specific within a 10 km(2) area. Thus, on the limited geographical range of this analysis, P. cartilagineum displays only modest genetic radiation, but its secondary metabolome was found to have experienced more extensive radiation. Such metabogenomic divergence demonstrated on the larger geographical scale of the Antarctic Peninsula, or perhaps even continent-wide, may contribute to the discovery of cryptic speciation.

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