Chemistry Commons^™

Connexin-46/50 In A Dynamic Lipid Environment Resolved By Cryoem At 1.9 Å, Jonathan A. Flores, Bassam G. Haddad, Kimberly A. Dolan, Janette Bernadette Myers, Craig C. Yoshioka, Jeremy Yoshioka, Daniel M. Zuckerman, Steve L. Reichow

Chemistry Faculty Publications and Presentations

Gap junctions establish direct pathways for cells to transfer metabolic and electrical messages. The local lipid environment is known to affect the structure, stability and intercellular channel activity of gap junctions; however, the molecular basis for these effects remains unknown. Here, we incorporate native connexin-46/50 (Cx46/50) intercellular channels into a dual lipid nanodisc system, mimicking a native cell-to-cell junction. Structural characterization by CryoEM reveals a lipid-induced stabilization to the channel, resulting in a 3D reconstruction at 1.9 Å resolution. Together with all-atom molecular dynamics simulations, it is shown that Cx46/50 in turn imparts long-range stabilization to the dynamic local lipid …

Size-Dependent Interactions Of Lipid-Coated Gold Nanoparticles: Developing A Better Mechanistic Understanding Through Model Cell Membranes And In Vivo Toxicity, Arek M. Engstrom, Ryan A. Faase, Joe E. Baio, Marilyn R. Mackiewicz, Stacey L. Harper

Chemistry Faculty Publications and Presentations

Introduction: Humans are intentionally exposed to gold nanoparticles (AuNPs) where they are used in variety of biomedical applications as imaging and drug delivery agents as well as diagnostic and therapeutic agents currently in clinic and in a variety of upcoming clinical trials. Consequently, it is critical that we gain a better understanding of how physiochemical properties such as size, shape, and surface chemistry drive cellular uptake and AuNP toxicity in vivo. Understanding and being able to manipulate these physiochemical properties will allow for the production of safer and more efficacious use of AuNPs in biomedical applications.
Methods and Materials: Here, …

Limited Sequence Diversity Within A Population Supports Prebiotic Rna Reproduction, Ryo Mizuuchi, Niles Lehman

Chemistry Faculty Publications and Presentations

The origins of life require the emergence of informational polymers capable of reproduction. In the RNA world on the primordial Earth, reproducible RNA molecules would have arisen from a mixture of compositionally biased, poorly available, short RNA sequences in prebiotic environments. However, it remains unclear what level of sequence diversity within a small subset of population is required to initiate RNA reproduction by prebiotic mechanisms. Here, using a simulation for template-directed recombination and ligation, we explore the effect of sequence diversity in a given population for the onset of RNA reproduction. We show that RNA reproduction is improbable in low …

The T6a Modification Acts As A Positive Determinant For The Anticodon Nuclease Prrc, And Is Distinctively Nonessential In Streptococcus Mutans, Jo Marie Bacusmo, Silvia S. Orsini, Jennifer Hu, Michael Demott, Patrick C. Thiaville, Ameer Elfarash, Mellie June Paulines, Diego Rojas-Benítez, Birthe Meineke, Christopher Deutsch, Dirk Iwata-Reuyl, Patrick A. Limbach, Peter C. Dedon, Kelly C. Rice, Stewart Shuman, Valérie De Crécy-Lagard

Chemistry Faculty Publications and Presentations

Endoribonuclease toxins (ribotoxins) are produced by bacteria and fungi to respond to stress, eliminate non-self competitor species, or interdict virus infection. PrrC is a bacterial ribotoxin that targets and cleaves tRNALys UUU in the anticodon loop. In vitro studies suggested that the post-transcriptional modification threonylcarbamoyl adenosine (t⁶A) is required for PrrC activity but this prediction had never been validated in vivo. Here, by using t⁶A-deficient yeast derivatives, it is shown that t⁶A is a positive determinant for PrrC proteins from various bacterial species. Streptococcus mutans is one of the few bacteria where the t …

Nmr-Based Structural Analysis Of Threonylcarbamoyl-Amp Synthase And Its Substrate Interactions, Kimberly A. Harris, Benjamin G. Bobay, Kathryn L. Sarachan, Alexis F. Sims, Yann Bilbille, Christopher Deutsch, Dirk Iwata-Reuyl, Paul F. Agris

Chemistry Faculty Publications and Presentations

The hypermodified nucleoside N(6)-threonylcarbamoyladenosine (t(6)A37) is present in many distinct tRNA species and has been found in organisms in all domains of life. This post-transcriptional modification enhances translation fidelity by stabilizing the anticodon/codon interaction in the ribosomal decoding site. The biosynthetic pathway of t(6)A37 is complex and not well understood. In bacteria, the following four proteins have been discovered to be both required and sufficient for t(6)A37 modification: TsaC, TsaD, TsaB, and TsaE. Of these, TsaC and TsaD are members of universally conserved protein families. Although TsaC has been shown to catalyze the formation of L-threonylcarbamoyl-AMP, a key intermediate in …

Hydrophobic Surfactant Proteins Strongly Induce Negative Curvature, Mariya Chavarha, Ryan W. Loney, Shankar B. Rananavare, Stephen B. Hall

Chemistry Faculty Publications and Presentations

The hydrophobic surfactant proteins SP-B and SP-C greatly accelerate the adsorption of vesicles containing the surfactant lipids to form a film that lowers the surface tension of the air/water interface in the lungs. Pulmonary surfactant enters the interface by a process analogous to the fusion of two vesicles. As with fusion, several factors affect adsorption according to how they alter the curvature of lipid leaflets, suggesting that adsorption proceeds via a rate-limiting structure with negative curvature, in which the hydrophilic face of the phospholipid leaflets is concave. In the studies reported here, we tested whether the surfactant proteins might promote …

Differential Effects Of The Hydrophobic Surfactant Proteins On The Formation Of Inverse Bicontinuous Cubic Phases, Mariya Chavarha, Ryan W. Loney, Kamlesh Kumar, Shankar B. Rananavare, Stephen B. Hall

Chemistry Faculty Publications and Presentations

Prior studies have shown that the biological mixture of the two hydrophobic surfactant proteins, SP-B and SP-C, produces faster adsorption of the surfactant lipids to an air/water interface, and that they induce 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE) to form inverse bicontinuous cubic phases. SP-B has a much greater effect than SP-C on adsorption. If the two proteins induce formation of the bicontinuous structures and faster adsorption by similar mechanisms, then they should also have differential ability to form the cubic phases. To test this hypothesis, we measured small angle X-ray scattering on the individual proteins combined with POPE. SP-B replicated the doserelated …

A Fast Response Highly Selective Probe For The Detection Of Glutathione In Human Blood Plasma, Yixing Guo, Xiaofeng Yang, Lovemore Hakuna, Aabha Barve, Jorge O. Escobedo, Mark Lowry, Robert M. Strongin

Chemistry Faculty Publications and Presentations

A fluorescent probe for glutathione (GSH) detection was developed. Our study indicates a possible mechanism which couples a conjugate addition and micelle-catalyzed large membered ring formation/elimination sequence. This method enables excellent selectivity towards GSH over other biological thiols such as cysteine (Cys) and homocysteine (Hcy). The proposed method is precise with a relative standard deviation (R.S.D) lower than 6% (n = 3) and has been successfully applied to determine GSH in human plasma with recoveries between 99.2% and 102.3%.

Tension Directly Stabilizes Reconstituted Kinetochore-Microtubule Attachments, Bungo Akiyoshi, Krishna K. Sarangapani, Andrew F. Powers, Christian R. Nelson, Steve Reichow, Hugo Arellano-Santoyo, Tamir Gonen, Jeffrey A. Ranish, Charles L. Asbury, Sue Biggins

Chemistry Faculty Publications and Presentations

Kinetochores are macromolecular machines that couple chromosomes to dynamic microtubule tips during cell division, thereby generating force to segregate the chromosomes. Accurate segregation depends on selective stabilization of correct ‘bi-oriented’ kinetochore-microtubule attachments, which come under tension due to opposing forces exerted by microtubules. Tension is thought to stabilize these bi-oriented attachments indirectly, by suppressing the destabilizing activity of a kinase, Aurora B. However, a complete mechanistic understanding of the role of tension requires reconstitution of kinetochore-microtubule attachments for biochemical and biophysical analyses in vitro. Here we show that native kinetochore particles retaining the majority of kinetochore proteins can be …

Organometallic Iron(Iii)-Salophene Exerts Cytotoxic Properties In Neuroblastoma Cells Via Mapk Activation And Ros Generation, Kyu Kwang Kim, Rakesh K. Singh, Robert M. Strongin, Richard G. Moore, Laurent Brard, Thilo S. Lange

Chemistry Faculty Publications and Presentations

The objective of the present study was to investigate the specific effects of Iron(III)-salophene (Fe-SP) on viability, morphology, proliferation, cell cycle progression, ROS generation and pro-apoptotic MAPK activation in neuroblastoma (NB) cells. A NCI-DTP cancer screen revealed that Fe-SP displayed high toxicity against cell lines of different tumor origin but not tumor type-specificity. In a viability screen Fe-SP exhibited high cytotoxicity against all three NB cell lines tested. The compound caused cell cycle arrest in G1 phase, suppression of cells progressing through S phase, morphological changes, disruption of the mitochondrial membrane depolarization potential, induction of apoptotic markers as well as …

Scoring Function To Predict Solubility Mutagenesis, Ye Tian, Christopher Deutsch, Bala Krishnamoorthy

Chemistry Faculty Publications and Presentations

Background: Mutagenesis is commonly used to engineer proteins with desirable properties not present in the wild type (WT) protein, such as increased or decreased stability, reactivity, or solubility. Experimentalists often have to choose a small subset of mutations from a large number of candidates to obtain the desired change, and computational techniques are invaluable to make the choices. While several such methods have been proposed to predict stability and reactivity mutagenesis, solubility has not received much attention. Results: We use concepts from computational geometry to define a three body scoring function that predicts the change in protein solubility due to …

Structure Of The Cholera Toxin Secretion Channel In Its Closed State, Steve Reichow, Konstantin V. Korotkov, Wim Gj Hol, Tamir Gonen

Chemistry Faculty Publications and Presentations

The type II secretion system (T2SS) is a macromolecular complex spanning the inner and outer membranes of Gram-negative bacteria. Remarkably, the T2SS secretes folded proteins including multimeric assemblies like cholera toxin and heat-labile enterotoxin from Vibrio cholerae and enterotoxigenic Escherichia coli, respectively. The major outer membrane T2SS protein is the “secretin” GspD. Electron cryomicroscopy reconstruction of the V. cholerae secretin at 19 Å resolution reveals a dodecameric structure reminiscent of a barrel with a large channel at its center that appears to contain a closed periplasmic gate. The GspD periplasmic domain forms a vestibule with a conserved constriction, and binds …

Cooperative Interaction Of Transcription Termination Factors With The Rna Polymerase Ii C-Terminal Domain, Bradley M. Lunde, Steve Reichow, Minkyu Kim, Hyunsuk Suh, Thomas C. Leeper, Fan Yang, Hannes Mutschler, Stephen Buratowski, Anton Meinhart, Gabriele Varani

Chemistry Faculty Publications and Presentations

Phosphorylation of the C-terminal domain of RNA polymerase II controls the co-transcriptional assembly of RNA processing and transcription factors. Recruitment relies on conserved CTDinteracting domains that recognize different CTD phosphoisoforms during the transcription cycle, but the molecular basis for their specificity remains unclear. We show that the CTD-interacting domains of two transcription termination factors, Rtt103 and Pcf11, achieve high affinity and specificity both by specifically recognizing the phosphorylated CTD and by cooperatively binding to neighboring CTD repeats. Single amino acid mutations at the protein-protein interface abolish cooperativity and affect recruitment at the 3′-end processing site in vivo. We suggest that …

Quasispecies-Like Behavior Observed In Catalytic Rna Populations Evolving In A Test Tube, Carolina Diaz Arenas, Niles Lehman

Chemistry Faculty Publications and Presentations

Background: During the RNA World, molecular populations were probably very small and highly susceptible to the force of strong random drift. In conjunction with Muller's Ratchet, this would have imposed difficulties for the preservation of the genetic information and the survival of the populations. Mechanisms that allowed these nascent populations to overcome this problem must have been advantageous.

Results: Using continuous in vitro evolution experimentation with an increased mutation rate imposed by MnCl2, it was found that clonal 100-molecule populations of ribozymes clearly exhibit certain characteristics of a quasispecies. This is the first time this has been seen …

Lipid-Protein Interactions Probed By Electron Crystallography, Steve L. Reichow, Tamir Gonen

Chemistry Faculty Publications and Presentations

Electron crystallography is arguably the only electron cryomicroscopy (cryoEM) technique able to deliver an atomic-resolution structure of membrane proteins embedded in the lipid-bilayer. In the electron crystallographic structures of the light driven ion pump, bacteriorhodopsin, and the water channel, aquaporin-0, sufficiently high resolution was obtained and both lipid and protein were visualized, modeled and described in detail. An extensive network of lipid-protein interactions mimicking native membranes is established and maintained in two-dimensional (2D) crystalline vesicles used for structural analysis by electron crystallography. Lipids are tightly integrated into the protein's architecture where they can affect the function, structure, quaternary assembly and …

Non-Canonical Binding Of Calmodulin To Aquaporin-0: Implications For Channel Regulation, Steve Reichow, Tamir Gonen

Chemistry Faculty Publications and Presentations

Aquaporins (AQPs) are a family of ubiquitous membrane channels that conduct water across cell membranes. AQPs form homo-tetramers containing four functional and independent water pores. Aquaporin-0 (AQP0) is expressed in the eye lens where its water permeability is regulated by calmodulin (CaM). Here we use a combination of biochemical methods and NMR spectroscopy to probe the interaction between AQP0 and CaM. We show CaM binds the AQP0 C-terminal domain in a calcium dependent manner. We demonstrate that only two CaM molecules bind a single AQP0 tetramer in a non-canonical fashion, suggesting a form of co-operativity between AQP0 monomers. Based on …

Electron Crystallography Of Aquaporins, Simeon Andrews, Steve Reichow, Tamir Gonen

Chemistry Faculty Publications and Presentations

Aquaporins are a family of ubiquitous membrane proteins that form a pore for the permeation of water. Both electron and X-ray crystallography played major roles in determining the atomic structures of a number of aquaporins. This review focuses on electron crystallography, and its contribution to the field of aquaporin biology. We briefly discuss electron crystallography and the two-dimensional crystallization process. We describe features of aquaporins common to both electron and X-ray crystallographic structures; as well as some structural insights unique to electron crystallography, including aquaporin junction formation and lipid-protein interactions.

Electrical Detection Of The Temperature Induced Melting Transition Of A Dna Hairpin Covalently Attached To Gold Interdigitated Microelectrodes, Greg P. Brewood, Yaswanth Rangineni, Daniel J. Fish, Ashwini Bhandiwad, David R. Evans, Raj Solanki, Albert S. Benight

Chemistry Faculty Publications and Presentations

The temperature induced melting transition of a self-complementary DNA strand covalently attached at the 5' end to the surface of a gold interdigitated microelectrode (GIME) was monitored in a novel, label-free, manner. The structural state of the hairpin was assessed by measuring four different electronic properties of the GIME (capacitance, impedance, dissipation factor and phase angle) as a function of temperature from 25 degrees C to 80 degrees C. Consistent changes in all four electronic properties of the GIME were observed over this temperature range, and attributed to the transition of the attached single-stranded DNA (ssDNA) from an intramolecular, folded …

Chiral Recognition Of Amino Acids By Use Of A Fluorescent Resorcinarene, Gerald I. Richard, Hadi M. Marwani, Shan Jiang, Sayo O. Fakayode, Mark Lowry, Robert M. Strongin, Isiah M. Warner

Chemistry Faculty Publications and Presentations

The spectroscopic properties of a chiral boronic acid based resorcinarene macrocycle employed for chiral analysis were investigated. Specifically, the emission and excitation characteristics of tetraarylboronate resorcinarene macrocycle (TBRM) and its quantum yield were evaluated. The chiral selector TBRM was investigated as a chiral reagent for the enantiomeric discrimination of amino acids using steady-state fluorescence spectroscopy. Chiral recognition of amino acids in the presence of the macrocycle was based on diastereomeric complexes. Results demonstrated that TBRM had better chiral discrimination ability for lysine as compared to the other amino acids. Partial least squares regression modeling (PLS-1) of spectral data for macrocycle-lysine …

The Structure And Function Of Small Nucleolar Ribonucleoproteins, Steve Reichow, Tomoko Hamma, Adrian R. Ferré-D’Amaré, Gabriele Varani

Chemistry Faculty Publications and Presentations

Eukaryotes and archaea use two sets of specialized ribonucleoproteins (RNPs) to carry out sequencespecific methylation and pseudouridylation of RNA, the two most abundant types of modifications of cellular RNAs. In eukaryotes, these protein–RNA complexes localize to the nucleolus and are called small nucleolar RNPs (snoRNPs), while in archaea they are known as small RNPs (sRNP). The C/D class of sno(s)RNPs carries out ribose- 20-O-methylation, while the H/ACA class is responsible for pseudouridylation of their RNA targets. Here, we review the recent advances in the structure, assembly and function of the conserved C/D and H/ACA sno(s)RNPs. Structures of each of the …

Dna Multiplex Hybridization On Microarrays And Thermodynamic Stability In Solution: A Direct Comparison, Daniel J. Fish, M. Todd Horne, Greg P. Brewood, Jim P. Goodarzi, Saba Alemayehu, Ashwini Bhandiwad, Robert P. Searles, Albert S. Benight

Chemistry Faculty Publications and Presentations

Hybridization intensities of 30 distinct short duplex DNAs measured on spotted microarrays, were directly compared with thermodynamic stabilities measured in solution. DNA sequences were designed to promote formation of perfect match, or hybrid duplexes containing tandem mismatches. Thermodynamic parameters DeltaH degrees , DeltaS degrees and DeltaG degrees of melting transitions in solution were evaluated directly using differential scanning calorimetry. Quantitative comparison with results from 63 multiplex microarray hybridization experiments provided a linear relationship for perfect match and most mismatch duplexes. Examination of outliers suggests that both duplex length and relative position of tandem mismatches could be important factors contributing to …