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Tardigrade Intrinsically Disordered Proteins as Potential Excipients for Biologics
512a
Wednesday, February 15, 2017
in terms of its partial specific volume. A negative preferential interaction coefficient shows that protecting osmolytes are preferentially excluded from ELP’s surface and enhance its aggregation out of solution, while a positive preferential interaction coefficient shows that denaturants bind to the ELP backbone and destabilize its aggregation. The results show that protecting osmolytes like TMAO and Betaine preferentially hydrate the ELP out of solution as the ELP’s TT linearly decreases with increasing osmolyte concentrations. Arginine, however, a weak denaturant, preferentially, yet weakly interacts with the ELP backbone at lower concentrations and suppresses aggregation by increasing its TT and solubility in solution. Using different molecular weights PEG we show that inert additives enhance ELP aggregation consistent with an excluded volume effect. These results show that ELP aggregation is driven by solvent interactions and that changes in the solvent environment upon addition of different osmolytes enhance or diminish the aggregation of ELP. Future experiments will use complimentary VPO measurements to look at the changes in water activity in the presence of osmolytes. 2520-Pos Board B127 Tardigrade Intrinsically Disordered Proteins as Potential Excipients for Biologics Samantha Piszkiewicz1, Aakash Mehta1, Thomas C. Boothby1, William Daniel1, Sergei Sheiko1, Bob Goldstein2, Gary J. Pielak1. 1 Chemistry, UNC Chapel Hill, Chapel Hill, NC, USA, 2Biology, UNC Chapel Hill, Chapel Hill, NC, USA. Protein-based ‘biologics’ — drugs derived from living organisms — are among the most effective therapeutic treatments on the market. However, protein-based biologics are unstable, have short half-lives and require low temperature storage, which often makes them prohibitively expensive. Although some drugs can be stabilized by formulation with excipients, most still require low temperature storage. In our search for new, more robust excipients, we turned to the tardigrade, a microscopic animal that synthesizes a unique family of intrinsically disordered proteins (IDPs) to protect its cellular components during desiccation. Oscillatory shear experiments demonstrate that at high concentrations and low temperatures, these tardigrade IDPs form reversible hydrogels. Encapsulating a globular test protein in this IDP hydrogel stabilizes the folded state of that protein. Additionally, these IDPs are an order of magnitude better than the FDA-approved excipient trehalose at protecting enzyme activity during desiccation and rehydration. These protective properties show the potential of tardigrade IDPs to serve as novel excipients for stabilizing biologics. 2521-Pos Board B128 Exploring the Unexpected Gelation of Tripeptides in a Binary Mixture of Water and Ethanol David DiGuiseppi1, Stefanie Farrell1, Nicolas Alvarez2, Reinhard Schweitzer-Stenner1. 1 Chemistry, Drexel University, Philadelphia, PA, USA, 2Chemical and Biological Engineering, Drexel University, Philadelphia, PA, USA. Hydrogels formed by peptides of different length are a special class of materials that have been heavily researched in the past due to their inherent biodegradability. In this context, some low molecular weight di- or tripeptides with aromatic residues and terminal groups have been shown to form gels after self-assembling into large supramolecular structures above critical concentrations in the centimolar range. Contrary to expectations, our group recently discovered that cationic glycylanalylglycine (GAG), a tripeptide of rather limited hydrophobicity, forms a gel in 55 mol% ethanol/45 mol% water at room temperature if the concentration exceeds 200 mM. The underlying structure is comprised of unusually long crystalline fibrils (in the 105m range), which do not exhibit the canonical b-sheet structure. Rheological measurements revealed a particularly strong gel with G0 and G00 values in the upper 104 Pa range. Still, the gel melts if the temperature is increased above 36 C. Melting curves measured by increasing and decreasing temperature revealed a significant hysteresis. Kinetic experiments revealed that the gelation of GAG is a multi-step mechanism that can be disentangled by spectroscopic means. By using UV circular dichroism as a novel indicator of gelation we are currently performing measurements that are aimed at constructing a threedimensional phase diagram of the peptide with regard to its melting temperature, peptide concentration and cosolvent fraction. Rheological measurements and optical microscopy will allow us to identify and characterize different gel phases. Thus, we will identify conditions which make the gel usable for practical applications such as ointments, sensors, and drug release systems.
2522-Pos Board B129 Exploring the Effects on the Conformational Propensity of Alanine in the Unblocked Tripeptide Glycyl-Analyl-Glycine in Water/Ethanol Mixtures David DiGuiseppi1, Nina Kubatova2, Gabrielle Lewis3, Harald Schwalbe2, Reinhard Schweitzer-Stenner1. 1 Chemistry, Drexel University, Philadelphia, PA, USA, 2Institut f€ur Organische Chemie und Chemische Biologie, Johann Wolfgang GoetheUniversit€at, Frankfurt am Main, Germany, 3Biology, Drexel University, Philadelphia, PA, USA. Short peptides have been shown to be model systems for obtaining the conformational propensities of individual amino acids. Recent studies on cationic GxG (x: different guest amino acid residues) peptides in water revealed the Ramachandran plots for 16 amino acid residues. Out of the natural amino acids, alanine in GAG has been shown to stand out owing to its unusually high propensity for polyproline II (pPII) conformations (0.72). Thus, its conformational distribution differs substantially from the classical ones of Ramachandran and Flory. Peptide-water interactions have been proposed as the major determinant of alanine’s preference for pPII. To elucidate how peptide hydration affects conformational preferences of alanine we used FTIR, vibrational circular dichroism, polarized Raman and NMR spectroscopy to determine Ramachandran plots of GAG in aqueous solutions with 3, 12, and 42mol% ethanol. We observed a modest decrease of the pPII fraction from 0.72 to 0.65 and an increase in b-strand population from 0.15 to 0.20 from pure water to 12 mol% ethanol. Interestingly, the corresponding changes of the enthalpic and entropic differences between pPII and b -strand by far exceed changes in Gibbs energy at room temperature owing to enthalpyentropy compensation effects. Our thermodynamic data reveal that the cosolvent stabilizes pPII enthalpically (10 kJ/mol in water to 18 kJ/mol in 12 mol% ethanol) but destabilizes it even more entropically (23 to 50 J/mol*K). Comparisons of IR and NMR spectra of GAG and N- methylacetamide in the same ethanol/water mixtures reveal changes of peptide hydration which causes the observed destabilization of pPII of alanine and the concomitant increase of its conformational entropy.
DNA Replication, Recombination, and Repair 2523-Pos Board B130 Near-Atomic Structural Model for Bacterial DNA Replication Initiation Complex and its Functional Insights Masahiro Shimizu1, Yasunori Noguchi2, Yukari Sakiyama2, Hironori Kawakami2, Tsutomu Katayama2, Shoji Takada1. 1 Kyoto University, Kyoto, Japan, 2Kyushu University, Fukuoka, Japan. Formation of the DNA replication initiation complex precedes chromosomal DNA replication. In Escherichia coli, this complex forms on the replication origin oriC, and is composed of about 11 DnaA molecules and one IHF dimer. This complex initiates DNA replication by unwinding specific DNA sequence in oriC and by loading DNA helicases there. While the researches on this initiation process have been accumulating, the entire structure of the oriCDnaA-IHF complex has not been elucidated. In order to reveal the structure of the replication initiation complex, we employed hybrid approaches of computational modeling and biochemical assay. By coarse-grained molecular dynamics simulations, we tested several possibilities of the oriC-DnaA-IHF complex. Obtained structural models were further tested by biochemical assays using reconstituted system. Coarse-grained structures were backtransferred into atomistic model. Finally we obtained the near-atomic model of crucial core part of E. coli replication initiation complex. The complex is composed of three parts. Left subcomplex contains 5 DnaA molecules and IHF, middle subcomplex is a single DnaA molecule, and right subcomplex contains 5 DnaA molecules. In the left and right subcomplexes, AAAþ domain of DnaA formed homo pentamer in a head-to-tail manner. We also investigated the significance of the spaces between protein binding sites by similar hybrid approaches. The space between IHF binding site and adjacent DnaA box was crucial for oriC-DnaA-IHF complex formation. In addition, the space between the left subcomplex and the right subcomplex was important for proper DNA helicase loading. 2524-Pos Board B131 Upf1-Like Helicaes - Same Subfamily, Yet so Different Behavior Saurabh Raj1, Joanne Kanaan2, Herve´ Le Hir2, Vincent Croquette1. 1 CNRS, Paris, France, 2IBENS, CNRS, Paris, France. Helicases are ubiquitous enzymes that are involved in all kinds of DNA/RNA metabolisms including transcription, recombination and replication. Although the most basic function of most helicases is to unwind a double stranded nucleic acid substrate, their properties vary a lot from one helicase family to another.
Wednesday, February 15, 2017
in terms of its partial specific volume. A negative preferential interaction coefficient shows that protecting osmolytes are preferentially excluded from ELP’s surface and enhance its aggregation out of solution, while a positive preferential interaction coefficient shows that denaturants bind to the ELP backbone and destabilize its aggregation. The results show that protecting osmolytes like TMAO and Betaine preferentially hydrate the ELP out of solution as the ELP’s TT linearly decreases with increasing osmolyte concentrations. Arginine, however, a weak denaturant, preferentially, yet weakly interacts with the ELP backbone at lower concentrations and suppresses aggregation by increasing its TT and solubility in solution. Using different molecular weights PEG we show that inert additives enhance ELP aggregation consistent with an excluded volume effect. These results show that ELP aggregation is driven by solvent interactions and that changes in the solvent environment upon addition of different osmolytes enhance or diminish the aggregation of ELP. Future experiments will use complimentary VPO measurements to look at the changes in water activity in the presence of osmolytes. 2520-Pos Board B127 Tardigrade Intrinsically Disordered Proteins as Potential Excipients for Biologics Samantha Piszkiewicz1, Aakash Mehta1, Thomas C. Boothby1, William Daniel1, Sergei Sheiko1, Bob Goldstein2, Gary J. Pielak1. 1 Chemistry, UNC Chapel Hill, Chapel Hill, NC, USA, 2Biology, UNC Chapel Hill, Chapel Hill, NC, USA. Protein-based ‘biologics’ — drugs derived from living organisms — are among the most effective therapeutic treatments on the market. However, protein-based biologics are unstable, have short half-lives and require low temperature storage, which often makes them prohibitively expensive. Although some drugs can be stabilized by formulation with excipients, most still require low temperature storage. In our search for new, more robust excipients, we turned to the tardigrade, a microscopic animal that synthesizes a unique family of intrinsically disordered proteins (IDPs) to protect its cellular components during desiccation. Oscillatory shear experiments demonstrate that at high concentrations and low temperatures, these tardigrade IDPs form reversible hydrogels. Encapsulating a globular test protein in this IDP hydrogel stabilizes the folded state of that protein. Additionally, these IDPs are an order of magnitude better than the FDA-approved excipient trehalose at protecting enzyme activity during desiccation and rehydration. These protective properties show the potential of tardigrade IDPs to serve as novel excipients for stabilizing biologics. 2521-Pos Board B128 Exploring the Unexpected Gelation of Tripeptides in a Binary Mixture of Water and Ethanol David DiGuiseppi1, Stefanie Farrell1, Nicolas Alvarez2, Reinhard Schweitzer-Stenner1. 1 Chemistry, Drexel University, Philadelphia, PA, USA, 2Chemical and Biological Engineering, Drexel University, Philadelphia, PA, USA. Hydrogels formed by peptides of different length are a special class of materials that have been heavily researched in the past due to their inherent biodegradability. In this context, some low molecular weight di- or tripeptides with aromatic residues and terminal groups have been shown to form gels after self-assembling into large supramolecular structures above critical concentrations in the centimolar range. Contrary to expectations, our group recently discovered that cationic glycylanalylglycine (GAG), a tripeptide of rather limited hydrophobicity, forms a gel in 55 mol% ethanol/45 mol% water at room temperature if the concentration exceeds 200 mM. The underlying structure is comprised of unusually long crystalline fibrils (in the 105m range), which do not exhibit the canonical b-sheet structure. Rheological measurements revealed a particularly strong gel with G0 and G00 values in the upper 104 Pa range. Still, the gel melts if the temperature is increased above 36 C. Melting curves measured by increasing and decreasing temperature revealed a significant hysteresis. Kinetic experiments revealed that the gelation of GAG is a multi-step mechanism that can be disentangled by spectroscopic means. By using UV circular dichroism as a novel indicator of gelation we are currently performing measurements that are aimed at constructing a threedimensional phase diagram of the peptide with regard to its melting temperature, peptide concentration and cosolvent fraction. Rheological measurements and optical microscopy will allow us to identify and characterize different gel phases. Thus, we will identify conditions which make the gel usable for practical applications such as ointments, sensors, and drug release systems.
2522-Pos Board B129 Exploring the Effects on the Conformational Propensity of Alanine in the Unblocked Tripeptide Glycyl-Analyl-Glycine in Water/Ethanol Mixtures David DiGuiseppi1, Nina Kubatova2, Gabrielle Lewis3, Harald Schwalbe2, Reinhard Schweitzer-Stenner1. 1 Chemistry, Drexel University, Philadelphia, PA, USA, 2Institut f€ur Organische Chemie und Chemische Biologie, Johann Wolfgang GoetheUniversit€at, Frankfurt am Main, Germany, 3Biology, Drexel University, Philadelphia, PA, USA. Short peptides have been shown to be model systems for obtaining the conformational propensities of individual amino acids. Recent studies on cationic GxG (x: different guest amino acid residues) peptides in water revealed the Ramachandran plots for 16 amino acid residues. Out of the natural amino acids, alanine in GAG has been shown to stand out owing to its unusually high propensity for polyproline II (pPII) conformations (0.72). Thus, its conformational distribution differs substantially from the classical ones of Ramachandran and Flory. Peptide-water interactions have been proposed as the major determinant of alanine’s preference for pPII. To elucidate how peptide hydration affects conformational preferences of alanine we used FTIR, vibrational circular dichroism, polarized Raman and NMR spectroscopy to determine Ramachandran plots of GAG in aqueous solutions with 3, 12, and 42mol% ethanol. We observed a modest decrease of the pPII fraction from 0.72 to 0.65 and an increase in b-strand population from 0.15 to 0.20 from pure water to 12 mol% ethanol. Interestingly, the corresponding changes of the enthalpic and entropic differences between pPII and b -strand by far exceed changes in Gibbs energy at room temperature owing to enthalpyentropy compensation effects. Our thermodynamic data reveal that the cosolvent stabilizes pPII enthalpically (10 kJ/mol in water to 18 kJ/mol in 12 mol% ethanol) but destabilizes it even more entropically (23 to 50 J/mol*K). Comparisons of IR and NMR spectra of GAG and N- methylacetamide in the same ethanol/water mixtures reveal changes of peptide hydration which causes the observed destabilization of pPII of alanine and the concomitant increase of its conformational entropy.
DNA Replication, Recombination, and Repair 2523-Pos Board B130 Near-Atomic Structural Model for Bacterial DNA Replication Initiation Complex and its Functional Insights Masahiro Shimizu1, Yasunori Noguchi2, Yukari Sakiyama2, Hironori Kawakami2, Tsutomu Katayama2, Shoji Takada1. 1 Kyoto University, Kyoto, Japan, 2Kyushu University, Fukuoka, Japan. Formation of the DNA replication initiation complex precedes chromosomal DNA replication. In Escherichia coli, this complex forms on the replication origin oriC, and is composed of about 11 DnaA molecules and one IHF dimer. This complex initiates DNA replication by unwinding specific DNA sequence in oriC and by loading DNA helicases there. While the researches on this initiation process have been accumulating, the entire structure of the oriCDnaA-IHF complex has not been elucidated. In order to reveal the structure of the replication initiation complex, we employed hybrid approaches of computational modeling and biochemical assay. By coarse-grained molecular dynamics simulations, we tested several possibilities of the oriC-DnaA-IHF complex. Obtained structural models were further tested by biochemical assays using reconstituted system. Coarse-grained structures were backtransferred into atomistic model. Finally we obtained the near-atomic model of crucial core part of E. coli replication initiation complex. The complex is composed of three parts. Left subcomplex contains 5 DnaA molecules and IHF, middle subcomplex is a single DnaA molecule, and right subcomplex contains 5 DnaA molecules. In the left and right subcomplexes, AAAþ domain of DnaA formed homo pentamer in a head-to-tail manner. We also investigated the significance of the spaces between protein binding sites by similar hybrid approaches. The space between IHF binding site and adjacent DnaA box was crucial for oriC-DnaA-IHF complex formation. In addition, the space between the left subcomplex and the right subcomplex was important for proper DNA helicase loading. 2524-Pos Board B131 Upf1-Like Helicaes - Same Subfamily, Yet so Different Behavior Saurabh Raj1, Joanne Kanaan2, Herve´ Le Hir2, Vincent Croquette1. 1 CNRS, Paris, France, 2IBENS, CNRS, Paris, France. Helicases are ubiquitous enzymes that are involved in all kinds of DNA/RNA metabolisms including transcription, recombination and replication. Although the most basic function of most helicases is to unwind a double stranded nucleic acid substrate, their properties vary a lot from one helicase family to another.