bluuebunny/crossref_metadata_2025_split huggingface dataset:
Purification and Biophysical Characterization of the Photosystem I Complex from Botryococcus braunii
Authors: Joaquín Ovalle Freisa M. , Guihurt Grace , Torres‐Martínez Zally , Morales‐Lozada Yermary , Griebenow Kai 
Published: June 2021

Botryococcus braunii (B. braunii) is a green photosynthetic microalga that is highly investigated due to its capacity of hydrocarbon accumulation, secretion, and synthesis. However, its photosynthetic apparatus has not been elucidated. Photosystem I (PSI) is a membrane‐bound pigment‐protein complex that mediates the electron transfer from plastocyanin on the lumenal side to ferredoxin on the cytoplasmic side. In this study, our purpose was to isolate, purify and characterize PSI core subunits and light harvesting proteins. After the initial isolation via differential ultracentrifugation and solubilization of thylakoid membranes with 1% of n‐dodecyl β‐D‐maltoside, the resulting supernatant was loaded onto a Toyopearl DEAE‐650 ion exchange chromatography column. The purified PSI fraction obtained was pooled and concentrated using an Amicon centrifugal filter unit. The resulting protein concentration was 1 mg/ml, and the PSI characteristic 677 nm absorption maximum was attained. The biophysical characterization was performed via UV/Vis and fluorescence spectroscopy, native and SDS‐polyacrylamide gel electrophoresis, and MALDI‐MS, with the characteristic peaks and molecular weight results of the proteins and subunits found in PSI. Our characterization results show that PSI has been successfully purified, which may provide the fundamental knowledge of the function and possible applications of this integral membrane protein complex in B. braunii.Support or Funding InformationThis research was funded by NIH Research Initiative for Scientific Enhancement (RISE) Program Grant No. 5R25GM061151‐14 and DOD Contract No. W911NF‐11‐1‐0218.
Effects of Mutation at Position 557 on the Catalytic Properties of Soybean Lipoxygenase‐1
Authors: Bassett Natasha F. , Clapp Charles H. 
Published: June 2021

Soybean lipoxygenase‐1 (SBLO‐1) catalyzes the oxygenation of linoleic acid to give primarily 13‐hydroperoxy‐9,11‐octadecadienoic acid (13‐HPOD) plus a small amount (10% at pH 7.5) of 9‐hydroperoxy‐10,12‐octadecadienoic acid (9‐HPOD). Replacement of phenylalanine‐557 with valine increases the amount of 9‐HPOD formed to 30–40%. Following earlier suggestions by Hornung et al. (Proc. Natl. Acad. Sci. USA 96 (1999) 4192–4197), our working hypothesis is that 13‐HPOD arises from tail‐first binding with the methyl terminus of linoleic acid near phe‐557, and that replacement of phe‐557, with smaller residues enables linoleic acid to bind in a head‐first manner leading to 9‐HPOD. To test this model, the F557S mutant was prepared, and its activity was determined with linoleic acid and 20‐hydroxy‐5,8,11,14‐eicosatetraenoic acid (20‐HETE), which has a hydroxyl group on the terminal carbon that might form a hydrogen bond with the hydroxyl group of serine‐557. Kinetics studies on the mutant were complicated by pronounced substrate inhibition at high substrate concentration. At low substrate concentration, the activity of the F557S mutant is 25% of wild‐type with linoleic acid as substrate and 65% of wild‐type with 20‐HETE as substrate. Oxygenation of linoleic acid by the F557S mutant produced only 8% of 9‐HPOD, considerably less than the percentage of 9‐HPOD produced by the F557V mutant. This result indicates that the size of the residue at position 557 is not the only factor that affects the regiochemistry of the reaction. To test the hypothesis that 9‐oxygenation results from head‐first binding, linoleoyl‐L‐valine (LLV) and linoleoyl‐D‐valine (LDV) have been investigated as substrates of the F557V mutant. The percentage of 9‐oxygenation with these substrates is comparable to what is observed with linoleic acid and shows little or no dependence on the stereochemistry of the valine moiety. These findings suggest that the 9‐oxygenation of LLV and LDV does not involve head‐first binding.Support or Funding InformationNSF CHE‐1213262
Relating Intein Flexibility to the Temperature Dependence of Activity
Authors: Yakely Anthony E. , Bonano Alexander A. , Jaramillo Mario V. , Williams Jennie E. , Reitter Julie N. , Mills Kenneth V. 
Published: June 2021

Protein splicing is the self‐catalyzed removal of an intein, or intervening protein, in coordination with the ligation of the exteins, the flanking polypeptides. We are examining the DNA Pol II inteins from the extremophiles Pyrococcus abyssi and Pyrococcus horikoshii. The inteins from P. abyssi and P. horikoshii are largely homologous except for the presence of a large, disordered loop in the P. abyssi intein, in contrast to a smaller loop in the P. horikoshii intein. We have observed temperature dependence of the activity of the two inteins, which may be due to the difference in their structures. The smaller loop may allow for a more rigid structure in the P. horikoshii intein resulting in a higher temperature needed for protein splicing. The P. abyssi intein has a longer loop resulting in a less rigid structure, so a lower temperature is needed for protein splicing. To test this hypothesis, we used recombinant DNA to make a chimera of the P. abyssi intein with the P. horikoshii loop, and vice versa, to determine whether the change in loop alters the temperature dependence of the inteins’ activity. We successfully created the chimeric genes for both chimeric inteins and discovered that the proteins expressed well and were capable of splicing. For the P. abyssi intein with the P. horokoshii loop, we found that the chimeric intein spliced more than the native intein at a lower temperature but less at a higher temperature. For the P. horokoshii intein with the P. abyssi loop, we found that it displayed less splicing than the original protein at various temperatures. Comparative activity assays will be run to examine the temperature dependence of activity among the native and chimeric inteins.Support or Funding InformationThis material is based upon work supported by the National Science Foundation under grants MCB‐1244089 and MCB‐1517138 and by a Henry Dreyfus Teacher‐Scholar Award (KVM).
Influence of Intein Flexibility on Temperature Dependence of Protein Splicing
Authors: Long Joshua D. , Pierre Isabelle , Reitter Julie , Mills Kenneth V. 
Published: June 2021

Protein splicing is a post‐translational modification by which intervening polypeptides called inteins catalyze their own excision from two flanking polypeptides, the exteins, concomitant with the ligation of the exteins. We studied the activity of DNA Polymerase II (PolII) inteins from the deep‐sea thermophiles Thermococcus kodakarensis (Tko), Thermococcus barophilus (Tba), Thermococcus sibiricus (Tsib), and Methanococcus infernus (Minf). Tko and Tba PolII inteins contain a homing endonuclease domain, while a flexible loop replaces this region in the Tsib and Minf inteins. This flexible loop, also found in Pyrococcus abyssi (Pab) and Pyrococcus horikoshii (Pho) PolII inteins, was hypothesized to be linked to the temperature dependent activity of these inteins. However, unlike Pab and Pho PolII inteins, we have shown that protein splicing and cleavage side reactions in the Tsib and Minf inteins occur in vivo in E. coli without the need for high temperature incubation in vitro. On the other hand, the Tba and Tko inteins splice poorly in E. coli and can be induced to splice in vitro at higher temperatures. We also have demonstrated that the Tba intein splices while the Tko intein undergoes a mixture of splicing and N‐terminal cleavage.Support or Funding InformationThis material is based upon work supported by the National Science Foundation under grants MCB‐0950245, MCB‐1244089 and MCB‐1517138 and by a Henry Dreyfus Teacher‐Scholar Award (KVM).
An Investigation of Salt‐Dependent Protein Splicing
Authors: Cousin Christopher E. , Reitter Julie N. , Nicastri Michael , Jaramillo Mario S. , Mills Kenneth V. 
Published: June 2021

Protein splicing is a post‐translational modification where an internal polypeptide, called an intein, catalyzes its excision from two flanking polypeptides, called exteins, concomitant with extein ligation. Usually, when intein fusion proteins from other organisms are overexpressed in E. coli, splicing is spontaneous in vivo. However, despite a very similar sequence to the Mma PolII intein, which is capable of splicing, the Halobacterium salinarum (Hsa) PolII intein did not splice upon in vivo expression in E. coli. Halobacterium salinarum is a halophilic organism, so it lives in an environment with extremely high salt concentrations. In an effort to induce reactivity, two fusion proteins of the Hsa intein were incubated at 28°C for 16 hours with increasing amounts of NaCl. The fusion protein with a wild type intein has glutamine as the last residue of the intein, whereas the second intein, termed MIHQN, replaces this glutamine with asparagine. After incubation, in vitro splicing was observed in both intein fusion proteins on incubation with 2 M and 2.5 M NaCl.Support or Funding InformationThis material is based upon work supported by the National Science Foundation under grants MCB‐1244089 and MCB‐1517138 (KVM), by a Henry Dreyfus Teacher‐Scholar Award (KVM), and the Summer Research Fellowships from Renee and Anthony M. Marlon, M.D (CC).
9(Z),12(Z)‐Eicosadienoic Acid as a Probe for Substrate Binding by Soybean Lipoxygenase‐1
Authors: Hinnenkamp David J. , Clapp Charles H. 
Published: June 2021

Soybean lipoxygenase is a non‐heme iron protein that catalyzes the oxygenation of linoleic acid and other polyunsaturated fatty acids to produce conjugated diene hydroperoxides. The manner in which substrates bind to this enzyme is uncertain. One hypothesis is that the terminal methyl group of substrates binds near phenylalanine‐557. To test this hypothesis, 9(Z),12(Z)‐eicosadienoic acid (9,12‐EDA) has been synthesized and studied as a substrate of wild‐type SBLO‐1 and the F557A mutant, in which the smaller residue at position 557 might allow accommodation of a fatty acid with a longer alkyl terminus. The kinetics are complicated by pronounced substrate inhibition at high concentrations of 9,12‐EDA. At low substrate concentration, 9,12‐EDA exhibits lower activity than linoleic acid with wild type SBLO‐1, but the activities of the two fatty acids are comparable with the F557A mutant. The results are consistent with the hypothesis that phe‐557 is located at the binding site for the methyl terminus of substrates.Support or Funding InformationNSF CHE‐1213262
Analysis of the Calyces from the Jamaica Species of Hibiscus Sabdariffa for Adrogenic Compounds
Authors: Butler Lakeisha Y , Morgan Melisa , Gray Wesley , Chin Kit , Snowden Janana 
Published: June 2021

The leaves and flowers of Hibiscus sabdariffa are known to have many pharmacological properties. Hibiscus sabdariffa leaf extracts (HLE) have been documented to have anti‐cancer activities in different cancer cell lines. HLE has been shown to inhibit cell proliferation, migration, and metastasis in human prostate cancer cells suggesting that this flow may be a source for anti‐androgenic compounds. However, the anti‐androgen effect has not been examined in the calyx. The objective of this study was to obtain a unique bioactive fraction from the calyx of H. sabdariffa and to demonstrate its ability to inhibit prostate cancer cell growth. To accomplish our objective, we generated an aqueous and a methanol extraction of the calyces of the H. sabdariffa. The extracts were then examined by HPLC chromatography and for anti‐proliferative activity using the LNCaP cancer cells. Extraction of 22g of the calyces for 48hrs at room temperature resulted in 5.56mg and 2.75mg of analyte in aqueous and methanol extraction respectively. We obtained high yields of extracts (253mg per kg of dry weight) using water as compared to methanol as the solvent. An HPLC chromatogram profile of each extract was generated to characterize the number of respective chemicals present in the analytes. Analysis of the methanol extract revealed the presence of at least two distinct peaks with a retention times of 3.12min and 3.27min. We observed that the aqueous extract resulted only in a single peak with a retention time of 3.02min. Next, we examined the ability of both extracts to inhibit the growth of LNCaP‐GFP cancer cell line. Treatment of these cells for 1–5 days resulted in dose and time dependent inhibition of cell growth. We found that the methanol extract was capable of inducing cytotoxicity in 24hrs as compared to aqueous extracts which induced cytotoxicity after 72hrs. Our results demonstrated that both methanol and aqueous extracts of H. sabdariffa contained growth inhibitory compounds.Support or Funding InformationNIH R15AT006821(WG) and USDA/NIFA 2012‐38821‐20092.
Biological Activity of Outer Membrane Vesicles from Clinical Isolates of Escherichia coli
Authors: Morales‐Lozada Yermary , Báez‐Bravo Gabriela , Ferrer‐Acosta Yancy , Gómez‐Moreno Ramón , Baerga‐Ortiz Abel 
Published: June 2021

All gram‐negative bacteria secrete outer membrane vesicles (OMV) that have several functions, among them the transportation of bacterial virulence and survival factors. It has been found by other groups that certain OMV promote aberrant growth in mammalian cells upon contact. OMV provide an insoluble secretion pathway for gram‐negative bacteria and are responsible for the release of outer membrane and periplasm content out of the cell. The main objective of this investigation is to compare the composition and biological activity of OMV isolated from diverse clinical strains of E. coli. We will assess morphological effects of OMV from selected clinical isolates of E. coli on HeLa cells. Furthermore, we will also measure the lipid composition of OMV for the different strains and mutants by GC/MS. As preliminary results, we observed a significant difference in the lipid composition of the IHE3034 (pks+ strain) OMV and the IHE3034‐ΔclbP OMV. The clbP gene is presumed to be involved in the biosynthesis of colibactin. We observed that the deletion of clbP peptidase causes a decrease in the percentage of 12:0 fatty acids and an increase of 16:0 fatty acids in OMV, but not in the cellular membrane. These results begin to establish a correlation between the presence of colibactin genes and events at the bacterial cell surface.Support or Funding InformationNSF grant CHE0953254 NIH grant R25GM061838 (MBRS‐RISE program) to Ramón Gómez‐Moreno
Crystallographic Study of Wild‐type and Mg2+‐free K‐Ras GTPase
Authors: Parker Jillian , Mattos Carla 
Published: June 2021

The small GTPase Ras has been widely studied due to its relevance in cancer and other developmental disorders, termed RASopothies. When bound to GTP, Ras conveys signals to downstream effectors to control cellular signaling processes such as proliferation, differentiation, and apoptosis. Hydrolysis of GTP to GDP and inorganic phosphate (Pi) halts Ras signaling. This switching mechanism is tightly regulated by GEFs (guanine nucleotide exchange factors) and GAPs (GTPase activating proteins), where oncogenic mutations in Ras lead to uncontrolled Ras cell cycling and tumorigenesis. To date, structures of the most oncogenic isoform, K‐Ras, in its activated GTP‐bound form have been difficult to obtain, with only a couple of its mutants currently deposited in the Protein Data Bank (PDB). The first crystal structure of wild‐type K‐Ras bound to the GTP‐analogue GppCH2p has been obtained to 1.89Å resolution. This structure shows K‐Ras in the state 1 conformation, with switch I away from the active site without threonine 35 interacting with the Mg2+ or nucleotide. Additionally, structures of active K‐Ras obtained from lower pH conditions show perturbed active site elements, the most striking of which is the absence of the canonical Mg2+ ion in the nucleotide binding pocket. The Mg2+‐less structures exhibit both local and long‐range alterations in residues located within conserved nucleotide‐binding motifs and involved in sensing the state of the nucleotide bound in the active site. Additionally, similarities are seen between Mg2+‐less K‐Ras and nucleotide‐less Ras found in complex with Sos, the most common Ras GEF. These results shed light on why the active form of K‐Ras has been structurally difficult to characterize, as well as provide a structural basis for more focused targeting of the K‐Ras isoform.Support or Funding InformationNSF MCB‐1244203
Allosteric role of R97 on the NRas active site
Authors: Reid Derion F , Mattos Carla 
Published: June 2021

Ras proteins are small GTPases that are involved in signal transduction pathways that controls cellular proliferation, differentiation, and survival. Ras is constitutively activated in roughly 20% of human cancers. The Ras subfamily is made up of H‐, K‐, and NRas, where NRas is the least studied. NRas functions as a molecular switch that exchanges guanosine diphosphate (GDP) for guanosine triphosphate (GTP) rendering activation, and hydrolyzes GTP to GDP for deactivation. Once activated, NRas interacts with Raf, an effector molecule that our lab has hypothesized to allosterically modulate intrinsic hydrolysis. The catalytic G domain of NRas comprises an effector lobe (residues 1–86) and allosteric lobe (residues 87–166). In our allosteric model, switch II of Ras becomes ordered when a negatively charged ligand binds between helix 3 and loop 7 allowing for intrinsic hydrolysis. Molecular dynamics studies of Ras bound to Raf revealed R97 of helix 3 becomes less dynamic upon binding of Raf, coordinating the binding of a negatively charged ligand at the allosteric site. Interestingly, R97G is a rare mutation of NRas, found in a human tumor. Here, we present the structure of NRasR97G bound to the non‐hydrolysable GTP analogue, GppNHp. The structure and hydrolysis rates of NRasR97G are compared to those of wild‐type NRas.Support or Funding InformationThis research was funded by NSF Grant Number MCB‐1244203.
Mechanistic Implications of the Unique Structural Features of the Anti‐Sigma Domain of the Pseudomonas Sigma Regulator, PupR
Authors: Jensen Jaime , Balbo Andrea , Neau David , Chakravarthy Srinivas , Zhao Huaying , Sinha Sangita , Colbert Christopher 
Published: June 2021

Gram‐negative bacteria tightly regulate intracellular iron, an essential nutrient. To ensure this strict control, some outer membrane TonB‐dependent transporters (TBDTs) responsible for iron import stimulate their own transcription in response to extracellular binding of ferric siderophore. This process is mediated by an inner membrane sigma regulator protein that transduces an unknown periplasmic signal from the TBDT to release an intracellular sigma factor from the inner membrane, which ultimately upregulates TBDT transcription. Here, we employ the Pseudomonas putida ferric‐pseudobactin BN7/BN8 sigma regulator, PupR, as a model system to understand the molecular mechanism of this conserved class of sigma regulators. We have determined the X‐ray crystal structure of the cytoplasmic anti‐sigma domain (ASD) of PupR to 2.0 Å. Size exclusion chromatography, small‐angle X‐ray scattering, and sedimentation velocity analytical ultracentrifugation (SV‐AUC) all indicate that, in contrast to other ASDs, the PupR‐ASD exists as a dimer in solution. Mutagenesis of residues at the dimer interface identified from the crystal structure disrupts dimerization and protein stability, as determined by SV‐AUC and thermal denaturation circular dichroism spectroscopy. Additionally, we have demonstrated that the helical content of the PupR‐ASD increases in the presence of trifluoroethanol, and are investigating the interaction of the PupR‐ASD with its cognate sigma factor, PupI. These combined results suggest that this type of inner membrane sigma regulator may utilize an unusual mechanism to sequester their cognate sigma factors and prevent transcriptional activation.Support or Funding InformationFunded by NSF ND EPSCoR DDA #FAR0025216 to JJ; NIH NINDS 1R03 NS090939 and NSF MCB‐1413525 to SS; NIH NIGMS 1R15 GM113227, NIH NCRR 2P20 RR015566, and NIH NIGMS P30 GM103332 to CC.
Structural Biology of Aromatic Amino Acid Biosynthesis in Plants: The Prephenate Branch Point
Authors: Holland Cynthia K , Jez Joseph M 
Published: June 2021

Plants are chemists that have spent the last 425 million years evolving unique secondary metabolites to communicate with their surroundings, defend themselves against pathogens, insects, and herbivores, and grow and develop. Many secondary metabolites are biosynthesized from the aromatic amino acids tryptophan, tyrosine, and phenylalanine, and the shikimate and aromatic amino acid biosynthetic pathways link primary and specialized metabolism. Prephenate aminotransferase (PAT) is a key enzyme in aromatic amino acid biosynthesis because its product, arogenate, is the immediate precursor for both tyrosine and phenylalanine. Interestingly, the enzyme has a dual role in metabolism and can also function as a classical aspartate aminotransferase. AtPAT is pyridoxal 5′‐phosphate‐dependent (PLP) enzyme belonging to the AAT‐like aspartate aminotransferase domain and follows a ping‐pong bi‐bi reaction mechanism through Schiff‐base formation to a catalytic K306 in the active site. Each subunit of the AtPAT dimer consists of a large domain and a small domain that belong to the α‐β class of protein fold. Despite the structural similarities to aspartate aminotransferases, the molecular basis of prephenate binding specificity remains elusive. To gain an understanding of the enzyme's chemistry and substrate selectivity, a 2.5 Å x‐ray structure of PAT from the model plant Arabidopsis thaliana (thale cress) was solved in complex with the PLP. To our knowledge, this is the first structure of a bona fide prephenate aminotransferase. Amino acid sequence comparisons of all known functional PATs shows that T84 and K169 are highly conserved; however, some enzymes annotated as PAT display sequence variations at these positions. Biochemical analysis of T84V, K169V, and T84V/K169V PAT reveals that the double mutant gained tyrosine aminotransferase activity and was able to convert 4‐hydroxyphenylpyruvate to tyrosine. T84 is on the opposite side of the active site pocket, while the amine backbone of K169 hydrogen bonds to PLP. We hypothesize that Thr84 and Lys169 are important for substrate recognition in the active site and will use these mutants in future crystallographic experiments. Overall, our structural studies will assist in understanding the role of this bi‐functional enzyme in the context of aromatic amino acid biosynthesis in plants.Support or Funding InformationThis material is based upon work supported by the National Science Foundation Graduate Research Fellowship under Grant No. DGE‐1143954. Any opinion, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation. Results shown in this report are derived from work performed at Argonne National Laboratory, Structural Biology Center at the Advanced Photon Source. Argonne is operated by UChicago Argonne, LLC, for the U.S. Department of Energy, Office of Biological and Environmental Research under contract DE‐AC02‐06CH11357.