The document summarizes research characterizing the radical S-adenosyl-L-methionine (SAM) epimerase NeoN. NeoN catalyzes the specific epimerization of carbon C-5''' in neomycin C to produce neomycin B. The goal is to determine NeoN's structure using X-ray crystallography to understand its catalytic activity. Initial experiments cloning the neoN gene from Streptomyces fradiae and expressing the protein are presented, though yields were low. Future work will optimize cloning and crystallization to elucidate NeoN's structure and characterize residues involved in substrate recognition.
Opportunities for probing the structure and mechanism of porphobilinogen synt...John Clarkson
J. Clarkson, E.K. Jaffe, R.M. Petrovich, J. Dong & P.R. Carey, “Opportunities for Probing the Structure and Mechanism of Porphobilinogen Synthase by Raman Spectroscopy.” J. Am. Chem. Soc., 119, 11556-11557, 1997.
A novel class of pyrimido[4,5-b]-1,4-benzoxazepines is described as inhibitors of epidermal growth factor receptor (EGFR) tyrosine kinase. Two compounds display potent EGFR inhibitory activity of less than 1 μM in cellular phosphorylation assays (IC50 0.47–0.69 μM) and are highly selective against a small kinase panel. Structure-activity relationship studies identified substitutions on the aniline ring and benzoxazepine core that improved potency, with meta-bromo and bis-methoxy substitutions yielding the most active compounds. These tricyclic azepine derivatives represent a new scaffold for EGFR kinase inhibitors with desired selectivity.
The document describes research to design degenerate primers to amplify a segment of the putative ACE-1 transcription factor gene in Peniophora sp. using bioinformatics tools. Researchers identified a conserved copper-fist DNA binding domain in the ACE-1 protein sequence of Phanerochaete chrysosporium that is about 50 amino acids long and 80-90% similar to other sequences. Degenerate primers targeting this conserved region were designed and analyzed in silico. The results suggest the conserved domain is likely important for function and differences are due to synonymous substitutions, supporting using online tools to accelerate molecular biology research.
Structure-Function Analysis of POR MutantsAYang999
This document summarizes protein structure and function, with a focus on mutations in the protein cytochrome P450 oxidoreductase (POR). It describes how mutations can alter a protein's amino acid sequence and structure. Specifically, it investigates the S102P and R550Q mutations in POR, which were found in humans. Experiments expressed and purified these mutant POR proteins, and will test their electron transfer activity compared to the wild type protein using a cytochrome c reduction assay. This will help determine if these mutations impair POR function.
Structure-Function Analysis of POR MutantsAYang999
This document summarizes protein structure and function, with a focus on mutations in the protein Cytochrome P450 Reductase (POR). It describes how: (1) POR's amino acid sequence determines its primary structure and folding; (2) genetic mutations can alter structure and compromise function; and (3) specific POR mutations are associated with Antley-Bixler Syndrome. The document outlines experiments to characterize the S102P and R550Q POR mutants using protein expression, purification, and a cytochrome c reduction assay to analyze structural and functional effects compared to wild type POR.
Computational methods and crystallography have been coupled to study the structure-function relationships of two heme enzymes: cytochrome c peroxidase (CCP) and nitric oxide synthase (NOS). For NOS, computational approaches were used to resolve ambiguities in the orientation of an inhibitor, 7-nitroindazole, bound in the active site. Calculations of interaction energies between the inhibitor and protein supported one orientation over the other. For CCP, site-directed mutagenesis, crystallography, and computation have helped explain why CCP uniquely stabilizes a tryptophan cation radical during catalysis, unlike other peroxidases.
Spin labeling is a technique to introduce stable paramagnetic centers into biomolecules like proteins and nucleic acids to study their structure and dynamics using electron paramagnetic resonance (EPR) spectroscopy. The most common method uses methanethiosulfonate spin labels that form disulfide bonds with engineered cysteine residues in the target protein. Alternative methods include spin labels attached via chemical ligation or nonsense suppression techniques. EPR data from spin-labeled sites can provide information about side chain mobility, solvent accessibility, and intra- or intermolecular distances within the biomolecule.
Opportunities for probing the structure and mechanism of porphobilinogen synt...John Clarkson
J. Clarkson, E.K. Jaffe, R.M. Petrovich, J. Dong & P.R. Carey, “Opportunities for Probing the Structure and Mechanism of Porphobilinogen Synthase by Raman Spectroscopy.” J. Am. Chem. Soc., 119, 11556-11557, 1997.
A novel class of pyrimido[4,5-b]-1,4-benzoxazepines is described as inhibitors of epidermal growth factor receptor (EGFR) tyrosine kinase. Two compounds display potent EGFR inhibitory activity of less than 1 μM in cellular phosphorylation assays (IC50 0.47–0.69 μM) and are highly selective against a small kinase panel. Structure-activity relationship studies identified substitutions on the aniline ring and benzoxazepine core that improved potency, with meta-bromo and bis-methoxy substitutions yielding the most active compounds. These tricyclic azepine derivatives represent a new scaffold for EGFR kinase inhibitors with desired selectivity.
The document describes research to design degenerate primers to amplify a segment of the putative ACE-1 transcription factor gene in Peniophora sp. using bioinformatics tools. Researchers identified a conserved copper-fist DNA binding domain in the ACE-1 protein sequence of Phanerochaete chrysosporium that is about 50 amino acids long and 80-90% similar to other sequences. Degenerate primers targeting this conserved region were designed and analyzed in silico. The results suggest the conserved domain is likely important for function and differences are due to synonymous substitutions, supporting using online tools to accelerate molecular biology research.
Structure-Function Analysis of POR MutantsAYang999
This document summarizes protein structure and function, with a focus on mutations in the protein cytochrome P450 oxidoreductase (POR). It describes how mutations can alter a protein's amino acid sequence and structure. Specifically, it investigates the S102P and R550Q mutations in POR, which were found in humans. Experiments expressed and purified these mutant POR proteins, and will test their electron transfer activity compared to the wild type protein using a cytochrome c reduction assay. This will help determine if these mutations impair POR function.
Structure-Function Analysis of POR MutantsAYang999
This document summarizes protein structure and function, with a focus on mutations in the protein Cytochrome P450 Reductase (POR). It describes how: (1) POR's amino acid sequence determines its primary structure and folding; (2) genetic mutations can alter structure and compromise function; and (3) specific POR mutations are associated with Antley-Bixler Syndrome. The document outlines experiments to characterize the S102P and R550Q POR mutants using protein expression, purification, and a cytochrome c reduction assay to analyze structural and functional effects compared to wild type POR.
Computational methods and crystallography have been coupled to study the structure-function relationships of two heme enzymes: cytochrome c peroxidase (CCP) and nitric oxide synthase (NOS). For NOS, computational approaches were used to resolve ambiguities in the orientation of an inhibitor, 7-nitroindazole, bound in the active site. Calculations of interaction energies between the inhibitor and protein supported one orientation over the other. For CCP, site-directed mutagenesis, crystallography, and computation have helped explain why CCP uniquely stabilizes a tryptophan cation radical during catalysis, unlike other peroxidases.
Spin labeling is a technique to introduce stable paramagnetic centers into biomolecules like proteins and nucleic acids to study their structure and dynamics using electron paramagnetic resonance (EPR) spectroscopy. The most common method uses methanethiosulfonate spin labels that form disulfide bonds with engineered cysteine residues in the target protein. Alternative methods include spin labels attached via chemical ligation or nonsense suppression techniques. EPR data from spin-labeled sites can provide information about side chain mobility, solvent accessibility, and intra- or intermolecular distances within the biomolecule.
Enzyme Discovery for Natural Product BiosynthesisHongnan Cao
A poster presentation of collaborative work on the NIH funded project of Enzyme Discovery for Natural Product Biosynthesis at 2015 American Crystallography Association Meeting at Philadelphia, PA. Thanks to Rice University, University of Wisconsin-Madison, The Scripps Research Institute, University of Kentucky, The Midwest Center for Structural Genomics, The Northeast Center for Structural Genomics, APS synchrotron at Argonne National Lab
Exploring Proteins and Proteomes. Stryer,CHAPTER 3 pptkhair ullah
Methods in Protein Chemistry
This chapter discusses several methods used to isolate, purify, detect, degrade, analyze, and synthesize proteins. It describes techniques such as centrifugation, solubility, dialysis, gel filtration, affinity chromatography, HPLC, electrophoresis, and mass spectrometry. It also covers determining a protein's amino acid sequence through methods like Edman degradation, solid phase synthesis, chemical and enzymatic cleavage, and the use of DNA sequencing. The goal of these methods is to obtain a protein's amino acid sequence and gain functional information about proteins and proteomes.
The document summarizes research investigating the interface phenomena of poly(o-ethoxyaniline) (POEA) films using atomic force spectroscopy. The study found that POEA films consist of conducting islands surrounded by a less conductive matrix. The conducting islands were characterized by the presence of double-layer forces and visualized using transmission electron microscopy. The conducting islands were only 15 nm in diameter and could only be identified using adhesion mapping, not contact mode atomic force microscopy which showed larger aggregates. The degree of doping and pH affected the morphology and interactions observed, with fully dedoped POEA at pH 5 and partially conducting polyaniline.
Structural Modeling of the Ptf1a C2 Sequence Bound to Mammalian Rbpj and RbjlWard Coats
In this study, the researchers:
1) Generated structural models of the C2 sequence of Ptf1a bound to the β-trefoil domains of mammalian Rbpj and Rbpjl.
2) Found that the C2 sequence binds in an extended conformation, maintaining interactions seen in the Notch-IC/Rbpj complex.
3) Observed conserved residues in the hydrophobic pocket that maintain binding of proteins with a conserved tryptophan motif.
Proteomic Strategies for purification of lactate dehydrogenase ...Gaurav Dwivedi
The document summarizes proteomic strategies for purifying lactate dehydrogenase (LDH) from chicken muscle. Key steps included homogenizing muscle tissue, removing debris via centrifugation, precipitating proteins with ammonium sulfate, dialyzing to remove salts, and purifying LDH using blue sepharose affinity chromatography. SDS-PAGE and activity assays confirmed the isolation of a single 35 kDa protein band corresponding to LDH. The multi-step process successfully extracted and purified LDH from chicken muscle for further study.
The document discusses the purification of a Sitag/RGD/His-tag fusion protein for use as a scaffold in tissue engineering. It examines different lysis and purification methods to extract the protein from E. coli cells. Lysing methods tested include freeze-thaw, detergents, and enzymes. Purification was initially attempted using nickel affinity chromatography via the His-tag, but residual proteins remained. Purification using silica and the silica-binding Sitag was then explored, but elution with L-lysine was ineffective. Increased washes and incubation improved elution, but some bacterial proteins still remained. Future work may use a silica gel system or alter purification conditions.
This document summarizes an experiment on isolating and characterizing the enzyme alkaline phosphatase (AP) from E. coli bacteria. Key steps included purifying AP using dialysis, salting-in/salting-out, and DEAE cellulose chromatography. SDS-PAGE was used to analyze purity and molecular weight. Kinetic experiments at varying pH levels used the substrate PNPP and spectrophotometry to generate Michaelis-Menten and Lineweaver-Burk plots, allowing determination of kinetic parameters like Vmax and Km. The goal was to understand AP enzymatic activity and affinity for substrate under different conditions.
This document summarizes a study that explored using RNA nucleosides as chiral sensing agents in NMR spectroscopy. Adenosine, guanosine, uridine, and cytidine were used as chiral derivatizing agents to differentiate between chiral primary amines. A three-component protocol was adopted to form complexes between the nucleosides and amines. The chiral differentiation ability of the nucleosides was examined using the 1H NMR chemical shift differences (ΔδR,S) between diastereomers formed from R- and S-amines. Adenosine and guanosine showed large chiral differentiation due to their purine rings. The measured diastereomeric excess using adenos
This document summarizes the discovery and optimization of a new class of positive allosteric modulators of AMPA receptors. Key points:
- A novel series was identified from a high throughput screen and optimized from an initial hit to a clinical candidate.
- Unusually for an ion channel target, optimization was integrated with regular generation of ligand-bound crystal structures, which uncovered a novel chemotype with a conserved trifluoromethyl interaction site.
- The hit was optimized through various modifications including changing substituents on aromatic rings, modifying amide linkages, adding or removing fluorine atoms, and altering fused ring systems to improve properties like developability, tolerability, and efficacy.
- Crystal
Voss et al. - 2006 - Identification and characterization of riproximin,Cristina Voss
1. Researchers purified and characterized a new type II ribosome-inactivating protein called riproximin from the plant Ximenia americana.
2. Riproximin was found to potently inhibit protein synthesis and cancer cell growth in vitro with picomolar IC50 values, and inhibit tumor growth in vivo after intraperitoneal or oral administration in a rat model.
3. The researchers identified the riproximin protein through mass spectrometry and cDNA sequencing. Molecular modeling showed riproximin has structural similarity to other toxic type II ribosome-inactivating proteins and an active site for its RNA N-glycosidase activity.
Chemical protein engineering synthetic and semisyntheticAli Hatami
This document summarizes various methods for chemically synthesizing and modifying peptides and proteins. It discusses solid phase peptide synthesis, native chemical ligation using peptide thioesters, and fragment condensation strategies. It also covers chemoselective ligations using oxime and hydrazone bonds and decarboxylative amide formation. Additionally, the document outlines chemical modifications like PEGylation, phosphorylation, and backbone modifications. Finally, it examines enzyme-mediated ligation techniques like sortase and biotin ligase that can link proteins and peptides in a sequence-specific manner.
This document discusses protein structure and bioinformatics. It begins by explaining the rationale for understanding protein structure and function, including determining protein sequences, structures, and relating this to function. It then covers levels of protein structure from primary to quaternary, methods for determining protein structures like X-ray crystallography, and uses of protein modeling and databases. The document provides examples of protein domains, folds, and membrane protein topology. It emphasizes that sequence determines conformation and that structure implies function.
This document presents the results of a study aiming to develop selective inhibitors of BACE2 using computer-aided drug design. BACE2 is homologous to BACE1 but is predominantly expressed in peripheral tissues and may serve as an alternative protease in APP processing. The study validated docking programs for reproducing binding poses of BACE1 and BACE2 inhibitors and evaluated scoring functions for virtual screening. A BACE2 homology model produced better virtual screening results than crystal structures. Future work will involve virtual screening larger libraries and designing new scaffolds for selective BACE2 inhibition.
Multinuclear liquid and solid-state NMR of Fructoborate complexJohn Edwards
This study used 1H, 13C, and 11B NMR spectroscopy to analyze the chemical structure, composition, and stability of Fruitex-B, a calcium fructoborate dietary supplement. NMR methods were developed to quantify the mono-complex, di-complex, free borate, and free fructose present in Fruitex-B. The results showed the complex is predominantly a di-ester form with borate coordinated to two fructose molecules. NMR analysis of multiple product batches demonstrated consistency in the relative concentrations of complex components. The study also examined the molecular stability of Fruitex-B when exposed to temperatures from 35-70°C.
The document discusses various topics in bioinformatics and protein structure. It provides an overview of ongoing thesis topics at Biobix including biomarker prediction, methylation, metabolomics, peptidomics, and more. It also discusses the rationale for understanding protein structure and function, levels of protein structure from primary to quaternary, methods for determining structure like X-ray crystallography, and approaches to secondary structure prediction including Chou-Fasman.
1) The document describes molecular dynamics simulations of the leucine transporter protein (LeuT) and the dopamine transporter protein (DAT) embedded in a lipid bilayer membrane to study substrate movement.
2) Key differences observed between LeuT and DAT include DAT's external gate forming less readily and its fourth extracellular loop unwinding more in the presence of substrate.
3) While LeuT and DAT dynamics were largely similar, some differences could provide insights into how DAT-specific inhibitors like cocaine interact with the transporter.
Dynamic modification of PMMA chips using PVA for GAG disaccharide isomer sepa...Yong Zhang
This document describes a microchip electrophoresis (MCE) method for separating unsaturated disaccharides from glycosaminoglycans (GAGs) using poly(methyl methacrylate) (PMMA) microchips dynamically coated with poly(vinyl alcohol) (PVA). PVA coating was shown to increase the hydrophilicity of the PMMA surface and reduce nonspecific adsorption. Using PVA-coated PMMA chips, two pairs of GAG disaccharide isomers (nDi-diSB/nDi-diSD and nDi-0S/nDi-HA) were baseline separated within 130 seconds by MCE for the first time. The dynamic PVA coating approach improves MCE resolution for
This document describes experiments to design potential protein antigens for a Pseudomonas aeruginosa vaccine. A homology model of type IV fimbrial precursor pilin was generated and showed good structural similarity to the reference protein despite some differences in binding region residues. Five redesigns of truncated PAK pilin were simulated: untruncated designs stabilized the binding region while truncated designs destabilized it. The untruncated designs are recommended for further vaccine development work.
This document describes a new method for selective N-methylation of peptide backbone amides on resin using the Mitsunobu reaction. The key aspects are:
1) N-trifluoroacetamide (Tfa) was used as the protecting group on resin-bound peptides, which can generate a nucleophilic anion for methylation via Mitsunobu conditions.
2) Tfa-protected peptides on resin underwent efficient (80-99%) and selective N-methylation of the backbone amide using the Mitsunobu reaction with triphenylphosphine, methanol and diisopropyl azodicarboxylate.
3) Unlike other reports, the T
Oracle Research Fellowship NeoN poster presentation_Spring 2015Samender Randhawa
- NeoN is a radical S-adenosyl-L-methionine (SAM) enzyme that epimerizes neomycin C to neomycin B through its two [4Fe-4S] clusters, playing a vital role in neomycin B antibiotic biosynthesis.
- The goal is to crystallize NeoN to understand its structure and catalytic activity at high resolution in order to modify it as a biocatalyst to recognize different substrates.
- NeoN will be cloned, expressed, purified, concentrated, and crystallized for X-ray diffraction to determine its structure and identify active site residues involved in substrate recognition and epimerization.
Structural and biochemical studies of cold shock domain containing proteins.
This thesis examines cold shock domain containing proteins through three chapters:
[1] A novel DNA microarray approach is developed to determine the sequence specificity of single-stranded nucleic acid binding proteins. Using this method, the major cold shock protein CspB from Bacillus subtilis is shown to bind preferentially to pyrimidine-rich sequences, with a high affinity for the consensus sequence 5'-GTCTTTG/T-3'.
[2] Six cold shock proteins from Salmonella typhimurium (CspA, B, C, D, E, and H) are cloned, expressed, and purified.
This document summarizes a study that found neomycin, an aminoglycoside antibiotic, is capable of binding tightly to single-stranded poly(A) RNA with a Kd in the micromolar range. Circular dichroism experiments showed neomycin forms a complex with poly(A) and increases its melting temperature from 44°C to 61°C, suggesting neomycin strongly stabilizes the poly(A) duplex. Isothermal titration calorimetry found neomycin binds oligo(A)30 with a binding constant of 5.3×10^6 M^-1 and a stoichiometry of one neomycin per 10 adenine bases. This study demonstrates that neomycin can target single
Enzyme Discovery for Natural Product BiosynthesisHongnan Cao
A poster presentation of collaborative work on the NIH funded project of Enzyme Discovery for Natural Product Biosynthesis at 2015 American Crystallography Association Meeting at Philadelphia, PA. Thanks to Rice University, University of Wisconsin-Madison, The Scripps Research Institute, University of Kentucky, The Midwest Center for Structural Genomics, The Northeast Center for Structural Genomics, APS synchrotron at Argonne National Lab
Exploring Proteins and Proteomes. Stryer,CHAPTER 3 pptkhair ullah
Methods in Protein Chemistry
This chapter discusses several methods used to isolate, purify, detect, degrade, analyze, and synthesize proteins. It describes techniques such as centrifugation, solubility, dialysis, gel filtration, affinity chromatography, HPLC, electrophoresis, and mass spectrometry. It also covers determining a protein's amino acid sequence through methods like Edman degradation, solid phase synthesis, chemical and enzymatic cleavage, and the use of DNA sequencing. The goal of these methods is to obtain a protein's amino acid sequence and gain functional information about proteins and proteomes.
The document summarizes research investigating the interface phenomena of poly(o-ethoxyaniline) (POEA) films using atomic force spectroscopy. The study found that POEA films consist of conducting islands surrounded by a less conductive matrix. The conducting islands were characterized by the presence of double-layer forces and visualized using transmission electron microscopy. The conducting islands were only 15 nm in diameter and could only be identified using adhesion mapping, not contact mode atomic force microscopy which showed larger aggregates. The degree of doping and pH affected the morphology and interactions observed, with fully dedoped POEA at pH 5 and partially conducting polyaniline.
Structural Modeling of the Ptf1a C2 Sequence Bound to Mammalian Rbpj and RbjlWard Coats
In this study, the researchers:
1) Generated structural models of the C2 sequence of Ptf1a bound to the β-trefoil domains of mammalian Rbpj and Rbpjl.
2) Found that the C2 sequence binds in an extended conformation, maintaining interactions seen in the Notch-IC/Rbpj complex.
3) Observed conserved residues in the hydrophobic pocket that maintain binding of proteins with a conserved tryptophan motif.
Proteomic Strategies for purification of lactate dehydrogenase ...Gaurav Dwivedi
The document summarizes proteomic strategies for purifying lactate dehydrogenase (LDH) from chicken muscle. Key steps included homogenizing muscle tissue, removing debris via centrifugation, precipitating proteins with ammonium sulfate, dialyzing to remove salts, and purifying LDH using blue sepharose affinity chromatography. SDS-PAGE and activity assays confirmed the isolation of a single 35 kDa protein band corresponding to LDH. The multi-step process successfully extracted and purified LDH from chicken muscle for further study.
The document discusses the purification of a Sitag/RGD/His-tag fusion protein for use as a scaffold in tissue engineering. It examines different lysis and purification methods to extract the protein from E. coli cells. Lysing methods tested include freeze-thaw, detergents, and enzymes. Purification was initially attempted using nickel affinity chromatography via the His-tag, but residual proteins remained. Purification using silica and the silica-binding Sitag was then explored, but elution with L-lysine was ineffective. Increased washes and incubation improved elution, but some bacterial proteins still remained. Future work may use a silica gel system or alter purification conditions.
This document summarizes an experiment on isolating and characterizing the enzyme alkaline phosphatase (AP) from E. coli bacteria. Key steps included purifying AP using dialysis, salting-in/salting-out, and DEAE cellulose chromatography. SDS-PAGE was used to analyze purity and molecular weight. Kinetic experiments at varying pH levels used the substrate PNPP and spectrophotometry to generate Michaelis-Menten and Lineweaver-Burk plots, allowing determination of kinetic parameters like Vmax and Km. The goal was to understand AP enzymatic activity and affinity for substrate under different conditions.
This document summarizes a study that explored using RNA nucleosides as chiral sensing agents in NMR spectroscopy. Adenosine, guanosine, uridine, and cytidine were used as chiral derivatizing agents to differentiate between chiral primary amines. A three-component protocol was adopted to form complexes between the nucleosides and amines. The chiral differentiation ability of the nucleosides was examined using the 1H NMR chemical shift differences (ΔδR,S) between diastereomers formed from R- and S-amines. Adenosine and guanosine showed large chiral differentiation due to their purine rings. The measured diastereomeric excess using adenos
This document summarizes the discovery and optimization of a new class of positive allosteric modulators of AMPA receptors. Key points:
- A novel series was identified from a high throughput screen and optimized from an initial hit to a clinical candidate.
- Unusually for an ion channel target, optimization was integrated with regular generation of ligand-bound crystal structures, which uncovered a novel chemotype with a conserved trifluoromethyl interaction site.
- The hit was optimized through various modifications including changing substituents on aromatic rings, modifying amide linkages, adding or removing fluorine atoms, and altering fused ring systems to improve properties like developability, tolerability, and efficacy.
- Crystal
Voss et al. - 2006 - Identification and characterization of riproximin,Cristina Voss
1. Researchers purified and characterized a new type II ribosome-inactivating protein called riproximin from the plant Ximenia americana.
2. Riproximin was found to potently inhibit protein synthesis and cancer cell growth in vitro with picomolar IC50 values, and inhibit tumor growth in vivo after intraperitoneal or oral administration in a rat model.
3. The researchers identified the riproximin protein through mass spectrometry and cDNA sequencing. Molecular modeling showed riproximin has structural similarity to other toxic type II ribosome-inactivating proteins and an active site for its RNA N-glycosidase activity.
Chemical protein engineering synthetic and semisyntheticAli Hatami
This document summarizes various methods for chemically synthesizing and modifying peptides and proteins. It discusses solid phase peptide synthesis, native chemical ligation using peptide thioesters, and fragment condensation strategies. It also covers chemoselective ligations using oxime and hydrazone bonds and decarboxylative amide formation. Additionally, the document outlines chemical modifications like PEGylation, phosphorylation, and backbone modifications. Finally, it examines enzyme-mediated ligation techniques like sortase and biotin ligase that can link proteins and peptides in a sequence-specific manner.
This document discusses protein structure and bioinformatics. It begins by explaining the rationale for understanding protein structure and function, including determining protein sequences, structures, and relating this to function. It then covers levels of protein structure from primary to quaternary, methods for determining protein structures like X-ray crystallography, and uses of protein modeling and databases. The document provides examples of protein domains, folds, and membrane protein topology. It emphasizes that sequence determines conformation and that structure implies function.
This document presents the results of a study aiming to develop selective inhibitors of BACE2 using computer-aided drug design. BACE2 is homologous to BACE1 but is predominantly expressed in peripheral tissues and may serve as an alternative protease in APP processing. The study validated docking programs for reproducing binding poses of BACE1 and BACE2 inhibitors and evaluated scoring functions for virtual screening. A BACE2 homology model produced better virtual screening results than crystal structures. Future work will involve virtual screening larger libraries and designing new scaffolds for selective BACE2 inhibition.
Multinuclear liquid and solid-state NMR of Fructoborate complexJohn Edwards
This study used 1H, 13C, and 11B NMR spectroscopy to analyze the chemical structure, composition, and stability of Fruitex-B, a calcium fructoborate dietary supplement. NMR methods were developed to quantify the mono-complex, di-complex, free borate, and free fructose present in Fruitex-B. The results showed the complex is predominantly a di-ester form with borate coordinated to two fructose molecules. NMR analysis of multiple product batches demonstrated consistency in the relative concentrations of complex components. The study also examined the molecular stability of Fruitex-B when exposed to temperatures from 35-70°C.
The document discusses various topics in bioinformatics and protein structure. It provides an overview of ongoing thesis topics at Biobix including biomarker prediction, methylation, metabolomics, peptidomics, and more. It also discusses the rationale for understanding protein structure and function, levels of protein structure from primary to quaternary, methods for determining structure like X-ray crystallography, and approaches to secondary structure prediction including Chou-Fasman.
1) The document describes molecular dynamics simulations of the leucine transporter protein (LeuT) and the dopamine transporter protein (DAT) embedded in a lipid bilayer membrane to study substrate movement.
2) Key differences observed between LeuT and DAT include DAT's external gate forming less readily and its fourth extracellular loop unwinding more in the presence of substrate.
3) While LeuT and DAT dynamics were largely similar, some differences could provide insights into how DAT-specific inhibitors like cocaine interact with the transporter.
Dynamic modification of PMMA chips using PVA for GAG disaccharide isomer sepa...Yong Zhang
This document describes a microchip electrophoresis (MCE) method for separating unsaturated disaccharides from glycosaminoglycans (GAGs) using poly(methyl methacrylate) (PMMA) microchips dynamically coated with poly(vinyl alcohol) (PVA). PVA coating was shown to increase the hydrophilicity of the PMMA surface and reduce nonspecific adsorption. Using PVA-coated PMMA chips, two pairs of GAG disaccharide isomers (nDi-diSB/nDi-diSD and nDi-0S/nDi-HA) were baseline separated within 130 seconds by MCE for the first time. The dynamic PVA coating approach improves MCE resolution for
This document describes experiments to design potential protein antigens for a Pseudomonas aeruginosa vaccine. A homology model of type IV fimbrial precursor pilin was generated and showed good structural similarity to the reference protein despite some differences in binding region residues. Five redesigns of truncated PAK pilin were simulated: untruncated designs stabilized the binding region while truncated designs destabilized it. The untruncated designs are recommended for further vaccine development work.
This document describes a new method for selective N-methylation of peptide backbone amides on resin using the Mitsunobu reaction. The key aspects are:
1) N-trifluoroacetamide (Tfa) was used as the protecting group on resin-bound peptides, which can generate a nucleophilic anion for methylation via Mitsunobu conditions.
2) Tfa-protected peptides on resin underwent efficient (80-99%) and selective N-methylation of the backbone amide using the Mitsunobu reaction with triphenylphosphine, methanol and diisopropyl azodicarboxylate.
3) Unlike other reports, the T
Oracle Research Fellowship NeoN poster presentation_Spring 2015Samender Randhawa
- NeoN is a radical S-adenosyl-L-methionine (SAM) enzyme that epimerizes neomycin C to neomycin B through its two [4Fe-4S] clusters, playing a vital role in neomycin B antibiotic biosynthesis.
- The goal is to crystallize NeoN to understand its structure and catalytic activity at high resolution in order to modify it as a biocatalyst to recognize different substrates.
- NeoN will be cloned, expressed, purified, concentrated, and crystallized for X-ray diffraction to determine its structure and identify active site residues involved in substrate recognition and epimerization.
Structural and biochemical studies of cold shock domain containing proteins.
This thesis examines cold shock domain containing proteins through three chapters:
[1] A novel DNA microarray approach is developed to determine the sequence specificity of single-stranded nucleic acid binding proteins. Using this method, the major cold shock protein CspB from Bacillus subtilis is shown to bind preferentially to pyrimidine-rich sequences, with a high affinity for the consensus sequence 5'-GTCTTTG/T-3'.
[2] Six cold shock proteins from Salmonella typhimurium (CspA, B, C, D, E, and H) are cloned, expressed, and purified.
This document summarizes a study that found neomycin, an aminoglycoside antibiotic, is capable of binding tightly to single-stranded poly(A) RNA with a Kd in the micromolar range. Circular dichroism experiments showed neomycin forms a complex with poly(A) and increases its melting temperature from 44°C to 61°C, suggesting neomycin strongly stabilizes the poly(A) duplex. Isothermal titration calorimetry found neomycin binds oligo(A)30 with a binding constant of 5.3×10^6 M^-1 and a stoichiometry of one neomycin per 10 adenine bases. This study demonstrates that neomycin can target single
The document describes the process of purifying the elongation factor LepA/EF4 protein from E. coli. The gene for EF4 was transformed into E. coli cells using a plasmid. The cells were then lysed using sonication and the EF4 protein was purified from the cell lysate using affinity chromatography and its hexahistidine tag. The concentration of the purified EF4 protein was determined to be 0.57 μg/μL using a Bradford assay and its molecular weight was found to be ~69 kDa by SDS-PAGE. Secondary and tertiary structural analysis using circular dichroism and fluorescence spectroscopy yielded thermodynamic values for EF4 protein denaturation.
GRAS proteins expression and purification Mesele Tilahun
The document summarizes the production and analysis of the GRAS protein Os-SCL7. It describes how the gene encoding the GRAS domain of Os-SCL7 was cloned and expressed in E. coli. The protein was then purified using nickel affinity chromatography and size exclusion chromatography. Sequence analysis revealed the protein is 378 amino acids with a predicted molecular weight of 41.5 kDa. Potential cleavage sites for specific proteases were also identified from the amino acid sequence.
This document discusses assembly-assisted peptide ligation in native conditions. It proposes that peptide fragments can assemble into protein-like structures through non-covalent interactions in native conditions, bringing peptide termini into close proximity. This proximity could allow the termini to ligate without the need for cysteine residues, as seen in native chemical ligation. The document outlines an experiment to test this hypothesis using two fragments of a zinc finger protein, which would assemble due to zinc ion binding and potentially ligate using the coupling reagent PMSF in native conditions.
This document summarizes the chemical synthesis of DNA through the phosphoramidite method. It describes the evolution of DNA synthesis from early phosphodiester and phosphotriester methods to the current phosphoramidite approach. The key steps of the phosphoramidite method are detritylation, activation and coupling of nucleotides, capping, and oxidation. Automated DNA synthesizers utilize this method to programmatically assemble DNA strands from nucleotide building blocks. Chemically synthesized DNA oligonucleotides have many applications, including gene cloning, sequencing, and engineering genes with novel properties.
1. Lysozyme crystals were grown using the hanging drop vapor diffusion method with varying concentrations of lysozyme and four precipitating agents. The best crystals formed at 15 mg/mL lysozyme, 30% PEG 8000, 1 M NaCl, pH 4.5.
2. X-ray diffraction data from the crystals revealed the unit cell dimensions to be a=129.8 Å, b=129.8 Å, c=91.4 Å, space group P43212, indicating a tetragonal crystal system.
3. The electron density map generated from diffraction data allowed amino acid residues to be fitted and verified using software tools, elucidating the lysozyme structure at high resolution.
Living cell surface labeling of exposed amine groups by membrane impermeable ...Ujwal Patil
Cell surface proteomics has seen momentous developments in the past two decades but still faces major challenges in location verification of identified cell surface proteins (CSPs). Recent approaches focus on modification/labeling of CSPs by chemical reagents followed by mass spectrometric analysis of labeled CSPs. Popular biotinylation regents have shown some intrinsic disadvantages such as internalization in the cell cytoplasm, poor recovery of biotinylated proteins, presence of endogenous biotin and non-specific interactions between avidin and proteins. In our study, silica coated iron oxide (Fe3O4@SiO2) superparamagnetic nanoparticles (MNPs) of 100-150 nm were utilized to prepare an impermeable and magnetically separable cell surface labeling reagent. Sulfo-N-hydroxysuccinimidyl (NHS) ester group was conjugated to the surface of Fe3O4@SiO2 MNPs via a disulfide bond to facilitate removal of the magnetic nanoparticle moiety after separation. The surface exposed amine groups of Saccharomyces cerevisae were modified at physiological pH on ice to preserve the native structure of CSPs. Electron microscopic analysis of MNPs conjugated to the S. cerevisae cell surface confirmed the impermeable nature of sulfo-NHS ester Fe3O4@SiO2 MNPs. The labeled CSPs were easily separated by using a magnet and eluted from MNPs by cleaving a disulfide bond. The LC-MS/MS analysis of labeled peptides revealed 30 surface proteins located on solvent exposed surface of the S cerevisae. The sulfo-NHS ester modified Fe3O4@SiO2 MNPs offers benefits such as impermeability, quick magnetic separation of labeled peptides and labeling under physiological conditions.
Gene sequencing is the process of determining the order of nucleotides in a gene or genome. It allows scientists to understand how alterations in DNA sequences can cause genetic conditions or affect protein function. There are several methods for gene sequencing, including Sanger sequencing which uses chain termination with dideoxynucleotides, and next generation sequencing techniques like pyrosequencing. Gene sequencing provides insight into genetic variations and metabolic pathways that can aid disease research and treatment development.
Computational simulations were used to investigate possible dimer structures of the neuronal protein alpha-synuclein. Molecular docking and molecular dynamics simulations were performed on dimers formed from alpha-helical and beta-sheet conformations of alpha-synuclein monomers. Binding energies and interactions between monomers in the dimer structures were analyzed. Both hydrophobic and electrostatic interactions contributed significantly to dimer stability, even though alpha-synuclein is highly charged. The central hydrophobic region of alpha-synuclein formed the majority of the interface between monomers in the dimer structures.
This document summarizes research characterizing the mumps virus nucleocapsid-binding domain (NBD) protein via fluorescence spectroscopy and circular dichroism. Researchers created a variant of the mumps NBD protein called F366W by introducing a tryptophan mutation using site-directed mutagenesis. This allowed stability measurements using fluorescence spectroscopy. The mutated protein was expressed in E. coli and purified. Fluorescence spectroscopy and circular dichroism were used to monitor the protein's tertiary structure as it was subjected to thermal and chemical denaturation conditions. Results from these experiments provided insights into the stability and folding of the mumps NBD protein.
This document provides the timetable and protocols for a practical course on making and analyzing tRNA synthetases in vivo and using cell-free protein synthesis. Over two weeks, students will perform site-directed mutagenesis to produce mutant aminoacyl-tRNA synthetases, express their proteins in E. coli cells and purify the proteins, and use cell-free synthesis to attempt incorporating a phosphotyrosine analogue into a target protein using their mutant synthetases and suppressor tRNA. The document outlines the experimental steps, including mutagenesis, transformation, plasmid preparation, sequencing, protein expression and purification, cell-free reaction set up, and analysis by SDS-PAGE. Safety procedures are also described to handle
This document discusses various gene sequencing methods. It begins by introducing DNA and the importance of sequencing the genetic code. It then describes several early sequencing techniques like Sanger sequencing using chain termination or chemical cleavage. It discusses the need for sequencing to understand genetic conditions. The document also covers topics like genome sequencing, genomics, and high-throughput sequencing techniques like dye-terminator sequencing which replaced radioactive labels with fluorescent labels to automate the process.
Gene synthesis involves chemically synthesizing DNA without a template by adding nucleotides one by one. It is the basis of synthetic biology. The key steps in gene synthesis are sequence design, oligo synthesis, gene assembly, sequence verification, and preparing the synthetic DNA. Errors can be introduced at each step, so sequences must be verified before use. Gene synthesis is used widely in research to study biological functions and develop applications like DNA vaccines.
The document describes a student project on bisulfite modification and methylation-specific PCR. It includes an introduction to epigenetics and DNA methylation, as well as objectives, materials and methods for DNA extraction from blood, quantification, bisulfite modification, and methylation-specific PCR. The student thanks their professors and department for guidance. Sections cover introduction, objectives, materials and methods, observations and results, discussion, conclusions, and references.
The document discusses various topics related to the genetic code, including:
1. The genetic code is degenerate, meaning many amino acids are specified by more than one codon. Wobble in the anticodon allows one tRNA to recognize multiple codons.
2. Three rules govern the genetic code: codons are read in groups of three in the 5' to 3' direction without gaps or overlaps.
3. Suppressor mutations can reside in the same gene or a different gene and suppress the effects of mutations by producing functional proteins.
The Assembly, Structure and Activation of Influenza a M2 Transmembrane Domain...Haley D. Norman
This document summarizes two research papers on computational methods for analyzing protein structures and interactions. The first paper describes a Bayesian method for determining protein structures from sparse single-molecule X-ray diffraction data. The second paper presents xMDFF, a new molecular dynamics flexible fitting approach for refining low-resolution protein structures determined by X-ray crystallography. The third paper introduces i-ATTRACT, a new flexible protein-protein docking method that combines rigid body and flexible interface residue energy minimization for predicting protein complex structures.
Understanding the role and evolution of internal symmetry in protein structure is a fundamental question in structural biology. We present here CE-Symm 2.0, a key tool to address that question, which is able to detect all types of protein internal symmetry and provides a robust and intuitive sequence-to-structure analysis of all repeats. Notable features compared to the previous version include an optimized multiple alignment between repeats, determination of the full point group, and identification of multiple symmetry axes. We expect CE-Symm to find ample use in evolutionary studies, functional annotation, and structural classification of proteins.
This work was presented at the 3DSIG 2016 conference in Orlando, FL, on July 8, 2016.
See also the poster form: http://www.slideshare.net/sbliven/3dsig-2016-poster-exploring-internal-symmetry-and-structural-repeats-with-cesymm
Oligonucleotides therapeutics work by altering gene expression through complementary binding to mRNA. There are three major classes: antisense oligonucleotides inhibit protein expression through hybridization to mRNA, siRNA induce mRNA degradation, and miRNA inhibit translation. Oligonucleotides are modified through conjugation or encapsulation to improve delivery and stability, and resist nuclease degradation. Antisense oligonucleotides specifically designed to hybridize with mRNA can function through RNase H degradation or steric blocking of translation.
1. Characterization of Radical S-adenosyl-L-methionine
Epimerase, NeoN
Samender Randhawa1, John Zhang1, Daniel P. Dowling1
1Department of Chemistry, University of Massachusetts Boston, Boston, MA
Funding for this research was provided by the Oracle Education Foundation grant to the CSM
Abstract
A vast number of enzymes are being characterized that belong to a
superfamily known as radical S-adenosyl-L-methionine (SAM) enzymes, whose
members contain a [4Fe–4S] cluster ligated by three cysteine residues. Radical SAM
enzymes generally initiate catalysis by reductively cleaving SAM, yielding a
molecule of L-methionine and the 5′-deoxyadenosyl radical species. This radical
species can then be utilized in Nature to initiate a numerous amount of radical
chemistry through substrate hydrogen atom abstraction. A number of radical SAM
enzymes, including the Neomycin C epimerase (NeoN), contain additional iron–
sulfur clusters that are required for the reactions they catalyze.
The goal of this work is to understand the underlying biochemical basis for
NeoN catalytic activity. To accomplish this, the technique of X-ray crystallography
will be utilized to obtain atomic resolution information for this system. We are
interested in how NeoN is capable of specifically catalyzing the epimerization of
one, stereospecific site of neomycin C, which contains multiple possible sites that
could be acted upon. Future studies of this system may lead to the development of
bioengineered epimerases, and we hypothesize that NeoN could be used as a
biocatalyst tool in the future for developing specific epimerases that could produce
novel compounds. Here, initial experiments to clone the neoN gene from
Streptomyces fradiae into an expression platform are presented.
Introduction
Radical S-adenosyl-L-methionine (SAM) enzymes (RS enzymes) carryout a
variety of biological functions, such as synthesis of cofactors, and antibiotics1. To
date 113,744 RS functional units have been characterized both biochemically and
structurally2. The majority of the RS enzymes adopt a full or partial triosephosphate
isomerase barrel (TIM) fold, with one of the first reported RS enzyme structures,
that of biotin synthase (BioB), adopting a full TIM barrel fold3 (Fig.1). The presence
of a [4Fe- 4S] cluster bound to a conserved cysteine triad is common to all RS
enzymes, and the cluster is positioned at the carboxy terminus of the protein barrel.
SAM binds to the cluster within the barrel, and the substrate (dethiobiotin for biotin
synthase4) binds proximal to SAM, poised for catalysis. The RS enzymes use a
common mechanism: the generation of a primary carbon- centered radical
intermediate, the 5′ deoxyadenosyl radical (dAdo●) (Fig.2).
NeoN is a recently characterized epimerase that plays a vital role in the last
biosynthetic step of neomycin B, an aminoglycoside antibiotic produced by
Streptomyces fradiae1. NeoN is a RS enzyme that selectively epimerizes the C-5'''
carbon of Neomycin C1 (Fig.3). The auxiliary clusters in SAM enzymes, including
NeoN, may have multiple uses, such as accepting or donating electrons during the
course of the reaction, or they may be anchoring points for substrates to bind. Their
utility expands the radical SAM enzyme functionality, enabling radical SAM
enzymes to perform added chemical reactions. We are interested in how NeoN is
capable of specifically catalyzing the epimerization of one, stereospecific site of
neomycin C, which contains multiple possible sites that could be acted upon.
Aims:
• To obtain atomic resolution information in order to characterize and understand
how NeoN functions.
• To identify important residues within the active site that play a role in catalytic
epimerization.
• To characterize substrate recognition in order to bind other non-substrate
molecules for specific epimerization reactions.
Future Work
• Cloning of NeoN genome, expressing, and crystallizing the protein using a
different strain of Streptomyces fradiae.
• Gibson assembly method of molecular cloning will be preferred over colony
PCR for correct expression of the gene.
• Ensuring that the expressed crystallized protein is soluble and functionally
active.
• Analyzing the active site residues to explore incorporating non-substrate
molecules for possible epimerization by the enzyme.
Methods, Results, and Development
X-ray crystallography is a tool used to identify the atomic and molecular
structure of a crystal which is a highly ordered structure. The crystalline arrangement of
atoms causes the incident X-rays to diffract into many specific directions. The
diffraction pattern can then be analyzed using computational programs to obtain an
electron density map, which reveals the protein’s structure and the binding of cofactors
and ligands. The overall scheme of crystallizing NeoN is shown in Figure 6.
The main idea behind crystallizing a protein is that most proteins are soluble at
physiological conditions, but as the concentration of solutes rises, the protein becomes
less soluble, leading it to crystallize or precipitate. The goal of crystallization is to
produce a well-ordered crystal lattice that is able to provide a diffraction pattern on
exposure to X-rays.
The amplification of neoN gene using the ATCC 19760 strain of Streptomyces
fradiae resulted in the production of two fairly close bands in the 900bp region (Fig.4).
Multiple annealing temperatures were tested to understand this causation (Fig.5). Due
to the difficulties in obtaining a positive clone using the standard ligation protocol, a
different method called the Gibson Assembly was used with the pET28a vector (Fig.7).
The amplification of neoN did yield a single band in the 900bp region, but the band
intensity was not high to be conclusive as an effective method for neoN gene
amplification. Therefore, the results of the Gibson assembly, unfortunately, were not
successful.
Conclusions
• The primer design plays an important role in the complete expression of the
NeoN protein and its further optimization will yield better results in the future.
• Epimerization of Neomycin C to Neomycin B is accomplished by NeoN through
its two [4Fe-4S] clusters at C-5′′′.
• A thorough understanding of the NeoN structure for Neomycin B synthesis will
open up the possibility of modifying the enzyme to recognize different types of
substrates, making NeoN a tractable tool as a biocatalyst.
References:
1. Fumitaka Kudo. et al. JACS (2014) 136, 13909-13915.
2. SFFLD - Superfamily List." SFLD - Superfamily List. Web. 26 Apr. 2015
3. Shisler, Krista. et al. Curr. Opin. Struct. Biol. (2012) 22, 701-710.
4. Berkovitch, F. et al. Science (2004) 303, 76–79.
Figure 2. Reductive cleavage of SAM.
RS enzymes use [4Fe-4S] clusters to bind
to SAM and transfer an electron to the
sulfonium of SAM, producing methionine
and dAdo● through homolytic cleavage of
S-5′C bond in SAM. The dAdo● then
abstracts an H-atom from the substrate to
initiate a radical-mediated transformation.
Figure 6. Protein X-ray
crystallography methods scheme.
The neoN gene was cloned from
Streptomyces fradiae (ATCC 19760)
into an expression vector containing N
terminal 6xHis tag (Hexa-histidine).
pET28 vector was selected for ligation
reactions with the neoN DNA as it
contains a selectable marker for
kanamycin resistance which is useful
for isolating cells with the correctly
inserted plasmid. Additionally, a
thrombin cleavage site is also a feature
of this vector whose role is crucial
during protein purification.
Figure 3. NeoN epimerization of
Neomycin C to Neomycin B. NeoN is a
putative RS enzyme which is encoded in
the neomycin gene cluster. This gene
cluster is structurally related to
aminoglycoside biosynthetic gene clusters
such as lividomycin B and paromomycin1.
Figure 1. Structure of BioB. Overall
TIM barrel structure is colored in red
(α helices) and yellow (β strands).
The cofactors and substrates are
arranged vertically as follows: [4Fe-
4S] cluster on the bottom, SAM,
dethiobiotin, and the [2Fe-2S] cluster.
Carbon atoms are colored cyan for
substrates. PDB accession code 1R30.
Figure 5. Colony PCR gel analysis. The
annealing temperature was varied to 62℃,
68℃, and 72℃ to account for the presence of
magnesium in the buffer which raises the
melting point of the primer as well as the
unwanted hairpin structures. A ten-fold dilution
(represented by ‘D’ next to temperature) was
also performed to examine any differences in
the amount of intensities obtained for neoN.
The agarose gel was analyzed and it was noted
that there was no impact of varying the
annealing temperature from the ideal
temperature of 55℃.
62 62D 68 68D 72 72D 1kb ladder
A1 B1
A2 B2
Figure 4. Colony PCR gel analysis. The
presence of neoN gene was confirmed in
Streptomyces fradiae cell line (ATCC 19760).
The PCR was performed at the ideal annealing
temperature of 55℃. Interestingly, there were
two bands present closely and this region of
gel was cut and the DNA was extracted from
it. The bands present closely in the 900bp
region indicated the possible amplification of
neoN in the colony PCR samples.
Figure 7. Gibson Assembly PCR gel analysis. (A) The Gibson assembly method of
cloning the neoN gene resulted in the presence of one band in the 900bp region when the
agarose gel was analyzed. However, the low intensity of the band suggested that the amount
of DNA was not high. (B) Further analysis of the gel with GelQuant software indicated that
the amount of DNA amplification was less than 5%. For the band in lane 6, the percentage
of neoN DNA in the sample against the background was 1362275/ 36190215=0.0376 or
3.76%
A B