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Oksana Lockridge - One of the best experts on this subject based on the ideXlab platform.
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Polyproline-Rich Peptides Organize Four Cholinesterase Subunits into a Tetramer; BChE and AChE Scavenge Polyproline Peptides Released during Metabolic Turnover
Proceedings, 2020Co-Authors: Oksana Lockridge, Lawrence M SchopferAbstract:The genes for acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) encode the proteins responsible for enzyme activity. Additional gene products, PRiMA and ColQ, anchor AChE and BChE proteins into membranes. Soluble AChE and BChE tetramers are composed of four identical subunits plus one Polyproline-rich peptide. Dilution does not release the Polyproline-rich peptide from tetramers. However, protein denaturation, for example, heating in a boiling water bath, dissociates the Polyproline-rich peptide. Using mass spectrometry to sequence peptides released from soluble AChE and BChE tetramers, we find sequences that correspond to proline-rich regions from a variety of proteins. A typical peptide sequence contains 20 consecutive prolines in a 23-residue peptide, LPPPPPPPPPPPPPPPPPPPPLP. There is no single, common consensus sequence, i.e., no specific gene appears to be responsible for the Polyproline-rich peptides found in soluble AChE and BChE tetramers. We propose that during metabolic turnover, protein fragments containing Polyproline-rich sequences are scavenged by AChE and BChE dimers, to make stable AChE and BChE tetramers. The 40-residue, alpha-helical C-terminus of AChE or BChE is the tetramerization domain that binds the Polyproline-rich peptide. Four parallel alpha helices wrap around a single antiparallel Polyproline peptide to lock the tetramer in place. This organization was established by classical X-ray crystallography for isolated C-termini in complex with a proline-rich peptide. The organization was confirmed for intact, tetrameric human BChE using cryoelectron microscopy. When 40 amino acids are deleted from the carboxy terminus, monomeric enzymes are created that retain full enzymatic activity.
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Polyproline-rich peptides associated with Torpedo californica acetylcholinesterase tetramers.
Chemico-biological interactions, 2020Co-Authors: Lilly Toker, Lawrence M Schopfer, Israel Silman, Tzviya Zeev-ben-mordehai, Joel L. Sussman, Oksana LockridgeAbstract:Acetylcholinesterase (AChE) terminates cholinergic neurotransmission by hydrolyzing acetylcholine. The collagen-tailed AChE tetramer is a product of 2 genes, ACHE and ColQ. The AChE tetramer consists of 4 identical AChE subunits and one Polyproline-rich peptide, whose function is to hold the 4 AChE subunits together. Our goal was to determine the amino acid sequence of the Polyproline-rich peptide(s) in Torpedo californica AChE (TcAChE) tetramers to aid in the analysis of images that will be acquired by cryo-EM. Collagen-tailed AChE was solubilized from Torpedo californica electric organ, converted to 300 kDa tetramers by digestion with trypsin, and purified by affinity chromatography. Polyproline-rich peptides were released by denaturing the TcAChE tetramers in a boiling water bath, and reducing disulfide bonds with dithiothreitol. Carbamidomethylated peptides were separated from TcAChE protein on a spin filter before they were analyzed by liquid chromatography tandem mass spectrometry on a high resolution Orbitrap Fusion Lumos mass spectrometer. Of the 64 identified collagen-tail (ColQ) peptides, 60 were from the Polyproline-rich region near the N-terminus of ColQ. The most abundant proline-rich peptides were SVNKCCLLTPPPPPMFPPPFFTETNILQE, at 40% of total mass-spectral signal intensity, and SVNKCCLLTPPPPPMFPPPFFTETNILQEVDLNNLPLEIKPTEPSCK, at 27% of total intensity. The high abundance of these 2 peptides makes them candidates for the principal form of the Polyproline-rich peptide in the trypsin-treated TcAChE tetramers.
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Tetramer organizing Polyproline-rich peptides identified by mass spectrometry after release of the peptides from Hupresin-purified butyrylcholinesterase tetramers isolated from milk of domestic pig (Sus scrofa).
Data in brief, 2018Co-Authors: Ashima Saxena, Lawrence M Schopfer, Tatyana Belinskaya, Oksana LockridgeAbstract:Abstract Milk of the domestic pig has 10 times more butyrylcholinesterase (BChE) per mL than porcine serum. We purified BChE from porcine milk by affinity chromatography on Hupresin-Sepharose. The pure porcine BChE (PoBChE) was a tetramer with a molecular weight of 340,000, similar to that of human BChE tetramers. The C-terminal 40 residues of PoBChE constitute the tetramerization domain. The glue that holds the 4 BChE subunits together is a Polyproline-rich peptide. Mass spectrometry analysis of trypsin-digested PoBChE identified a variety of Polyproline-rich peptides originating from 12 different proteins. The donor proteins exist in the nucleus or cytoplasm of cells and contribute their Polyproline-rich peptides after a cell is degraded. The secreted PoBChE scavenges the Polyproline-rich peptides and incorporates one Polyproline peptide per PoBChE tetramer, where the Polyproline peptide is bound noncovalently but very tightly with an estimated dissociation constant of 10–12 M. The most abundant Polyproline-rich peptides were derived from acrosin, homeobox protein HoxB4, lysine-specific demethylase 6B, proline-rich protein 12, and proline-rich membrane anchor 1 (PRiMA). The research article associated with the data in this report can be found in Saxena et al. (2018). The Data in Brief report lists all the Polyproline-rich peptides identified in PoBChE tetramers.
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Tetramer-organizing Polyproline-rich peptides differ in CHO cell-expressed and plasma-derived human butyrylcholinesterase tetramers.
Biochimica et biophysica acta, 2016Co-Authors: Lawrence M Schopfer, Oksana LockridgeAbstract:Tetrameric butyrylcholinesterase (BChE) in human plasma is the product of multiple genes, namely one BCHE gene on chromosome 3q26.1 and multiple genes that encode Polyproline-rich peptides. The function of the Polyproline-rich peptides is to assemble BChE into tetramers. CHO cells transfected with human BChE cDNA express BChE monomers and dimers, but only low quantities of tetramers. Our goal was to identify the Polyproline-rich peptides in CHO-cell derived human BChE tetramers. CHO cell-produced human BChE tetramers were purified from serum-free culture medium. Peptides embedded in the tetramerization domain were released from BChE tetramers by boiling and identified by liquid chromatography-tandem mass spectrometry. A total of 270 proline-rich peptides were sequenced, ranging in size from 6-41 residues. The peptides originated from 60 different proteins that reside in multiple cell compartments including the nucleus, cytoplasm, and endoplasmic reticulum. No single protein was the source of the Polyproline-rich peptides in CHO cell-expressed human BChE tetramers. In contrast, 70% of the tetramer-organizing peptides in plasma-derived BChE tetramers originate from lamellipodin. No protein source was identified for Polyproline peptides containing up to 41 consecutive proline residues. In conclusion, the use of Polyproline-rich peptides as a tetramerization motif is documented only for the cholinesterases, but is expected to serve other tetrameric proteins as well. The CHO cell data suggest that the BChE tetramer-organizing peptide can arise from a variety of proteins.
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Origin of Polyproline-rich peptides in human butyrylcholinesterase tetramers.
Chemico-biological interactions, 2016Co-Authors: Hong Peng, Lawrence M Schopfer, Oksana LockridgeAbstract:The human butyrylcholinesterase (HuBChE) tetramer is composed of 4 identical subunits and a noncovalently bound Polyproline-rich peptide. In a previous report we identified lamellipodin as the source of the Polyproline-rich peptides in HuBChE tetramers purified from plasma. Our current goal was to identify proteins in addition to lamellipodin that donate Polyproline-rich peptides to plasma HuBChE tetramers. Peptides were released from 1 mg of pure plasma-derived HuBChE tetramers by boiling. Mass spectrometry identified 74 Polyproline-rich peptides. MALDI-TOF mass spectra and spectral counting of the LC-MS/MS data supported the conclusion that lamellipodin accounted for 70% of the Polyproline-rich peptides. Additional precursor proteins were matched through BLASTp searches, suggesting but not proving, that 20 proteins including UDP-N-acetyl glucosamine transferase ALG13 homolog, leiomodin 2, and zinc finger homeobox protein 2 are sources of Polyproline-rich peptides found in HuBChE tetramers. Eighteen Polyproline-rich peptides had no match in the human protein database. In conclusion, HuBChE assembles into tetramers through interaction of its C-terminal domain with Polyproline peptides derived from a variety of proteins.
Lawrence M Schopfer - One of the best experts on this subject based on the ideXlab platform.
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Polyproline-Rich Peptides Organize Four Cholinesterase Subunits into a Tetramer; BChE and AChE Scavenge Polyproline Peptides Released during Metabolic Turnover
Proceedings, 2020Co-Authors: Oksana Lockridge, Lawrence M SchopferAbstract:The genes for acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) encode the proteins responsible for enzyme activity. Additional gene products, PRiMA and ColQ, anchor AChE and BChE proteins into membranes. Soluble AChE and BChE tetramers are composed of four identical subunits plus one Polyproline-rich peptide. Dilution does not release the Polyproline-rich peptide from tetramers. However, protein denaturation, for example, heating in a boiling water bath, dissociates the Polyproline-rich peptide. Using mass spectrometry to sequence peptides released from soluble AChE and BChE tetramers, we find sequences that correspond to proline-rich regions from a variety of proteins. A typical peptide sequence contains 20 consecutive prolines in a 23-residue peptide, LPPPPPPPPPPPPPPPPPPPPLP. There is no single, common consensus sequence, i.e., no specific gene appears to be responsible for the Polyproline-rich peptides found in soluble AChE and BChE tetramers. We propose that during metabolic turnover, protein fragments containing Polyproline-rich sequences are scavenged by AChE and BChE dimers, to make stable AChE and BChE tetramers. The 40-residue, alpha-helical C-terminus of AChE or BChE is the tetramerization domain that binds the Polyproline-rich peptide. Four parallel alpha helices wrap around a single antiparallel Polyproline peptide to lock the tetramer in place. This organization was established by classical X-ray crystallography for isolated C-termini in complex with a proline-rich peptide. The organization was confirmed for intact, tetrameric human BChE using cryoelectron microscopy. When 40 amino acids are deleted from the carboxy terminus, monomeric enzymes are created that retain full enzymatic activity.
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Polyproline-rich peptides associated with Torpedo californica acetylcholinesterase tetramers.
Chemico-biological interactions, 2020Co-Authors: Lilly Toker, Lawrence M Schopfer, Israel Silman, Tzviya Zeev-ben-mordehai, Joel L. Sussman, Oksana LockridgeAbstract:Acetylcholinesterase (AChE) terminates cholinergic neurotransmission by hydrolyzing acetylcholine. The collagen-tailed AChE tetramer is a product of 2 genes, ACHE and ColQ. The AChE tetramer consists of 4 identical AChE subunits and one Polyproline-rich peptide, whose function is to hold the 4 AChE subunits together. Our goal was to determine the amino acid sequence of the Polyproline-rich peptide(s) in Torpedo californica AChE (TcAChE) tetramers to aid in the analysis of images that will be acquired by cryo-EM. Collagen-tailed AChE was solubilized from Torpedo californica electric organ, converted to 300 kDa tetramers by digestion with trypsin, and purified by affinity chromatography. Polyproline-rich peptides were released by denaturing the TcAChE tetramers in a boiling water bath, and reducing disulfide bonds with dithiothreitol. Carbamidomethylated peptides were separated from TcAChE protein on a spin filter before they were analyzed by liquid chromatography tandem mass spectrometry on a high resolution Orbitrap Fusion Lumos mass spectrometer. Of the 64 identified collagen-tail (ColQ) peptides, 60 were from the Polyproline-rich region near the N-terminus of ColQ. The most abundant proline-rich peptides were SVNKCCLLTPPPPPMFPPPFFTETNILQE, at 40% of total mass-spectral signal intensity, and SVNKCCLLTPPPPPMFPPPFFTETNILQEVDLNNLPLEIKPTEPSCK, at 27% of total intensity. The high abundance of these 2 peptides makes them candidates for the principal form of the Polyproline-rich peptide in the trypsin-treated TcAChE tetramers.
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Tetramer organizing Polyproline-rich peptides identified by mass spectrometry after release of the peptides from Hupresin-purified butyrylcholinesterase tetramers isolated from milk of domestic pig (Sus scrofa).
Data in brief, 2018Co-Authors: Ashima Saxena, Lawrence M Schopfer, Tatyana Belinskaya, Oksana LockridgeAbstract:Abstract Milk of the domestic pig has 10 times more butyrylcholinesterase (BChE) per mL than porcine serum. We purified BChE from porcine milk by affinity chromatography on Hupresin-Sepharose. The pure porcine BChE (PoBChE) was a tetramer with a molecular weight of 340,000, similar to that of human BChE tetramers. The C-terminal 40 residues of PoBChE constitute the tetramerization domain. The glue that holds the 4 BChE subunits together is a Polyproline-rich peptide. Mass spectrometry analysis of trypsin-digested PoBChE identified a variety of Polyproline-rich peptides originating from 12 different proteins. The donor proteins exist in the nucleus or cytoplasm of cells and contribute their Polyproline-rich peptides after a cell is degraded. The secreted PoBChE scavenges the Polyproline-rich peptides and incorporates one Polyproline peptide per PoBChE tetramer, where the Polyproline peptide is bound noncovalently but very tightly with an estimated dissociation constant of 10–12 M. The most abundant Polyproline-rich peptides were derived from acrosin, homeobox protein HoxB4, lysine-specific demethylase 6B, proline-rich protein 12, and proline-rich membrane anchor 1 (PRiMA). The research article associated with the data in this report can be found in Saxena et al. (2018). The Data in Brief report lists all the Polyproline-rich peptides identified in PoBChE tetramers.
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Tetramer-organizing Polyproline-rich peptides differ in CHO cell-expressed and plasma-derived human butyrylcholinesterase tetramers.
Biochimica et biophysica acta, 2016Co-Authors: Lawrence M Schopfer, Oksana LockridgeAbstract:Tetrameric butyrylcholinesterase (BChE) in human plasma is the product of multiple genes, namely one BCHE gene on chromosome 3q26.1 and multiple genes that encode Polyproline-rich peptides. The function of the Polyproline-rich peptides is to assemble BChE into tetramers. CHO cells transfected with human BChE cDNA express BChE monomers and dimers, but only low quantities of tetramers. Our goal was to identify the Polyproline-rich peptides in CHO-cell derived human BChE tetramers. CHO cell-produced human BChE tetramers were purified from serum-free culture medium. Peptides embedded in the tetramerization domain were released from BChE tetramers by boiling and identified by liquid chromatography-tandem mass spectrometry. A total of 270 proline-rich peptides were sequenced, ranging in size from 6-41 residues. The peptides originated from 60 different proteins that reside in multiple cell compartments including the nucleus, cytoplasm, and endoplasmic reticulum. No single protein was the source of the Polyproline-rich peptides in CHO cell-expressed human BChE tetramers. In contrast, 70% of the tetramer-organizing peptides in plasma-derived BChE tetramers originate from lamellipodin. No protein source was identified for Polyproline peptides containing up to 41 consecutive proline residues. In conclusion, the use of Polyproline-rich peptides as a tetramerization motif is documented only for the cholinesterases, but is expected to serve other tetrameric proteins as well. The CHO cell data suggest that the BChE tetramer-organizing peptide can arise from a variety of proteins.
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Origin of Polyproline-rich peptides in human butyrylcholinesterase tetramers.
Chemico-biological interactions, 2016Co-Authors: Hong Peng, Lawrence M Schopfer, Oksana LockridgeAbstract:The human butyrylcholinesterase (HuBChE) tetramer is composed of 4 identical subunits and a noncovalently bound Polyproline-rich peptide. In a previous report we identified lamellipodin as the source of the Polyproline-rich peptides in HuBChE tetramers purified from plasma. Our current goal was to identify proteins in addition to lamellipodin that donate Polyproline-rich peptides to plasma HuBChE tetramers. Peptides were released from 1 mg of pure plasma-derived HuBChE tetramers by boiling. Mass spectrometry identified 74 Polyproline-rich peptides. MALDI-TOF mass spectra and spectral counting of the LC-MS/MS data supported the conclusion that lamellipodin accounted for 70% of the Polyproline-rich peptides. Additional precursor proteins were matched through BLASTp searches, suggesting but not proving, that 20 proteins including UDP-N-acetyl glucosamine transferase ALG13 homolog, leiomodin 2, and zinc finger homeobox protein 2 are sources of Polyproline-rich peptides found in HuBChE tetramers. Eighteen Polyproline-rich peptides had no match in the human protein database. In conclusion, HuBChE assembles into tetramers through interaction of its C-terminal domain with Polyproline peptides derived from a variety of proteins.
David E. Clemmer - One of the best experts on this subject based on the ideXlab platform.
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solvent mediation of peptide conformations Polyproline structures in water methanol ethanol and 1 propanol as determined by ion mobility spectrometry mass spectrometry
Journal of the American Society for Mass Spectrometry, 2019Co-Authors: Tarick J Elbaba, David H. Russell, Daniel R Fuller, David A Hales, David E. ClemmerAbstract:Ion mobility spectrometry and circular dichroism spectroscopy are used to examine the populations of the small model peptide, Polyproline-13 in water, methanol, ethanol, and 1-propanol over a range of solution temperatures (from 288 to 318 K). At low temperatures, the less-polar solvents (1-propanol and ethanol) favor the all-cis Polyproline I helix (PPI); as the temperature is increased, the trans-configured Polyproline II helix (PPII) is formed. In polar solvents (methanol and water), PPII is favored at all temperatures. From the experimental data, we determine the relative stabilities of the eight structures in methanol, ethanol, and 1-propanol, as well as four in water, all with respect to PPII. Although these conformers show relatively small differences in free energies, substantial variability is observed in the enthalpies and entropies across the structures and solvents. This requires that enthalpies and entropies be highly correlated: in 1-propanol, cis-configured PPI conformations are energetically favorable but entropically disfavored. In more polar solvents, PPI is enthalpically less favorable and entropy favors trans-configured forms. While either ΔH0 or ΔS0 can favor different structures, no conformation in any solvent is simultaneously energetically and entropically stabilized. These data present a rare opportunity to examine the origin of conformational stability.
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Characterizing Intermediates Along the Transition from Polyproline I to Polyproline II Using Ion Mobility Spectrometry-Mass Spectrometry
Journal of the American Chemical Society, 2014Co-Authors: Liuqing Shi, Alison E. Holliday, Huilin Shi, Feifei Zhu, Michael A. Ewing, David H. Russell, David E. ClemmerAbstract:Polyproline exists predominately as the all-cis Polyproline I (PPI) helix in aliphatic alcohols, whereas the all-trans Polyproline II (PPII) helix is favored in aqueous solutions. Previous ion mobility spectrometry-mass spectrometry (IMS-MS) work demonstrates that the gas-phase conformations of Polyproline ions can be related to the corresponding PPI and PPII helices in solution [J. Phys. Chem. B 2004, 108, 4885]. Here, we use IMS-MS to examine the detailed intermediate steps associated with the process of Polyproline-13 (Pro13) conversion from the PPI helix to the PPII helix upon solvent exchange. Collision cross section distributions of Pro13 [M + 2H](2+) ions obtained at different transition times indicate the presence of two major conformers, identified as the PPI and PPII helices, and six conformers that appear as subpopulations of Polyproline. Further analysis shows a transition mechanism with sequential cis-trans isomerizations followed by a parallel process to establish PPII and two smaller subpopulations at equilibrium. Temperature-dependent studies are used to obtain Arrhenius activation parameters for each step of the mechanism, and molecular dynamics simulations provide insight about the structures of the intermediates. It appears that prolines sequentially flip from cis to trans starting from the N-terminus. However, after the first few transitions, possible steps take place at the center of the peptide chain; subsequently, several pathways appear to be accessible at the same time. Our results reflect the existence of stable subpopulations in Polyprolines and provide new insight into the structural changes during the transition process of Polyproline peptides converting from PPI to PPII in aqueous solution.
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Anhydrous Polyproline Helices and Globules
The Journal of Physical Chemistry B, 2004Co-Authors: Anne E. Counterman, David E. ClemmerAbstract:Ion mobility/time-of-flight methods and molecular modeling calculations have been used to examine the conformations of a range of polymer lengths and charge states of Polyproline peptides, [Pron + zH]z+ (n = 3−56; z = 1−6). Ions formed from 1-propanol solutions {[Pron + H]+ (n = 5−11) and [Pron + 2H]2+ (n = 10−22)} favor extended forms of the classical Polyproline I helix. In these conformers, all proline residues are in the cis configuration, and protonation at the N-terminus allows hydrogen bonds to be formed with backbone carbonyl groups of the second and third proline residues in each polymer. Protonation of this all-cis form at the N-terminus also stabilizes the helix macrodipole. Singly charged ions formed from aqueous solutions favor globular and hairpin-like conformers that contain both cis- and trans-proline residues. Higher charge state ions (z = 3−6) formed from aqueous solutions favor relatively extended conformations, although these are not as extended as the Polyproline II structural limit. ...
Jia-cherng Horng - One of the best experts on this subject based on the ideXlab platform.
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Impacts of the Terminal Charged Residues on Polyproline Conformation
The journal of physical chemistry. B, 2018Co-Authors: Kuei-yen Huang, Jia-cherng HorngAbstract:Cis–trans isomerization of proline is involved in various biological processes, such as protein folding, cell signaling, and ion-channel gating. Polyproline is a useful system for better understanding proline isomerization because it exists predominantly as two forms, all-cis Polyproline I (PPI) and all-trans Polyproline II (PPII) helices. The stability of PPI and PPII can be modulated by various effects, including aromatic–proline interactions, terminal charges, and stereoelectronic effects. Here, we used a series of oligoproline peptides in which positively charged or negatively charged amino acids were incorporated into the termini to investigate their effects on Polyproline conformation. Circular dichroism measurements show that a cationic residue at the C-terminus or an anionic residue at the N-terminus increases the stability of a PPII helix; in particular, the C-terminal cationic residues impose an enormous impact on PPII stability. The electrostatic attractions between a cationic sidechain and the...
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Strategy and Effects of Polyproline Peptide Stapling by Copper(I)-Catalyzed Alkyne-Azide Cycloaddition Reaction.
Chembiochem : a European journal of chemical biology, 2018Co-Authors: Wen‐hsiu Tseng, Jia-cherng Horng, Sheng-kai WangAbstract:Polyproline is a unique type of peptide that has a stable, robust, and well-defined helical structure in an aqueous environment. These features have allowed Polyproline to be used as a nanosized scaffold for applications in chemical biology and related fields. To understand its structural properties and to expand the applications, this secondary structure was tested systematically by stapling the peptide at different locations with staples of various lengths. Using the efficient copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC), we successfully prepared stapled Polyproline and investigated the impact of this peptide macrocyclization through circular dichroism analysis. Whereas the stapling seems to have no significant effect on Polyproline helix II (PPII) conformation in water, the location and the length of the staple affect the transformation of conformation in n-propanol. These results provide valuable information for future research using peptide stapling to manipulate Polyproline conformation for various applications.
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Design of Polyproline-Based Catalysts for Ester Hydrolysis.
ACS omega, 2017Co-Authors: Pei-yu Hung, Kuei-yen Huang, Yu-han Chen, Jia-cherng HorngAbstract:A number of simple oligopeptides have been recently developed as minimalistic catalysts for mimicking the activity and selectivity of natural proteases. Although the arrangement of amino acid residues in natural enzymes provides a strategy for designing artificial enzymes, creating catalysts with efficient binding and catalytic activity is still challenging. In this study, we used the Polyproline scaffold and designed a series of 13-residue peptides with a catalytic dyad or triad incorporated to serve as artificial enzymes. Their catalytic efficiency on ester hydrolysis was evaluated by ultraviolet–visible spectroscopy using the p-nitrophenyl acetate assay, and their secondary structures were also characterized by circular dichroism spectroscopy. The results indicate that a well-formed Polyproline II structure may result in a much higher catalytic efficiency. This is the first report to show that a functional dyad or triad engineered into a Polyproline helix framework can enhance the catalytic activity on...
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Effects of the Terminal Aromatic Residues on Polyproline Conformation: Thermodynamic and Kinetic Studies.
The journal of physical chemistry. B, 2015Co-Authors: Yu-ju Lin, Li-kang Chu, Jia-cherng HorngAbstract:In a peptide or protein, the sequence of aromatic residue-proline or proline-aromatic residue shows a high propensity in forming cis prolyl bonds due to aromatic-proline interactions. In this work, we designed and prepared the Polyproline peptides with aromatic amino acids (F, Y, W) incorporated into their N-terminal or C-terminal end to investigate the effects of a terminal aromatic residue on Polyproline conformation and the transition kinetics of Polyproline I (PPI) to Polyproline II (PPII) helices. Circular dichroism measurements reveal that the N-terminal aromatic-proline interaction imposes a more pronounced consequence on the forming propensity of PPI conformation than does the C-terminal aromatic-proline interaction in n-propanol. The propensity of forming PPI is correlated with the strength of aromatic-proline interactions in the order of Tyr-Pro > Trp-Pro > Phe-Pro. In aqueous solution, kinetic studies indicate that aromatic-substitution effects are nondirectional and indistinct on the PPI → PPII conversion rates, suggesting that aromatic-proline interactions may not be an important factor in this process. In addition, the temperature-dependent kinetics shows that the hydrophobicity of aromatic side chain may play a critical role affecting the activation enthalpy and entropy of the conversion of PPI to PPII, providing new insights into the folding of Polyproline helices.
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The impact of 4-thiaproline on Polyproline conformation.
The journal of physical chemistry. B, 2014Co-Authors: Yu-ju Lin, Chiao-hsin Chang, Jia-cherng HorngAbstract:Proline is unique among the genetically coded amino acids; because of the presence of a saturated pyrrolidine ring, it favors a cis peptide bond more strongly than other amino acids. The prolyl peptide bond conformational preference can be modulated by alterations to the atoms or substitution groups on the ring. In the study of a simple Ac-Xaa-OMe system, (2R)-4-thiaproline (Thp) was shown to favor an endo ring pucker and a cis prolyl peptide bond. Herein, to investigate the effects of Thp on a more complex system, that is, the Polyproline structure, we prepared a series of Polyproline peptides with one or multiple proline residues substituted with Thp and used circular dichroism (CD) spectroscopy to characterize their structures. In contrast to the results obtained using the Ac-Xaa-OMe system, here we found that Thp not only destabilizes all-trans Polyproline II conformation, but also disfavors all-cis Polyproline I structure. On the basis of the hybrid density functional theory analysis, we demonstrate that this phenomenon could be due to the small transition barrier between an exo and an endo pucker for the thiazolidine ring of Thp in a PPI helix and a weak backbone n → π* interaction for Thp in PPII conformation. The combination of experimental and computational data allows us to gain new insights into the impact of 4-thiaproline on Polyproline conformation.
Jeffrey S. Chamberlain - One of the best experts on this subject based on the ideXlab platform.
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The Polyproline site in hinge 2 influences the functional capacity of truncated dystrophins.
PLoS genetics, 2010Co-Authors: Glen B. Banks, Luke M. Judge, James M. Allen, Jeffrey S. ChamberlainAbstract:Mutations in dystrophin can lead to Duchenne muscular dystrophy or the more mild form of the disease, Becker muscular dystrophy. The hinge 3 region in the rod domain of dystrophin is particularly prone to deletion mutations. In-frame deletions of hinge 3 are predicted to lead to BMD, however the severity of disease can vary considerably. Here we performed extensive structure-function analyses of truncated dystrophins with modified hinges and spectrin-like repeats in mdx mice. We found that the Polyproline site in hinge 2 profoundly influences the functional capacity of a microdystrophin(DeltaR4-R23/DeltaCT) with a large deletion in the hinge 3 region. Inclusion of Polyproline in microdystrophin(DeltaR4-R23/DeltaCT) led to small myofibers (12% smaller than wild-type), Achilles myotendinous disruption, ringed fibers, and aberrant neuromuscular junctions in the mdx gastrocnemius muscles. Replacing hinge 2 of microdystrophin(DeltaR4-R23/DeltaCT) with hinge 3 significantly improved the functional capacity to prevent muscle degeneration, increase muscle fiber area, and maintain the junctions. We conclude that the rigid alpha-helical structure of the Polyproline site significantly impairs the functional capacity of truncated dystrophins to maintain appropriate connections between the cytoskeleton and extracellular matrix.
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the Polyproline site in hinge 2 influences the functional capacity of truncated dystrophins
PLOS Genetics, 2010Co-Authors: Glen B. Banks, Luke M. Judge, James M. Allen, Jeffrey S. ChamberlainAbstract:Mutations in dystrophin can lead to Duchenne muscular dystrophy or the more mild form of the disease, Becker muscular dystrophy. The hinge 3 region in the rod domain of dystrophin is particularly prone to deletion mutations. In-frame deletions of hinge 3 are predicted to lead to BMD, however the severity of disease can vary considerably. Here we performed extensive structure-function analyses of truncated dystrophins with modified hinges and spectrin-like repeats in mdx mice. We found that the Polyproline site in hinge 2 profoundly influences the functional capacity of a microdystrophinΔR4-R23/ΔCT with a large deletion in the hinge 3 region. Inclusion of Polyproline in microdystrophinΔR4-R23/ΔCT led to small myofibers (12% smaller than wild-type), Achilles myotendinous disruption, ringed fibers, and aberrant neuromuscular junctions in the mdx gastrocnemius muscles. Replacing hinge 2 of microdystrophinΔR4-R23/ΔCT with hinge 3 significantly improved the functional capacity to prevent muscle degeneration, increase muscle fiber area, and maintain the junctions. We conclude that the rigid α-helical structure of the Polyproline site significantly impairs the functional capacity of truncated dystrophins to maintain appropriate connections between the cytoskeleton and extracellular matrix.
