Smart Poly(imidazoyl-l-lysine): Synthesis and Reversible Helix-to-Coil Transition at Neutral pH.
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ABSTRACT:
SUBMITTER: Piedra-Arroni E
PROVIDER: S-EPMC6432093 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Glycinamide ribonucleotide transformylase (GART) undergoes a pH-dependent coil-helix transition with pK(a) approximately 7. An alpha-helix is formed at high pH spanning 8 residues of a 21-residue-long loop, comprising the segment Thr120-His121-Arg122-Gln123-Ala124-Leu125-Glu126-Asn127. To understand the electrostatic nature of this loop-helix, called the activation loop-helix, which leads to the formation and stability of the alpha-helix, pK(a) values of all ionizable residues of GART have been calculated, using Poisson-Boltzmann electrostatic calculations and crystallographic data. Crystallographic structures of high and low pH E70A GART have been used in our analysis. Low pK(a) values of 5.3, 5.3, 3.9, 1.7, and 4.7 have been calculated for five functionally important histidines, His108, His119, His121, His132, and His137, respectively, using the high pH E70A GART structure. Ten theoretical single and double mutants of the high pH E70A structure have been constructed to identify pairwise interactions of ionizable residues, which have aided in elucidating the multiplicity of electrostatic interactions of the activation loop-helix, and the impact of the activation helix on the catalytic site. Based on our pK(a) calculations and structural data, we propose that: (1) His121 forms a molecular switch for the coil-helix transition of the activation helix, depending on its protonation state; (2) a strong electrostatic interaction between His132 and His121 is observed, which can be of stabilizing or destabilizing nature for the activation helix, depending on the relative orientation and protonation states of the rings of His121 and His132; (3) electrostatic interactions involving His119 and Arg122 play a role in the stability of the activation helix; and (4) the activation helix contains the helix-promoting sequence Arg122-Gln123-Ala124-Leu125-Glu126, but its alignment relative to the N and C termini of the helix is not optimal, and is possibly of a destabilizing nature. Finally, we provide electrostatic evidence that the formation and closure of the activation helix create a hydrophobic environment for catalytic-site residue His108, to facilitate catalysis.
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Project description:Self-regulated "smart" insulin administration system that mimic pancreatic endocrine function would be highly desirable for diabetes management. Here, a glucose-responsive continuous insulin delivery system is developed, where novel polyhedral oligosilsesquioxane (POSS) modified with 3-aminophenylboronic acid (APBA) were used to encapsulate insulin (insulin entrapment efficiency: 73.2%) to prepare a fast response, high stability, good distribution, and excellent biocompatible system. Due to the strong hydrophobicity of POSS, the POSS moiety is located at the core in aqueous solution and combines with the boronic group of APBA and the diol generated in PEG-insulin to form a nanomicelle structure, that is, nanoparticles naturally. Micelles self-assembled from these molecules possess glucose-responsiveness at varying glucose concentrations. The interaction of the PBA and diol containing insulin via boronate ester bond and its interchange with glucose was investigated by FT-IR, 1H NMR and XPS. Furthermore, the successful glucose-triggered release of insulin from the POSS-APBA micelles was investigated at neutral pH. A linear graph was plotted with the measured released insulin vs glucose concentrations, with a linear correlation coefficient (R2) value close to 1. Circular dichroism (CD) spectroscopy analysis was performed to measure insulin activity by comparing secondary structures of insulin, PEG-Insulin, and POSS-APBA@insulin. When confirming intracellular apoptosis signaling, cleaved caspase 3 and caspase 9 were not increased by 640 μg/ml POSS-APBA and POSS-APBA@insulin in HeLa, HDF and HUVE cells. Application in the biomedical field for controlled delivery of insulin appear to be promising.
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Project description:Poly(A)-specific ribonuclease (PARN) is a 3'-exoribonuclease that plays an important role in regulating the stability and maturation of RNAs. Recently, PARN has been found to regulate the maturation of the human telomerase RNA component (hTR), a noncoding RNA required for telomere elongation. Specifically, PARN cleaves the 3'-end of immature, polyadenylated hTR to form the mature, nonpolyadenylated template. Despite PARN's critical role in mediating telomere maintenance, little is known about how PARN's function is regulated by post-translational modifications. In this study, using shRNA- and CRISPR/Cas9-mediated gene silencing and knockout approaches, along with 3'-exoribonuclease activity assays and additional biochemical methods, we examined whether PARN is post-translationally modified by acetylation and what effect acetylation has on PARN's activity. We found PARN is primarily acetylated by the acetyltransferase p300 at Lys-566 and deacetylated by sirtuin1 (SIRT1). We also revealed how acetylation of PARN can decrease its enzymatic activity both in vitro, using a synthetic RNA probe, and in vivo, by quantifying endogenous levels of adenylated hTR. Furthermore, we also found that SIRT1 can regulate levels of adenylated hTR through PARN. The findings of our study uncover a mechanism by which PARN acetylation and deacetylation regulate its enzymatic activity as well as levels of mature hTR. Thus, PARN's acetylation status may play a role in regulating telomere length.
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Project description:The dynamics of peptide ?-helices have been studied extensively for many years, and the kinetic mechanism of the helix-coil dynamics has been discussed controversially. Recent experimental results have suggested that equilibrium helix-coil dynamics are governed by movement of the helix/coil boundary along the peptide chain, which leads to slower unfolding kinetics in the helix center compared with the helix ends and position-independent helix formation kinetics. We tested this diffusion of boundary model in helical peptides of different lengths by triplet-triplet energy transfer measurements and compared the data with simulations based on a kinetic linear Ising model. The results show that boundary diffusion in helical peptides can be described by a classical, Einstein-type, 1D diffusion process with a diffusion coefficient of 2.7?10(7) (amino acids)(2)/s or 6.1?10(-9) cm(2)/s. In helices with a length longer than about 40 aa, helix unfolding by coil nucleation in a helical region occurs frequently in addition to boundary diffusion. Boundary diffusion is slowed down by helix-stabilizing capping motifs at the helix ends in agreement with predictions from the kinetic linear Ising model. We further tested local and nonlocal effects of amino acid replacements on helix-coil dynamics. Single amino acid replacements locally affect folding and unfolding dynamics with a ?f-value of 0.35, which shows that interactions leading to different helix propensities for different amino acids are already partially present in the transition state for helix formation. Nonlocal effects of amino acid replacements only influence helix unfolding (?f = 0) in agreement with a diffusing boundary mechanism.
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Project description:Obtaining the correct balance of secondary structure propensities is a central priority in protein force-field development. Given that current force fields differ significantly in their alpha-helical propensities, a correction to match experimental results would be highly desirable. We have determined simple backbone energy corrections for two force fields to reproduce the fraction of helix measured in short peptides at 300 K. As validation, we show that the optimized force fields produce results in excellent agreement with nuclear magnetic resonance experiments for folded proteins and short peptides not used in the optimization. However, despite the agreement at ambient conditions, the dependence of the helix content on temperature is too weak, a problem shared with other force fields. A fit of the Lifson-Roig helix-coil theory shows that both the enthalpy and entropy of helix formation are too small: the helix extension parameter w agrees well with experiment, but its entropic and enthalpic components are both only about half the respective experimental estimates. Our structural and thermodynamic analyses point toward the physical origins of these shortcomings in current force fields, and suggest ways to address them in future force-field development.
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Project description:Bioorthogonal reactions that are fast and reversible under physiological conditions are in high demand for biological applications. Herein, it is shown that an ortho boronic acid substituent makes aryl ketones rapidly conjugate with ?-nucleophiles at neutral pH. Specifically, 2-acetylphenylboronic acid and derivatives were found to conjugate with phenylhydrazine with rate constants of 10(2) to 10(3) M(-1) s(-1) , comparable to the fastest bioorthogonal conjugations known to date. (11) B NMR analysis revealed the varied extent of iminoboronate formation of the conjugates, in which the imine nitrogen forms a dative bond with boron. The iminoboronate formation activates the imines for hydrolysis and exchange, rendering these oxime/hydrazone conjugations reversible and dynamic under physiological conditions. The fast and dynamic nature of the iminoboronate chemistry should find wide applications in biology.
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Project description:For a model deca-alanine peptide the cavity (ideal hydrophobic) contribution to hydration favors the helix state over extended states and the paired helix bundle in the assembly of two helices. The energetic contributions of attractive protein-solvent interactions are separated into quasi-chemical components consisting of a short-range part arising from interactions with solvent in the first hydration shell and the remaining long-range part that is well described by a Gaussian. In the helix-coil transition, short-range attractive protein-solvent interactions outweigh hydrophobic hydration and favor the extended coil states. Analysis of enthalpic effects shows that it is the favorable hydration of the peptide backbone that favors the unfolded state. Protein intramolecular interactions favor the helix state and are decisive in favoring folding. In the pairing of two helices, the cavity contribution outweighs the short-range attractive protein-water interactions. However, long-range, protein-solvent attractive interactions can either enhance or reverse this trend depending on the mutual orientation of the helices. In helix-helix assembly, change in enthalpy arising from change in attractive protein-solvent interactions favors disassembly. In helix pairing as well, favorable protein intramolecular interactions are found to be as important as hydration effects. Overall, hydrophilic protein-solvent interactions and protein intramolecular interactions are found to play a significant role in the thermodynamics of folding and assembly in the system studied.
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Project description:It is known that different amino acid residues have effects on the thermodynamic stability of an alpha-helix. The underlying mechanism for the thermodynamic helical propensity is not well understood. The major accepted hypothesis is the difference in the side-chain configurational entropy loss upon helix formation. However, the changes in the side-chain configurational entropy explain only part of the thermodynamic helical propensity, thus implying that there must be a difference in the enthalpy of helix-coil transition for different residues. This work provides an experimental test to this hypothesis. Direct calorimetric measurements of folding of a model host peptide in which the helix formation is induced by metal binding is applied to a wide range of residue types, both naturally occurring and nonnatural, at the guest site. Based on the calorimetric results for 12 peptides, it was found that indeed there is a difference in the enthalpy of helix-coil transition for different amino acid residues, and simple empirical rules that define these differences are presented. The obtained difference in the enthalpies of helix-coil transition complement the differences in configurational entropies and provide the complete thermodynamic characterization of the helix-forming tendencies.
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Project description:Current clinical treatment of Helicobacter pylori infection, the main etiological factor in the development of gastritis, gastric ulcers, and gastric carcinoma, requires a combination of at least two antibiotics and one proton pump inhibitor. However, such triple therapy suffers from progressively decreased therapeutic efficacy due to the drug resistance and undesired killing of the commensal bacteria due to poor selectivity. Here, we report the development of antimicrobial polypeptide-based monotherapy, which can specifically kill H. pylori under acidic pH in the stomach while inducing minimal toxicity to commensal bacteria under physiological pH. Specifically, we designed a class of pH-sensitive, helix-coil conformation transitionable antimicrobial polypeptides (HCT-AMPs) (PGA)m-r-(PHLG-MHH)n, bearing randomly distributed negatively charged glutamic acid and positively charged poly(?-6-N-(methyldihexylammonium)hexyl-l-glutamate) (PHLG-MHH) residues. The HCT-AMPs showed unappreciable toxicity at physiological pH when they adopted random coiled conformation. Under acidic condition in the stomach, they transformed to the helical structure and exhibited potent antibacterial activity against H. pylori, including clinically isolated drug-resistant strains. After oral gavage, the HCT-AMPs afforded comparable H. pylori killing efficacy to the triple-therapy approach while inducing minimal toxicity against normal tissues and commensal bacteria, in comparison with the remarkable killing of commensal bacteria by 65% and 86% in the ileal contents and feces, respectively, following triple therapy. This strategy renders an effective approach to specifically target and kill H. pylori in the stomach while not harming the commensal bacteria/normal tissues.
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Project description:Coiled-coil proteins contain a characteristic seven-residue sequence repeat whose positions are designated a to g. The interacting surface between alpha-helices in a classical coiled coil is formed by interspersing nonpolar side chains at the a and d positions with hydrophilic residues at the flanking e and g positions. To explore how the chemical nature of these core amino acids dictates the overall coiled-coil architecture, we replaced all eight e and g residues in the GCN4 leucine zipper with nonpolar alanine side chains. Surprisingly, the alanine-containing mutant forms a stable alpha-helical heptamer in aqueous solution. The 1.25-A resolution crystal structure of the heptamer reveals a parallel seven-stranded coiled coil enclosing a large tubular channel with an unusual heptad register shift between adjacent staggered helices. The overall geometry comprises two interleaved hydrophobic helical screws of interacting cross-sectional a and d layers that have not been seen before. Moreover, asparagines at the a positions play an essential role in heptamer formation by participating in a set of buried interhelix hydrogen bonds. These results demonstrate that heptad repeats containing four hydrophobic positions can direct assembly of complex, higher-order coiled-coil structures with rich diversity for close packing of alpha-helices.
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