RESUMEN
Octafluorocyclopentene (OFCP) engages linear, unprotected peptides in polysubstitution cascades that generate complex fluorinated polycycles. The reactions occur in a single flask at 0-25 °C and require no catalysts or heavy metals. OFCP can directly polycyclize linear sequences using native functionality, or fluorospiroheterocyclic intermediates can be intercepted with exogenous nucleophiles. The latter tactic generates molecular hybrids composed of peptides, sugars, lipids, and heterocyclic components. The platform can create stereoisomers of both single- and double-looped macrocycles. Calculations indicate that the latter can mimic diverse protein surface loops. Subsets of the molecules have low energy conformers that shield the polar surface area through intramolecular hydrogen bonding. A significant fraction of OFCP-derived macrocycles tested show moderate to high passive permeability in parallel artificial membrane permeability assays.
Asunto(s)
Membranas Artificiales , Péptidos , Péptidos/químicaRESUMEN
De novo enzyme design has sought to introduce active sites and substrate-binding pockets that are predicted to catalyse a reaction of interest into geometrically compatible native scaffolds1,2, but has been limited by a lack of suitable protein structures and the complexity of native protein sequence-structure relationships. Here we describe a deep-learning-based 'family-wide hallucination' approach that generates large numbers of idealized protein structures containing diverse pocket shapes and designed sequences that encode them. We use these scaffolds to design artificial luciferases that selectively catalyse the oxidative chemiluminescence of the synthetic luciferin substrates diphenylterazine3 and 2-deoxycoelenterazine. The designed active sites position an arginine guanidinium group adjacent to an anion that develops during the reaction in a binding pocket with high shape complementarity. For both luciferin substrates, we obtain designed luciferases with high selectivity; the most active of these is a small (13.9 kDa) and thermostable (with a melting temperature higher than 95 °C) enzyme that has a catalytic efficiency on diphenylterazine (kcat/Km = 106 M-1 s-1) comparable to that of native luciferases, but a much higher substrate specificity. The creation of highly active and specific biocatalysts from scratch with broad applications in biomedicine is a key milestone for computational enzyme design, and our approach should enable generation of a wide range of luciferases and other enzymes.
Asunto(s)
Aprendizaje Profundo , Luciferasas , Biocatálisis , Dominio Catalítico , Estabilidad de Enzimas , Calor , Luciferasas/química , Luciferasas/metabolismo , Luciferinas/metabolismo , Luminiscencia , Oxidación-Reducción , Especificidad por SustratoRESUMEN
Flavin-dependent halogenases (FDHs) catalyze selective halogenation of electron-rich aromatic compounds without the need for harsh oxidants required by conventional oxidative halogenation reactions. Predictive models for halogenase site selectivity could greatly improve their utility for chemical synthesis. Toward this end, we analyzed the structures and selectivity of three halogenase variants evolved to halogenate tryptamine with orthogonal selectivity. Crystal structures and reversion mutations revealed key residues involved in altering halogenase selectivity. Density functional theory calculations and molecular dynamics simulations are both consistent with hypohalous acid as the active halogenating species in FDH catalysis. This model was used to accurately predict the site selectivity of halogenase variants toward different synthetic substrates, providing a valuable tool for implementing halogenases in biocatalysis efforts.
RESUMEN
The assembly of proteins into fibrillar amyloid structures was once considered to be pathologic and essentially irreversible. Recent studies reveal amyloid-like structures that form reversibly, derived from protein low-complexity domains which function in cellular metabolism. Here, by comparing atomic-level structures of reversible and irreversible amyloid fibrils, we find that the ß-sheets of reversible fibrils are enriched in flattened (as opposed to pleated) ß-sheets formed by stacking of extended ß-strands. Quantum mechanical calculations show that glycine residues favor extended ß-strands which may be stabilized by intraresidue interactions between the amide proton and the carbonyl oxygen, known as C5 hydrogen-bonds. Larger residue side chains favor shorter strands and pleated sheets. These findings highlight a structural element that may regulate reversible amyloid assembly.
Asunto(s)
Amiloide , Proteínas Amiloidogénicas , Amiloide/química , Péptidos beta-Amiloides/química , Enlace de Hidrógeno , Conformación Proteica en Lámina beta , Dominios ProteicosRESUMEN
Via the design of a new, soluble poly(S-alkyl-l-cysteine) precursor, a route was developed for the successful preparation of long-chain poly(dehydroalanine), ADH, as well as the incorporation of dehydroalanine residues and ADH segments into copolypeptides. Based on experimental and computational data, ADH was found to adopt a previously unobserved "hybrid coil" structure, which combines the elements of 25-helical and 310-helical conformations. Analysis of the spectroscopic properties of ADH revealed that it possesses a strong inherent blue fluorescence, which may be amenable for use in imaging applications. ADH also contains reactive electrophilic groups that allowed its efficient modification to functionalized polypeptides after reactions under mild conditions with thiol and amine nucleophiles. The combined structural, spectroscopic, and reactivity properties of ADH make it a unique reactive and fluorescent polypeptide component for utilization in self-assembled biomaterials.
Asunto(s)
Alanina , Péptidos , Alanina/análogos & derivados , Alanina/química , Cisteína/química , Péptidos/química , Compuestos de SulfhidriloRESUMEN
We report the development of new side-chain amino acid-functionalized α-helical homopolypeptides that reversibly form coacervate phases in aqueous media. The designed multifunctional nature of the side-chains was found to provide a means to actively control coacervation via mild, biomimetic redox chemistry as well as allow response to physiologically relevant environmental changes in pH, temperature, and counterions. These homopolypeptides were found to possess properties that mimic many of those observed in natural coacervate forming intrinsically disordered proteins. Despite ordered α-helical conformations that are thought to disfavor coacervation, molecular dynamics simulations of a polypeptide model revealed a high degree of side-chain conformational disorder and hydration around the ordered backbone, which may explain the ability of these polypeptides to form coacervates. Overall, the modular design, uniform nature, and ordered chain conformations of these polypeptides were found to provide a well-defined platform for deconvolution of molecular elements that influence biopolymer coacervation and tuning of coacervate properties for downstream applications.
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Aminoácidos/química , Péptidos/química , Suspensiones/química , Interacciones Hidrofóbicas e Hidrofílicas , Simulación de Dinámica Molecular , Péptidos/síntesis química , Transición de Fase , Conformación Proteica en Hélice alfa , Temperatura de TransiciónRESUMEN
Catalytic enantioselection usually depends on differences in steric interactions between prochiral substrates and a chiral catalyst. We have discovered a carbene Si-H insertion in which the enantioselectivity depends primarily on the electronic characteristics of the carbene substrate, and the log(er) values are linearly related to Hammett parameters. A new class of chiral tetraphosphate dirhodium catalysts was developed; it shows excellent activity and enantioselectivity for the insertion of diarylcarbenes into the Si-H bond of silanes. Computational and mechanistic studies show how the electronic differences between the two aryls of the carbene lead to differences in energies of the diastereomeric transition states. This study provides a new strategy for asymmetric catalysis exploiting the electronic properties of the substrates.
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Hidrógeno/química , Metano/análogos & derivados , Silanos/síntesis química , Silicio/química , Electrónica , Metano/química , Estructura Molecular , Silanos/química , EstereoisomerismoRESUMEN
A dedicated enzyme for the formation of the central C ring in the tetracyclic ergoline of clinically important ergot alkaloids has never been found. Herein, we report a dual role catalase (EasC), unexpectedly using O2 as the oxidant, that catalyzes the oxidative cyclization of the central C ring from a 1,3-diene intermediate. Our study showcases how nature evolves the common catalase for enantioselective C-C bond construction of complex polycyclic scaffolds.
Asunto(s)
Catalasa/química , Ergolinas/síntesis química , Proteínas Fúngicas/química , Aspergillus fumigatus/enzimología , Aspergillus nidulans/enzimología , Ciclización , Radicales Libres/química , Modelos Químicos , Oxidación-Reducción , Saccharomyces cerevisiae/enzimologíaRESUMEN
Protein-nucleic acid interactions are central to a variety of biological processes, many of which involve large-scale conformational changes that lead to bending of the nucleic acid helix. Here, we focus on the nonsequence-specific protein TRBP, whose double-stranded RNA-binding domains (dsRBDs) interact with the A-form geometry of double-stranded RNA (dsRNA). Crystal structures of dsRBD-dsRNA interactions suggest that the dsRNA helix must bend in such a way that its major groove expands to conform to the dsRBD's binding surface. We show through isothermal titration calorimetry experiments that dsRBD2 of TRBP binds dsRNA with a temperature-independent observed binding affinity (KD â¼500 nM). Furthermore, a near-zero observed heat capacity change (ΔCp = 70 ± 40 cal·mol(-1)·K(-1)) suggests that large-scale conformational changes do not occur upon binding. This result is bolstered by molecular-dynamics simulations in which dsRBD-dsRNA interactions generate only modest bending of the RNA along its helical axis. Overall, these results suggest that this particular dsRBD-dsRNA interaction produces little to no change in the A-form geometry of dsRNA in solution. These results further support our previous hypothesis, based on extensive gel-shift assays, that TRBP preferentially binds to sites of nearly ideal A-form structure while being excluded from sites of local deformation in the RNA helical structure. The implications of this mechanism for efficient micro-RNA processing will be discussed.