RESUMEN
In this issue of Cell Chemical Biology, Peng and Weerapana1 report the combination of chemoproteomic and proximity-based labeling approaches to identify cysteines in nuclear proteins that are reactive toward electrophilic probe compounds. They apply this novel technology to identify proteins that are localized to the nucleus and chromatin upon probe labeling.
Asunto(s)
Cisteína , Proteínas , Cisteína/metabolismoRESUMEN
Histone post-translational modifications (PTMs) on lysine residues, including methylation, ubiquitylation, and sumoylation, have been studied using semisynthetic histones reconstituted into nucleosomes. These studies have revealed the in vitro effects of histone PTMs on chromatin structure, gene transcription, and biochemical crosstalk. However, the dynamic and transient nature of most enzyme-chromatin interactions poses a challenge toward identifying specific enzyme-substrate interactions. To address this, we report methodology for the synthesis of two ubiquitylated activity-based probe histones, H2BK120ub(G76C) and H2BK120ub(G76Dha), that may be used to trap enzyme active-site cysteines as disulfides or in the form of thioether linkages, respectively. The general synthetic method we report for converting ubiquitylated nucleosomes into activity-based probes may also be applied to other histone sites of ubiquitylation in order to facilitate the identification of enzyme-chromatin interactions.
Asunto(s)
Cromatina , Histonas , Histonas/metabolismo , Cromatina/genética , Nucleosomas/genética , Ubiquitinación , Procesamiento Proteico-PostraduccionalRESUMEN
An efficient total chemical synthesis of site-specifically sumoylated histone H4 was undertaken to generate homogenously modified mononucleosomes. These were tested as substrates in biochemical assays with the histone H2B-specific ubiquitin ligases Rad6 and Bre1, which revealed the strong inhibition of H2B ubiquitylation by SUMO. This novel negative biochemical crosstalk between SUMO and ubiquitin was also confirmed to exist in human cells.
Asunto(s)
Histonas , Proteínas de Saccharomyces cerevisiae , Humanos , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitinación , Ubiquitina/metabolismoRESUMEN
The tumor suppressor and master gene regulator protein p53 has been the subject of intense investigation for several decades due to its mutation in about half of all human cancers. However, mechanistic studies of p53 in cells are complicated by its many dynamic binding partners and heterogeneous post-translational modifications. The design of therapeutics that rescue p53 functions in cells requires a mechanistic understanding of its protein-protein interactions in specific protein complexes and identifying changes in p53 activity by diverse post-translational modifications. This review highlights the important roles that peptide and protein chemistry have played in biophysical and biochemical studies aimed at elucidating p53 regulation by several key binding partners. The design of various peptide inhibitors that rescue p53 function in cells and new opportunities in targeting p53-protein interactions are discussed. In addition, the review highlights the importance of a protein semisynthesis approach to comprehend the role of site-specific PTMs in p53 regulation.
Asunto(s)
Neoplasias , Proteína p53 Supresora de Tumor , Humanos , Mutación , Neoplasias/metabolismo , Péptidos/química , Procesamiento Proteico-Postraduccional , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The post-translational modification of histones by the small ubiquitin-like modifier (SUMO) protein has been associated with gene regulation, centromeric localization, and double-strand break repair in eukaryotes. Although sumoylation of histone H4 was specifically associated with gene repression, this could not be proven due to the challenge of site-specifically sumoylating H4 in cells. Biochemical crosstalk between SUMO and other histone modifications, such as H4 acetylation and H3 methylation, that are associated with active genes also remains unclear. We addressed these challenges in mechanistic studies using an H4 chemically modified at Lys12 by SUMO-3 (H4K12su) and incorporated into mononucleosomes and chromatinized plasmids for functional studies. Mononucleosome-based assays revealed that H4K12su inhibits transcription-activating H4 tail acetylation by the histone acetyltransferase p300, as well as transcription-associated H3K4 methylation by the extended catalytic module of the Set1/COMPASS (complex of proteins associated with Set1) histone methyltransferase complex. Activator- and p300-dependent in vitro transcription assays with chromatinized plasmids revealed that H4K12su inhibits both H4 tail acetylation and RNA polymerase II-mediated transcription. Finally, cell-based assays with a SUMO-H4 fusion that mimics H4 tail sumoylation confirmed the negative crosstalk between histone sumoylation and acetylation/methylation. Thus, our studies establish the key role for histone sumoylation in gene silencing and its negative biochemical crosstalk with active transcription-associated marks in human cells.
Asunto(s)
Histonas/metabolismo , ARN Polimerasa II/genética , Sumoilación , Transcripción Genética , Extractos Celulares , Humanos , ARN Polimerasa II/metabolismoRESUMEN
Tuberculosis is a global health problem caused by infection with the Mycobacterium tuberculosis (Mtb) bacteria. Although antibiotic treatment has dramatically reduced the impact of tuberculosis on the population, the existence and spreading of drug resistant strains urgently demands the development of new drugs that target Mtb in a different manner than currently used antibiotics. The prokaryotic ubiquitin-like protein (Pup) proteasome system is an attractive target for new drug development as it is unique to Mtb and related bacterial genera. Using a Pup-based fluorogenic substrate, we screened for inhibitors of Dop, the Mtb depupylating protease, and identified I-OMe-Tyrphostin AG538 (1) and Tyrphostin AG538 (2). The hits were validated and determined to be fast-reversible, non-ATP competitive inhibitors. We synthesized >25 analogs of 1 and 2 and show that several of the synthesized compounds also inhibit the depupylation actions of Dop on native substrate, FabD-Pup. Importantly, the pupylation activity of PafA, the sole Pup ligase in Mtb, was also inhibited by some of these compounds.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Desarrollo de Medicamentos , Inhibidores Enzimáticos/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Tirfostinos/farmacología , Ubiquitinas/antagonistas & inhibidores , Antibacterianos/síntesis química , Antibacterianos/química , Proteínas Bacterianas/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Mycobacterium tuberculosis/enzimología , Relación Estructura-Actividad , Tirfostinos/síntesis química , Tirfostinos/química , Ubiquitinas/metabolismoRESUMEN
We report the generation of gas-phase riboguanosine radicals that were tagged at ribose with a fixed-charge 6-(trimethylammonium)hexane-1-aminocarbonyl group. The radical generation relied on electron transfer from fluoranthene anion to noncovalent dibenzocrown-ether dication complexes which formed nucleoside cation radicals upon one-electron reduction and crown-ether ligand loss. The cation radicals were characterized by collision-induced dissociation (CID), photodissociation (UVPD), and UV-vis action spectroscopy. Identification of charge-tagged guanosine radicals was challenging because of spontaneous dissociations by loss of a hydrogen atom and guanine that occurred upon storing the ions in the ion trap without further excitation. The loss of H proceeded from an exchangeable position on N-7 in guanine that was established by deuterium labeling and was the lowest energy dissociation of the guanosine radicals according to transition-state energy calculations. Rate constant measurements revealed an inverse isotope effect on the loss of either hydrogen or deuterium with rate constants kH = 0.25-0.26 s-1 and kD = 0.39-0.54 s-1. We used time-dependent density functional theory calculations, including thermal vibronic effects, to predict the absorption spectra of several protomeric radical isomers. The calculated spectra of low-energy N-7-H guanine-radical tautomers closely matched the action spectra. Transition-state-theory calculations of the rate constants for the loss of H-7 and guanine agreed with the experimental rate constants for a narrow range of ion effective temperatures. Our calculations suggest that the observed inverse isotope effect does not arise from the isotope-dependent differences in the transition-state energies. Instead, it may be caused by the dynamics of post-transition-state complexes preceding the product separation.
RESUMEN
Mutations in the E3 ubiquitin ligase RING domains of BRCA1/BARD1 predispose carriers to breast and ovarian cancers. We present the structure of the BRCA1/BARD1 RING heterodimer with the E2 enzyme UbcH5c bound to its cellular target, the nucleosome, along with biochemical data that explain how the complex selectively ubiquitylates lysines 125, 127 and 129 in the flexible C-terminal tail of H2A in a fully human system. The structure reveals that a novel BARD1-histone interface couples to a repositioning of UbcH5c compared to the structurally similar PRC1 E3 ligase Ring1b/Bmi1 that ubiquitylates H2A Lys119 in nucleosomes. This interface is sensitive to both H3 Lys79 methylation status and mutations found in individuals with cancer. Furthermore, NMR reveals an unexpected mode of E3-mediated substrate regulation through modulation of dynamics in the C-terminal tail of H2A. Our findings provide insight into how E3 ligases preferentially target nearby lysine residues in nucleosomes by a steric occlusion and distancing mechanism.
Asunto(s)
Proteína BRCA1/química , Proteína BRCA1/metabolismo , Histonas/metabolismo , Nucleosomas/química , Nucleosomas/metabolismo , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Proteína BRCA1/ultraestructura , Sitios de Unión , Dominio Catalítico , Microscopía por Crioelectrón , Histonas/química , Histonas/ultraestructura , Humanos , Lisina/química , Lisina/metabolismo , Modelos Moleculares , Unión Proteica , Reproducibilidad de los Resultados , Proteínas Supresoras de Tumor/ultraestructura , Enzimas Ubiquitina-Conjugadoras/química , Enzimas Ubiquitina-Conjugadoras/metabolismo , Enzimas Ubiquitina-Conjugadoras/ultraestructura , Ubiquitina-Proteína Ligasas/ultraestructuraRESUMEN
The unmodified R5 peptide from silaffin in the diatom Cylindrotheca fusiformis rapidly precipitates silica particles from neutral aqueous solutions of orthosilicic acid. A range of post-translational modifications found in R5 contribute toward tailoring silica morphologies in a species-specific manner. We investigated the specific effect of R5 lysine side-chain trimethylation, which adds permanent positive charges, on silica particle formation. Our studies revealed that a doubly trimethylated R5K3,4me3 peptide has reduced maximum activity yet, surprisingly, generates larger silica particles. Molecular dynamics simulations of R5K3,4me3 binding by the precursor orthosilicate anion revealed that orthosilicate preferentially associates with unmodified lysine side-chain amines and the peptide N terminus. Thus, larger silica particles arise from reduced orthosilicate association with trimethylated lysine side chains and their redirection to the N terminus of the R5 peptide.
Asunto(s)
Fragmentos de Péptidos/química , Precursores de Proteínas/química , Ácido Silícico/química , Dióxido de Silicio/química , Sitios de Unión , Diatomeas/química , Metilación , Simulación de Dinámica Molecular , Tamaño de la PartículaRESUMEN
The essential human enzyme lysine specific demethylase 1 (LSD1) silences genes by demethylating mono- and dimethylated lysine 4 in histone H3 (H3K4me1/2). Studies of the minimal requirements for LSD1 activity are complicated by the heterogeneity of histone modification states in cells. We overcame this challenge by generating homogeneous mononucleosome substrates containing semisynthetic H3K4me2. Biophysical and biochemical assays with full-length LSD1 revealed its ability to bind and demethylate nucleosomes. Consistent with a requirement for nucleosome binding prior to demethylation, a competing nucleosome-binding peptide from the high-mobility group protein effectively inhibited LSD1 activity. Thus, our studies provide the first glimpse of nucleosome demethylation by LSD1 in the absence of other scaffolding proteins.
Asunto(s)
Histona Demetilasas/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Nucleosomas/metabolismo , Procesamiento Proteico-Postraduccional , Histona Demetilasas/química , Histona Demetilasas/aislamiento & purificación , Histonas/química , Humanos , Metilación , Modelos Moleculares , Nucleosomas/química , Unión ProteicaRESUMEN
The posttranslational modification of cellular proteins by ubiquitin (Ub), called ubiquitylation, is indispensable for the normal growth and development of eukaryotic organisms. In order to conduct studies that elucidate the precise mechanistic roles for Ub, access to site-specifically and homogenously ubiquitylated proteins and peptides is critical. However, the low abundance, heterogeneity, and dynamic nature of protein ubiquitylation are significant limitations toward such studies. Here we provide a facile expressed protein ligation method that does not require specialized apparatus and permits the rapid semisynthesis of ubiquitylated peptides by using the atom-efficient ligation auxiliary 2-aminooxyethanethiol.
Asunto(s)
Péptidos/química , Técnicas de Síntesis en Fase Sólida/métodos , Compuestos de Sulfhidrilo/química , Ubiquitina/química , Ubiquitinación , Cisteína/química , Escherichia coli/genética , Ésteres/química , Expresión Génica , Hidroxilaminas/química , Imidas/química , Inteínas , Espectroscopía de Resonancia Magnética , Péptidos/aislamiento & purificación , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Ubiquitina/biosíntesis , Ubiquitina/metabolismo , Proteínas Ubiquitinadas/biosíntesis , Proteínas Ubiquitinadas/química , Proteínas Ubiquitinadas/aislamiento & purificación , Ubiquitinas/biosíntesis , Ubiquitinas/química , Zinc/metabolismoRESUMEN
The COMPASS (complex of proteins associated with Set1) complex represents the prototype of the SET1/MLL family of methyltransferases that controls gene transcription by H3K4 methylation (H3K4me). Although H2B monoubiquitination (H2Bub) is well known as a prerequisite histone mark for COMPASS activity, how H2Bub activates COMPASS remains unclear. Here, we report the cryoelectron microscopy (cryo-EM) structures of an extended COMPASS catalytic module (CM) bound to the H2Bub and free nucleosome. The COMPASS CM clamps onto the nucleosome disk-face via an extensive interface to capture the flexible H3 N-terminal tail. The interface also sandwiches a critical Set1 arginine-rich motif (ARM) that autoinhibits COMPASS. Unexpectedly, without enhancing COMPASS-nucleosome interaction, H2Bub activates the enzymatic assembly by packing against Swd1 and alleviating the inhibitory effect of the Set1 ARM upon fastening it to the acidic patch. By delineating the spatial configuration of the COMPASS-H2Bub-nucleosome assembly, our studies establish the structural framework for understanding the long-studied H2Bub-H3K4me histone modification crosstalk.
Asunto(s)
Histona Metiltransferasas/ultraestructura , Histonas/ultraestructura , Cromatina/genética , Microscopía por Crioelectrón/métodos , Metilación de ADN/genética , Epigénesis Genética/genética , Epigenómica/métodos , Proteínas Fúngicas/química , Histona Metiltransferasas/química , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/química , Histonas/genética , Kluyveromyces/genética , Kluyveromyces/metabolismo , Metiltransferasas/metabolismo , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/ultraestructura , Nucleosomas/metabolismo , Subunidades de Proteína , Proteínas de Saccharomyces cerevisiae/metabolismo , UbiquitinaciónRESUMEN
The Cu(i)-mediated click reaction of proteins with affinity tags enables their selective isolation from complex mixtures. However, irreversible protein modification limits the interpretation of results from subsequent biophysical and biochemical assays. We report a facile and modular chemical strategy to reversibly modify peptides and proteins with biotin and FLAG affinity tags at a clickable glutamine (CliQ) residue.
Asunto(s)
Química Clic , Glutamina/química , Péptidos/química , Proteínas/química , Marcadores de Afinidad , Biotina/química , Oxidación-ReducciónRESUMEN
The SET1/MLL family of histone methyltransferases is conserved in eukaryotes and regulates transcription by catalyzing histone H3K4 mono-, di-, and tri-methylation. These enzymes form a common five-subunit catalytic core whose assembly is critical for their basal and regulated enzymatic activities through unknown mechanisms. Here, we present the crystal structure of the intact yeast COMPASS histone methyltransferase catalytic module consisting of Swd1, Swd3, Bre2, Sdc1, and Set1. The complex is organized by Swd1, whose conserved C-terminal tail not only nucleates Swd3 and a Bre2-Sdc1 subcomplex, but also joins Set1 to construct a regulatory pocket next to the catalytic site. This inter-subunit pocket is targeted by a previously unrecognized enzyme-modulating motif in Swd3 and features a doorstop-style mechanism dictating substrate selectivity among SET1/MLL family members. By spatially mapping the functional components of COMPASS, our results provide a structural framework for understanding the multifaceted functions and regulation of the H3K4 methyltransferase family.
Asunto(s)
Proteínas Fúngicas/química , N-Metiltransferasa de Histona-Lisina/química , Histonas/química , Kluyveromyces/química , Proteínas de Saccharomyces cerevisiae/química , Secuencia de Aminoácidos , Animales , Dominio Catalítico , Línea Celular , Cristalografía por Rayos X , Proteínas de Unión al ADN/química , Humanos , Insectos , Metilación , Proteínas Nucleares/química , Dominios Proteicos , Saccharomyces cerevisiae/química , Alineación de Secuencia , Especificidad por Sustrato , Factores de Transcripción/químicaRESUMEN
Reversible post-translational modifications of histone proteins in eukaryotic chromatin are closely tied to gene function and cellular development. Specific combinations of histone modifications, or marks, are implicated in distinct DNA-templated processes mediated by a range of chromatin-associated enzymes that install, erase and interpret the histone code. Mechanistic studies of the precise biochemical relationship between sets of marks and their effects on chromatin function are significantly complicated by the dynamic nature and heterogeneity of marks in cellular chromatin. Protein semisynthesis is a chemical technique that enables the piecewise assembly of uniformly and site-specifically modified histones in quantities sufficient for biophysical and biochemical analyses. Recent pioneering efforts in semisynthesis have yielded access to histones site-specifically modified by entire proteins, such as ubiquitin (Ub) and the small ubiquitin-like modifier (SUMO). Herein, we highlight key studies of biochemical crosstalk involving Ub and SUMO in chromatin that were enabled by histone semisynthesis.
Asunto(s)
Cromatina/química , Histonas/química , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/química , Ubiquitina/química , Animales , Código de Histonas , Histonas/síntesis química , Humanos , Modelos Moleculares , Nucleosomas/química , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/síntesis química , Sumoilación , Ubiquitina/síntesis química , UbiquitinaciónRESUMEN
Missense mutations that disrupt the RING domain of the tumor suppressor gene BRCA1 lead to increased risk of breast and ovarian cancer. The BRCA1 RING domain is a ubiquitin ligase, whose structure and function rely critically on forming a heterodimer with BARD1, which also harbors a RING domain. The function of the BARD1 RING domain is unknown. In families severely affected with breast cancer, we identified inherited BARD1 missense mutations Cys53Trp, Cys71Tyr, and Cys83Arg that alter three zinc-binding residues of the BARD1 RING domain. Each of these mutant BARD1 proteins retained the ability to form heterodimeric complexes with BRCA1 to make an active ubiquitin ligase, but the mutant BRCA1/BARD1 complexes were deficient in binding to nucleosomes and in ubiquitylating histone H2A. The BARD1 mutations also caused loss of transcriptional repression of BRCA1-regulated estrogen metabolism genes CYP1A1 and CYP3A4; breast epithelial cells edited to create heterozygous loss of BARD1 showed significantly higher expression of CYP1A1 and CYP3A4 Reintroduction of wild-type BARD1 into these cells restored CYP1A1 and CYP3A4 transcription to normal levels, but introduction of the cancer-predisposing BARD1 RING mutants failed to do so. These results indicate that an intact BARD1 RING domain is critical to BRCA1/BARD1 binding to nucleosomes and hence to ubiquitylation of histone H2A and also critical to transcriptional repression of BRCA1-regulated genes active in estrogen metabolism.
Asunto(s)
Estrógenos/metabolismo , Histonas/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Neoplasias de la Mama/genética , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Estrógenos/genética , Femenino , Regulación de la Expresión Génica , Histonas/genética , Humanos , Masculino , Mutación Missense , Nucleosomas/metabolismo , Dominios Proteicos , Proteínas Supresoras de Tumor/química , Ubiquitina-Proteína Ligasas/química , UbiquitinaciónRESUMEN
Designing new antimicrobial peptides (AMPs) focuses heavily on the activity of the peptide and less on the elements that stabilize the secondary structure of these peptides. Studies have shown that improving the structure of naturally occurring AMPs can affect activity and so here we explore the relationship between structure and activity of two non-naturally occurring AMPs. We have used a backbone-cyclized peptide as a template and designed an uncyclized analogue of this peptide that has antimicrobial activity. We focused on beta-hairpin-like structuring features. Improvements to the structure of this peptide reduced the activity of the peptide against gram-negative, Escherichia coli but improved the activity against gram-positive, Corynebacterium glutamicum. Distinctions in structuring effects on gram-negative versus gram-positive activity were also seen in a second peptide system. Structural improvements resulted in a peptide that was more active than the native against gram-positive bacterium but less active against gram-negative bacterium. Our results show that there is not always a correlation between improved hairpin-structuring and activity. Other factors such as the type of bacteria being targeted as well as net positive charge can play a role in the potency of AMPs. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.
Asunto(s)
Antibacterianos/química , Péptidos Catiónicos Antimicrobianos/química , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Antibacterianos/síntesis química , Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/síntesis química , Péptidos Catiónicos Antimicrobianos/farmacología , Corynebacterium glutamicum/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Estabilidad Proteica , Estructura Secundaria de Proteína , Relación Estructura-ActividadRESUMEN
Lysine-specific demethylase 1 (LSD1) downregulates eukaryotic gene activity by demethylating mono- and dimethylated Lys4 in histone H3. Elucidating the biochemical crosstalk of LSD1 with histone post-translational modifications (PTMs) is essential for developing LSD1-targeted therapeutics in human cancers. We interrogated the small ubiquitin-like modifier (SUMO)-driven regulation of LSD1 activity with semisynthetic nucleosomes containing site-specifically methylated and sumoylated histones. We discovered that nucleosomes containing sumoylated histone H4 (suH4), a modification associated with gene repression, stimulate LSD1 activity by a mechanism dependent upon the SUMO-interaction motif in CoREST. Furthermore, the stimulatory effect of suH4 was spatially limited and did not extend to the demethylation of adjacent nonsumoylated nucleosomes. Thus, we have identified histone modification by SUMO as the first PTM that stimulates intranucleosomal demethylation by the developmentally critical LSD1-CoREST complex.
Asunto(s)
Proteínas Co-Represoras/metabolismo , Histona Demetilasas/metabolismo , Histonas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Sumoilación , Humanos , Metilación , Simulación del Acoplamiento Molecular , Nucleosomas/metabolismoRESUMEN
The C-terminal electrophilic activation of peptides by α-thioesterification requires strongly acidic conditions or complex chemical manipulations, which ultimately limit functional group compatibility and broad utility. Herein, we report a readily accessible N-mercaptoethoxyglycinamide (MEGA) solid-phase linker for the facile synthesis of latent peptide α-thioesters. Incubating peptide-MEGA sequences with 2-mercaptoethanesulfonic acid at mildly acidic pH yielded α-thioesters that were directly used in NCL without purification. The MEGA linker yielded robust access to thioesters ranging in length from 4 to 35 amino acids, and greatly simplified the synthesis of cyclic peptides. Finally, the high utility of MEGA was demonstrated by the one-pot synthesis of a functional analog of the Sunflower Trypsin Inhibitor 1.
Asunto(s)
Ésteres/síntesis química , Gluconatos/química , Péptidos/síntesis química , Compuestos de Sulfhidrilo/síntesis química , Ciclización , Ésteres/química , Concentración de Iones de Hidrógeno , Estructura Molecular , Péptidos/química , Compuestos de Sulfhidrilo/químicaRESUMEN
Many naturally occurring antimicrobial peptides (AMPs) are amphipathic with a ß-hairpin conformation stabilized by cross-strand disulfides across the associated ß-strands. Here, we show that the disulfides are not essential. Other structuring means such as better ß-turns and noncovalent cross-strand interactions can, with proper design, replace the disulfides with no loss in antimicrobial activity. Our results also demonstrate that the hairpin turn region may play a role in membrane recognition for at least one member of this class, since a homodimeric turnless ß-sheet analog showed no antimicrobial activity. We also examined the effects of N-terminal fatty acid adducts on AMPs. Surprisingly, the large hydrophobic carboxylic moieties examined completely eliminated the antimicrobial activity of previously active ß-hairpin peptides.