RESUMEN
Disulfide-rich peptides and proteins are among the most fascinating bioactive molecules. The difficulties associated with the preparation of these targets have prompted the development of various chemical strategies. Nevertheless, the production of these targets remains very challenging or elusive. Recently, we introduced a strategy for one-pot disulfide bond formation, tackling most of the previous limitations. However, the effect of the order of oxidation remained an underexplored issue. Herein we report on the complete synthetic flexibility of the approach with respect to the order of oxidation of three disulfide bonds in targets that lack the knot motif. In contrast, our study reveals an essential order of disulfide bond formation in the EETI-II knotted miniprotein. This synthetic strategy was applied for the synthesis of novel analogues of the plectasin antimicrobial peptide with enhanced activities against methicillin-resistant Staphylococcus aureus (MRSA), a notorious human pathogen.
Asunto(s)
Péptidos Antimicrobianos/química , Cucurbitaceae/química , Disulfuros/síntesis química , Proteínas de Plantas/química , Disulfuros/química , HumanosRESUMEN
One of the applied synthetic strategies for correct disulfide bond formation relies on the use of orthogonal Cys protecting groups. This approach requires purification before and after the deprotection steps, which prolongs the entire synthetic process and lowers the yield of the reaction. A major challenge in using this approach is to be able to apply one-pot synthesis under mild conditions and aqueous media. In this study, we report the development of an approach for rapid disulfide bond formation by employing palladium chemistry and S-acetamidomethyl-cysteine [Cys(Acm)]. Oxidation of Cys(Acm) to the corresponding disulfide bond is achieved within minutes in a one-pot operation by applying palladium and diethyldithiocarbamate. The utility of this reaction was demonstrated by the synthesis of the peptide oxytocin and the first total chemical synthesis of the protein thioredoxin-1. Our investigation revealed a critical role of the Acm protecting group in the disulfide bond formation, apparently due to the generation of a disulfiram in the reaction pathway, which significantly assists the oxidation step.
RESUMEN
Understanding the determinants of α-conotoxin (α-CTX) selectivity for different nicotinic acetylcholine receptor (nAChR) subtypes is a prerequisite for the design of tool compounds to study nAChRs. However, selectivity optimization of these small, disulfide-rich peptides is difficult not only because of an absence of α-CTX/nAChR co-structures but also because it is challenging to predict how a mutation to an α-CTX will alter its potency and selectivity. As a prototypical system to investigate selectivity, we employed the α-CTX LvIA that is 25-fold selective for the α3ß2 nAChR over the related α3ß4 nAChR subtype, which is a target for nicotine addiction. Using two-electrode voltage clamp electrophysiology, we identified LvIA[D11R] that is 2-fold selective for the α3ß4 nAChR, reversing the subtype preference. This effect is specifically due to the change in charge and not shape of LvIA[D11R], as substitution of D11 with citrulline retains selectivity for the α3ß2 nAChR. Furthermore, LvIA[D11K] shows a stronger reversal, with 4-fold selectivity for the α3ß4 nAChR. Motivated by these findings, using site-directed mutagenesis, we found that ß2[K79A] (I79 on ß4), but not ß2[K78A] (N78 on ß4), largely restores the potency of basic mutants at position 11. Finally, to understand the structural basis of this effect, we used AlphaFold2 to generate models of LvIA in complex with both nAChR subtypes. Both models confirm the plausibility of an electrostatic mechanism to explain the data and also reproduce a broad range of potency and selectivity structure-activity relationships for LvIA mutants, as measured using free energy perturbation simulations. Our work highlights how electrostatic interactions can drive α-CTX selectivity and may serve as a strategy for optimizing the selectivity of LvIA and other α-CTXs.
Asunto(s)
Conotoxinas , Receptores Nicotínicos , Conotoxinas/genética , Conotoxinas/farmacología , Electricidad Estática , Receptores Nicotínicos/genética , Mutación/genética , Péptidos , Antagonistas Nicotínicos/farmacologíaRESUMEN
Despite six decades of efforts to synthesize peptides and proteins bearing multiple disulfide bonds, this synthetic challenge remains an unsolved problem in most targets (e.g., knotted mini proteins). Here we show a de novo general synthetic strategy for the ultrafast, high-yielding formation of two and three disulfide bonds in peptides and proteins. We develop an approach based on the combination of a small molecule, ultraviolet-light, and palladium for chemo- and regio-selective activation of cysteine, which enables the one-pot formation of multiple disulfide bonds in various peptides and proteins. We prepare bioactive targets of high therapeutic potential, including conotoxin, RANTES, EETI-II, and plectasin peptides and the linaclotide drug. We anticipate that this strategy will be a game-changer in preparing millions of inaccessible targets for drug discovery.
Asunto(s)
Disulfuros/química , Disulfuros/metabolismo , Péptidos/metabolismo , Proteínas/metabolismo , Cromatografía Líquida de Alta Presión , Péptidos/síntesis química , Péptidos/química , Proteínas/síntesis química , Proteínas/química , Espectrometría de Masa por Ionización de Electrospray , EstereoisomerismoRESUMEN
Histone modifications dynamically regulate chromatin structure and function, thereby mediating many processes that require access to DNA. Chemical protein synthesis has emerged as a powerful approach for generating homogeneously modified histone analogues in workable amounts for subsequent incorporation into nucleosome arrays for biochemical, functional and structural studies. This short review focuses on the strength of total chemical protein synthesis and semisynthetic approaches to generate ubiquitylated histones in their native or non-native forms and the utility of these analogues to decode the role of ubiquitylation in epigenetics.
Asunto(s)
Cromatina/química , Histonas/química , Ubiquitinación , Animales , Técnicas de Química Sintética/métodos , Epigénesis Genética , Histonas/síntesis química , Humanos , Modelos Moleculares , Nucleosomas/químicaRESUMEN
Organic chemistry allows for the modification and chemical preparation of protein analogues for various studies. The thiolate side chain of the Cys residue has been a key functionality in these ventures. In order to generate complex molecular targets, there is a particular need to incorporate orthogonal protecting groups of the thiolated amino acids to control the directionality of synthesis and modification site. Here, we demonstrate the tuning of palladium chemoselectivity in aqueous medium for on-demand deprotection of several Cys-protecting groups that are useful in protein synthesis and modification. These tools allow the preparation of highly complex analogues as we demonstrate in the synthesis of the copper storage protein and selectively modified peptides with multiple Cys residues. We also report the synthesis of an activity-based probe comprising ubiquitinated histone H2A and its incorporation into nucleosomes and demonstrate its reactivity with deubiquitinating enzyme to generate a covalent nucleosome-enzyme complex.
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Cisteína/química , Paladio/química , Procesamiento Proteico-Postraduccional , Proteínas/síntesis química , Secuencia de Aminoácidos , Aminoácidos , Cobre/química , Enzimas Desubicuitinizantes/metabolismo , Histonas/síntesis química , Nucleosomas/química , Coloración y Etiquetado , Tiazolidinas/química , Proteínas Ubiquitinadas/síntesis químicaRESUMEN
Chemical synthesis of histones allows precise control of the installation of post-translational modifications via the coupling of derivatized amino acids. Shortcomings of other approaches for obtaining modified histones for epigenetic studies include heterogeneity of the obtained product and difficulties in incorporating multiple modifications on the same histone. In this protocol, unprotected peptide fragments are prepared by Fmoc solid-phase synthesis and coupled in aqueous buffers via native chemical ligation (NCL; in NCL, a peptide bond is formed between a peptide with an N-terminal Cys and another peptide having a C-terminal thioester). This task is challenging, with obstacles relating to the preparation and ligation of hydrophobic peptides, as well as the requirement for multiple purification steps due to protecting-group manipulations during the polypeptide assembly process. To address this, our approach uses an easily removable solubilizing tag for the synthesis and ligation of hydrophobic peptides, as well as a more efficient and better-yielding method to remove Cys-protecting groups that uses palladium chemistry (specifically [Pd(allyl)Cl]2 and PdCl2 complexes). The utility of this approach is demonstrated in the syntheses of ubiquitinated H2B at Lys34, phosphorylated H2A at Tyr57 and unmodified H4. Each of these analogs can be prepared in milligram quantities within â¼20-30 d.
Asunto(s)
Técnicas de Química Sintética/métodos , Histonas/síntesis química , Paladio/química , Fragmentos de Péptidos/síntesis química , Aminoácidos , Fluorenos , Histonas/química , Fragmentos de Péptidos/químicaRESUMEN
Reversible attachment of solubilizing tags to hydrophobic peptides to facilitate their purification and ligation is an essential yet challenging task in chemical protein synthesis. The efficient palladium-assisted removal of the solubilizing tag linked to the Cys side chain is reported. The strategy was applied for the efficient preparation of histone protein H4 from two fragments via one-pot operation of ligation, removal of the solubilizing tag, and desulfurization.
Asunto(s)
Cisteína/química , Paladio/química , Proteínas/síntesis química , Histonas/síntesis química , Interacciones Hidrofóbicas e Hidrofílicas , Péptidos/síntesis química , Conformación Proteica , Solubilidad , SolventesRESUMEN
The cytoplasmic C-terminal domain (CTD) of KcsA, a bacterial homotetrameric potassium channel, is an amphiphilic domain that forms a helical bundle with four-fold symmetry mediated by hydrophobic and electrostatic interactions. Previously we have established that a CTD-derived 34-residue peptide associates into a tetramer in a pH-dependent manner (Kamnesky et al., JMB 2012;418:237-247). Here we further investigate the molecular determinants of tetramer formation in the CTD by characterizing the kinetics of monomer-tetramer equilibrium for 10 alanine mutants using NMR, sedimentation equilibrium (SE) and molecular dynamics simulation. NMR and SE concur in finding single-residue contributions to tetramer stability to be in the 0.5 to 3.5 kcal/mol range. Hydrophobic interactions between residues lining the tetramer core generally contributed more to formation of tetramer than electrostatic interactions between residues R147, D149 and E152. In particular, alanine replacement of residue R147, a key contributor to inter-subunit salt bridges, resulted in only a minor effect on tetramer dissociation. Mutations outside of the inter-subunit interface also influenced tetramer stability by affecting the tetramerization on-rate, possibly by changing the inherent helical propensity of the peptide. These findings are interpreted in the context of established paradigms of protein-protein interactions and protein folding, and lay the groundwork for further studies of the CTD in full-length KcsA channels.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Canales de Potasio/química , Canales de Potasio/genética , Alanina/metabolismo , Proteínas Bacterianas/metabolismo , Dicroismo Circular , Citoplasma/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Simulación de Dinámica Molecular , Mutación , Canales de Potasio/metabolismo , Unión Proteica , Pliegue de Proteína , Multimerización de Proteína , Estabilidad ProteicaRESUMEN
The intracellular C-terminal domain (CTD) of KcsA, a bacterial homotetrameric potassium channel, is a 40-residue-long segment that natively adopts a helical bundle conformation with 4-fold symmetry. A hallmark of KcsA behavior is pH-induced conformational change, which leads to the opening of the channel at acidic pH. Previous studies have reached conflicting conclusions as to the role of the CTD in this transition. Here, we investigate the involvement of this domain in pH-mediated channel opening by NMR using a soluble peptide corresponding to residues 128-160 of the CTD (CTD34). At neutral pH, CTD34 exhibits concentration-dependent spectral changes consistent with oligomer formation. We prove this slowly tumbling species to be a tetramer with a dissociation constant of (2.0±0.5)×10(-)(11) M(3) by NMR and sedimentation equilibrium experiments. Whereas monomeric CTD34 is only mildly helical, secondary chemical shifts prove that the tetrameric species adopts a tight native-like helical bundle conformation. The tetrameric species undergoes pH-dependent dissociation, and CTD34 is fully monomeric below pH 5.0. The structural basis for this phenomenon is the destabilization of the tetrameric CTD34 by protonation of residue H145 in the monomeric form of the peptide. We conclude that (i) the CTD34 peptide is independently capable of forming a tetrameric helical bundle, and (ii) this structurally significant conformational shift is modulated by the effects of solution pH on residue H145. Therefore, the involvement of this domain in the pH gating of the channel is strongly suggested.