RESUMO
Knotted peptides present a wealth of structurally diverse, biologically active molecules, with the inhibitor cystine knot/knottin class among the most ecologically common ones. Many of these natural products interact with extracellular targets such as voltage-gated ion channels with exquisite selectivity and potency, making them intriguing therapeutic modalities. Such compounds are often produced in low concentrations by intractable organisms, making structural and biological characterization challenging, which is frequently overcome by various expression strategies. Here, we sought to test a biosynthetic route for the expression and study of knotted peptides. We screened expression constructs for a biosynthesized knotted peptide to determine the most influential parameters for successful disulfide folding and used NMR spectroscopic fingerprinting to validate topological structures. We performed pharmacokinetic characterization, which indicated that the interlocking disulfide structure minimizes liabilities of linear peptide sequences, and propose a mechanism by which knotted peptides are cleared. We then developed an assay to monitor solution folding in real time, providing a strategy for studying the folding process during maturation, which provided direct evidence for the importance of backbone organization as the driving force for topology formation.
RESUMO
Advances in the design of permeable peptides and in the synthesis of large arrays of macrocyclic peptides with diverse amino acids have evolved on parallel but independent tracks. Less precedent combines their respective attributes, thereby limiting the potential to identify permeable peptide ligands for key targets. Herein, we present novel 6-, 7-, and 8-mer cyclic peptides (MW 774-1076 g·mol-1) with passive permeability and oral exposure that feature the amino acids and thioether ring-closing common to large array formats, including DNA- and RNA-templated synthesis. Each oral peptide herein, selected from virtual libraries of partially N-methylated peptides using in silico methods, reflects the subset consistent with low energy conformations, low desolvation penalties, and passive permeability. We envision that, by retaining the backbone N-methylation pattern and consequent bias toward permeability, one can generate large peptide arrays with sufficient side chain diversity to identify permeability-biased ligands to a variety of protein targets.
Assuntos
Peptídeos Cíclicos/farmacologia , Sulfetos/farmacologia , Administração Oral , Animais , Células CACO-2 , Permeabilidade da Membrana Celular , Cães , Humanos , Células Madin Darby de Rim Canino , Masculino , Metilação , Estrutura Molecular , Peptídeos Cíclicos/administração & dosagem , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/farmacocinética , Conformação Proteica , Ratos Sprague-Dawley , Bibliotecas de Moléculas Pequenas/administração & dosagem , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/farmacocinética , Bibliotecas de Moléculas Pequenas/farmacologia , Sulfetos/administração & dosagem , Sulfetos/síntese química , Sulfetos/farmacocinética , TermodinâmicaRESUMO
PURPOSE: Investigate the use of solution NMR spectroscopy to evaluate whether the general ΔpKa rule is valid in organic solvents. Such information may be useful in evaluation of acid-base reactions and solvent selection for salt formation. METHODS: (1)H NMR chemical shift changes in model bases during titration with acids, and separately, on the addition of acids at a molar ratio of 1:1 were determined in water, dimethyl sulfoxide, and methanol. The effect of acid strength on the fraction of ionized base was examined. RESULTS: (1)H NMR chemical shift changes indicated protonation (in situ salt formation). Different media affected the observed chemical shift changes. In all media investigated the data followed the ΔpKa (base-acid) general rule, that the pKa value of the acids should be 2-3 units lower than the pKa of the base to ensure proton transfer. The addition of water into organic solvents increased the fraction of ionized base. CONCLUSIONS: Protonation, as measured by chemical shift changes using solution NMR spectroscopy, provided novel insight on potential salt formation in different media. Even though pKa values change with the solvent, the general ΔpKa rule can be applied in different solvent systems. Solution NMR spectroscopy appears to be a useful tool to evaluate salt formation reaction and process control in different solvent systems.
Assuntos
Prótons , Solventes/química , Dimetil Sulfóxido/química , Etilenodiaminas/química , Compostos Heterocíclicos com 1 Anel/química , Espectroscopia de Ressonância Magnética/métodos , Metanol/química , Quinuclidinas/química , Sais/química , Soluções , Água/químicaRESUMO
Integral membrane proteins remain a significant challenge to structural studies by solution NMR spectroscopy. This is due not only to spectral complexity, but also because the effects of slow molecular reorientation are exacerbated by the need to solubilize the protein in aqueous detergent micelles. These assemblies can be quite large and require deuteration for optimal use of the TROSY effect. In principle, another approach is to employ reverse micelle encapsulation to solubilize the protein in a low-viscosity solvent in which the rapid tumbling of the resulting particle allows for use of standard triple-resonance methods. The preparation of such samples of membrane proteins is difficult. Using a 54 kDa construct of the homotetrameric potassium channel KcsA, we demonstrate a strategy that employs a hybrid surfactant to transfer the protein to the reverse micelle system.
Assuntos
Proteínas de Membrana/química , Micelas , Proteínas de Membrana/metabolismo , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Canais de Potássio/química , Canais de Potássio/metabolismo , Conformação ProteicaRESUMO
Theoretical considerations suggest that protein cold denaturation can potentially provide a means to explore the cooperative substructure of proteins. Protein cold denaturation is generally predicted to occur well below the freezing point of water. Here NMR spectroscopy of ubiquitin encapsulated in reverse micelles dissolved in low viscosity alkanes is used to follow cold-induced unfolding to temperatures below -25 degrees C. Comparison of cold-induced structural transitions in a variety of reverse micelle-buffer systems indicate that protein-surfactant interactions are negligible and allow the direct observation of the multistate cold-induced unfolding of the protein.
Assuntos
Temperatura Baixa , Ubiquitina/química , Modelos Moleculares , Conformação Proteica , Desnaturação ProteicaRESUMO
The modern view of protein thermodynamics predicts that proteins undergo cold-induced unfolding. Unfortunately, the properties of proteins and water conspire to prevent the detailed observation of this fundamental process. Here we use protein encapsulation to allow cold denaturation of the protein ubiquitin to be monitored by high-resolution NMR at temperatures approaching -35 degrees C. The cold-induced unfolding of ubiquitin is found to be highly noncooperative, in distinct contrast to the thermal melting of this and other proteins. These results demonstrate the potential of cold denaturation as a means to dissect the cooperative substructures of proteins and to provide a rigorous framework for testing statistical thermodynamic treatments of protein stability, dynamics and function.
Assuntos
Desnaturação Proteica , Proteínas/química , Ubiquitina/química , Algoritmos , Temperatura Baixa , Congelamento , Espectroscopia de Ressonância Magnética , Micelas , Modelos Moleculares , Conformação Proteica , Ubiquitina/metabolismoRESUMO
Encapsulating a protein in a reverse micelle and dissolving it in a low-viscosity solvent can lower the rotational correlation time of a protein and thereby provides a novel strategy for studying proteins in a variety of contexts. The preparation of the sample is a key element in this approach and is guided by a number of competing parameters. Here we examine the applicability of several strategies for the preparation and characterization of encapsulated proteins dissolved in low viscosity fluids that are suitable for high performance NMR spectroscopy. Ubiquitin is used as a model system to explore various issues such as the homogeneity of the encapsulation, characterization of the hydrodynamic performance of reverse micelles containing protein molecules, and the effective pH of the water environment of the reverse micelle.
Assuntos
Micelas , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Cápsulas , Difusão , Composição de Medicamentos , Concentração de Íons de Hidrogênio , Isótopos de Nitrogênio , Tamanho da Partícula , Rotação , Solubilidade , Soluções , Ubiquitina/química , ViscosidadeRESUMO
We have characterized a membrane protein containing residues P688-T762 of the integrin beta3 subunit, encompassing its transmembrane and cytoplasmic domains, by nuclear magnetic resonance spectroscopy. Under conditions in which it is monomeric in dodecylphosphocholine micelles, the protein consists mainly of alpha-helical structures. An amino-terminal helix corresponding to the beta3 transmembrane helix extends into the membrane-proximal region of the cytoplasmic domain. Moreover, following an apparent hinge at residues H722-D723, residues K725-A735 are mostly alpha-helical. In the presence of membrane-mimicking detergents, the cytoplasmic domain connected to the transmembrane helix is substantially ordered at pH 4.8 and 50 degrees C. Its carboxyl-terminal end takes on a turn-helix configuration characteristic of the immunoreceptor tyrosine-based activation motif. These structural features of the beta3 subunit should help to explain its interaction with numerous cytosolic interacting proteins and begin to illuminate the mechanism of integrin activation.
Assuntos
Citoplasma/química , Integrina beta3/química , Proteínas de Membrana/química , Ressonância Magnética Nuclear Biomolecular , Fosforilcolina/análogos & derivados , Subunidades Proteicas/química , Sequência de Aminoácidos , Sítios de Ligação , Dicroísmo Circular , Temperatura Alta , Concentração de Íons de Hidrogênio , Proteínas de Membrana/síntese química , Micelas , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular/métodos , Fosforilcolina/química , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/síntese química , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/química , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Subunidades Proteicas/síntese química , Proteínas Recombinantes de Fusão/síntese química , TermodinâmicaRESUMO
Application of triple-resonance and isotope-edited-NOE methods to the study of increasingly larger macromolecules and their complexes remains a central goal of solution NMR spectroscopy. The slow reorientational motion of larger molecules leads to rapid transverse relaxation and results in losses in both resolution and sensitivity of multidimensional-multinuclear solution NMR experiments. A recently described technique employs a physical approach to increase the tumbling rate of macromolecules in an attempt to preserve access to the full range of structural restraints available to studies of smaller systems. This technique involves encapsulation of a hydrated protein in a surfactant shell which is subsequently solubilized in a low viscosity solvent. A simple, efficient and cost effective NMR cell that accommodates the moderate liquefaction pressures required in the encapsulation method is described. Application of the method to the 56 kD triose phosphate isomerase homodimer is demonstrated.