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1.
J Org Chem ; 88(11): 6588-6598, 2023 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-37155983

RESUMO

Cyclic peptoids are macrocyclic oligomers of N-substituted glycines with specific folding abilities and excellent metal binding properties. In this work, we show how strategic positioning of chiral (S)- and (R)-(1-carboxyethyl)glycine units influences the conformational stability of water-soluble macrocyclic peptoids as sodium complexes. The reported results are based on nuclear magnetic resonance spectroscopy, extensive computational studies, and X-ray diffraction analysis using single crystals grown from aqueous solutions. The studies include 1H relaxometric investigations of hexameric cyclic peptoids in the presence of the Gd3+ ion to assess their thermodynamic stabilities and relaxivities.

2.
Angew Chem Int Ed Engl ; 61(27): e202201895, 2022 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-35415953

RESUMO

Despite recent progress, it remains challenging to program biomacromolecules to assemble into discrete nanostructures with pre-determined sizes and topologies. We report here a novel strategy to address this challenge. By using two orthogonal pairs of heterodimeric coiled coils as the building blocks, we constructed six discrete supramolecular assemblies, each composed of a prescribed number of coiled coil components. Within these assemblies, different coiled coils were connected via end-to-side covalent linkages strategically pre-installed between the non-complementary pairs. The overall topological features of two highly complex assemblies, a "barbell" and a "quadrilateral" form, were characterized experimentally and were in good agreement to the designs. This work expands the design paradigms for peptide-based discrete supramolecular assemblies and will provide a route for de novo fabrication of functional protein materials.


Assuntos
Nanoestruturas , Peptídeos , Fenômenos Biofísicos , Nanoestruturas/química , Peptídeos/química , Domínios Proteicos , Proteínas/química
3.
J Proteome Res ; 19(9): 3856-3866, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32786687

RESUMO

Aberrant protein synthesis and protein expression are a hallmark of many conditions ranging from cancer to Alzheimer's. Blood-based biomarkers indicative of changes in proteomes have long been held to be potentially useful with respect to disease prognosis and treatment. However, most biomarker efforts have focused on unlabeled plasma proteomics that include nonmyeloid origin proteins with no attempt to dynamically tag acute changes in proteomes. Herein we report a method for evaluating de novo protein synthesis in whole blood liquid biopsies. Using a modification of the "bioorthogonal noncanonical amino acid tagging" (BONCAT) protocol, rodent whole blood samples were incubated with l-azidohomoalanine (AHA) to allow incorporation of this selectively reactive non-natural amino acid within nascent polypeptides. Notably, failure to incubate the blood samples with EDTA prior to implementation of azide-alkyne "click" reactions resulted in the inability to detect probe incorporation. This live-labeling assay was sensitive to inhibition with anisomycin and nascent, tagged polypeptides were localized to a variety of blood cells using FUNCAT. Using labeled rodent blood, these tagged peptides could be consistently identified through standard LC/MS-MS detection of known blood proteins across a variety of experimental conditions. Furthermore, this assay could be expanded to measure de novo protein synthesis in human blood samples. Overall, we present a rapid and convenient de novo protein synthesis assay that can be used with whole blood biopsies that can quantify translational change as well as identify differentially expressed proteins that may be useful for clinical applications.


Assuntos
Alcinos , Azidas , Química Click , Reação de Cicloadição , Biossíntese de Proteínas
4.
Soft Matter ; 16(44): 10180-10186, 2020 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-33057563

RESUMO

The size of a probe bead reported by holographic particle characterization depends on the proportion of the surface area covered by bound target molecules and so can be used as an assay for molecular binding. We validate this technique by measuring the kinetics of irreversible binding for the antibodies immunoglobulin G (IgG) and immunoglobulin M (IgM) as they attach to micrometer-diameter colloidal beads coated with protein A. These measurements yield the antibodies' binding rates and can be inverted to obtain the concentration of antibodies in solution. Holographic molecular binding assays therefore can be used to perform fast quantitative immunoassays that are complementary to conventional serological tests.


Assuntos
Imunoglobulina G , Imunoensaio , Imunoglobulina M
5.
Org Biomol Chem ; 18(12): 2312-2320, 2020 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-32159574

RESUMO

We report a general approach to promote the folding of synthetic oligopeptides capable of forming homodimeric coiled coil assemblies. By pre-organizing the peptides on macrocyclic oligomer scaffolds, the stability of the coiled coils is enhanced with an observed increase in the melting temperature of 30 °C to 40 °C. Molecular dynamics simulations substantiate the hypothesis that the enhanced stability is established by constraining motion at the peptide termini and by pre-organizing intramolecular helix-helix contacts. We demonstrate the modularity of this approach by using a family of peptoid scaffolds to promote the folding of a dimeric coiled coil. Importantly, this strategy for templating coiled coils allows preservation of native amino acid sequences. Comparing a macrocyclic peptoid scaffold to its linear counterparts indicates that both types of assemblies are effective for organizing stable coiled coils. These results will guide future designs of coiled coil peptides for biomedical applications and as building blocks for more complex supramolecular assemblies.


Assuntos
Peptoides/química , Dobramento de Proteína , Dimerização , Simulação de Dinâmica Molecular , Estrutura Secundária de Proteína , Termodinâmica
6.
Biopolymers ; 110(6): e23266, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30835823

RESUMO

Like polypeptides, peptoids, or N-substituted glycine oligomers, have intrinsic conformational preferences due to their amide backbones and close spacing of side chain substituents. However, the conformations that peptoids adopt are distinct from polypeptides due to several structural differences: the peptoid backbone is composed of tertiary amide bonds that have trans and cis conformers similar in energy, they lack a backbone hydrogen bond donor, and have an N-substituent. To better understand how these differences manifest in actual peptoid structures, we analyzed 46 high quality, experimentally determined peptoid structures reported in the literature to extract their backbone conformational preferences. One hundred thirty-two monomer dihedral angle pairs were compared to the calculated energy landscape for the peptoid Ramachandran plot, and were found to fall within the expected minima. Interestingly, only two regions of the backbone dihedral angles ϕ and ψ were found to be populated that are mirror images of each other. Furthermore, these two conformers are present in both cis and trans forms. Thus, there are four primary conformers that are sufficient to describe almost all known backbone conformations for peptoid oligomers, despite conformational constraints imposed by a variety of side chains, macrocyclization, or crystal packing forces. Because these conformers are predominant in peptoid structure, and are distinct from those found in protein secondary structures, we propose a simple naming system to aid in the description and classification of peptoid structure.


Assuntos
Peptoides/química , Cristalografia por Raios X , Ligação de Hidrogênio , Peptídeos/química , Estrutura Secundária de Proteína , Estereoisomerismo
7.
Biochim Biophys Acta Biomembr ; 1860(6): 1414-1423, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29621496

RESUMO

Hydrophobic interactions govern specificity for natural antimicrobial peptides. No such relationship has been established for synthetic peptoids that mimic antimicrobial peptides. Peptoid macrocycles synthesized with five different aromatic groups are investigated by minimum inhibitory and hemolytic concentration assays, epifluorescence microscopy, atomic force microscopy, and X-ray reflectivity. Peptoid hydrophobicity is determined using high performance liquid chromatography. Disruption of bacterial but not eukaryotic lipid membranes is demonstrated on the solid supported lipid bilayers and Langmuir monolayers. X-ray reflectivity studies demonstrate that intercalation of peptoids with zwitterionic or negatively charged lipid membranes is found to be regulated by hydrophobicity. Critical levels of peptoid selectivity are demonstrated and found to be modulated by their hydrophobic groups. It is suggested that peptoids may follow different optimization schemes as compared to their natural analogues.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Lipídeos de Membrana/química , Ânions/química , Peptídeos Catiônicos Antimicrobianos/síntese química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Cromatografia Líquida de Alta Pressão , Eritrócitos/efeitos dos fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Bicamadas Lipídicas/química , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Microscopia de Força Atômica , Estrutura Molecular , Eletricidade Estática , Relação Estrutura-Atividade , Especificidade por Substrato
8.
J Am Chem Soc ; 138(5): 1543-50, 2016 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-26812069

RESUMO

The design of folded miniature proteins is predicated on establishing noncovalent interactions that direct the self-assembly of discrete thermostable tertiary structures. In this work, we describe how a network of cation-π interactions present in proteins containing "WSXWS motifs" can be emulated to stabilize the core of a miniature protein. This 19-residue protein sequence recapitulates a set of interdigitated arginine and tryptophan residues that stabilize a distinctive ß-strand:loop:PPII-helix topology. Validation of the compact fold determined by NMR was carried out by mutagenesis of the cation-π network and by comparison to the corresponding disulfide-bridged structure. These results support the involvement of a coordinated set of cation-π interactions that stabilize the tertiary structure.


Assuntos
Cátions/química , Proteínas/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Estrutura Terciária de Proteína , Alinhamento de Sequência
9.
Chembiochem ; 17(19): 1824-1828, 2016 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-27539882

RESUMO

Poly-proline type II (PPII) helical PXXP motifs are the recognition elements for a variety of protein-protein interactions that are critical for cellular signaling. Despite development of protocols for locking peptides into α-helical and ß-strand conformations, there remains a lack of analogous methods for generating mimics of PPII helical structures. We describe herein a strategy to enforce PPII helical secondary structure in the 19-residue TrpPlexus miniature protein. Through sequence variation, we showed that a network of cation-π interactions could drive the formation of PPII helical conformations for both peptide and N-substituted glycine peptoid residues. The achievement of chemically diverse PPII helical scaffolds provides a new route towards discovering peptidomimetic inhibitors of protein-protein interactions mediated by PXXP motifs.


Assuntos
Peptídeos/química , Peptidomiméticos , Cátions/química , Modelos Moleculares , Ligação Proteica , Estrutura Secundária de Proteína
10.
Langmuir ; 32(48): 12905-12913, 2016 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-27793068

RESUMO

The peptidomimetic approach has emerged as a powerful tool for overcoming the inherent limitations of natural antimicrobial peptides, where the therapeutic potential can be improved by increasing the selectivity and bioavailability. Restraining the conformational flexibility of a molecule may reduce the entropy loss upon its binding to the membrane. Experimental findings demonstrate that the cyclization of linear antimicrobial peptoids increases their bactericidal activity against Staphylococcus aureus while maintaining high hemolytic concentrations. Surface X-ray scattering shows that macrocyclic peptoids intercalate into Langmuir monolayers of anionic lipids with greater efficacy than for their linear analogues. It is suggested that cyclization may increase peptoid activity by allowing the macrocycle to better penetrate the bacterial cell membrane.


Assuntos
Antibacterianos/farmacologia , Peptoides/farmacologia , Membrana Celular/efeitos dos fármacos , Ciclização , Staphylococcus aureus/efeitos dos fármacos
11.
Biopolymers ; 103(4): 227-36, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25403751

RESUMO

Antimicrobial peptides (AMPs) are critical components of the innate immune system and exhibit bactericidal activity against a broad spectrum of bacteria. We investigated the use of N-substituted glycine peptoid oligomers as AMP mimics with potent antimicrobial activity. The antimicrobial mechanism of action varies among different AMPs, but many of these peptides can penetrate bacterial cell membranes, causing cell lysis. We previously hypothesized that amphiphilic cyclic peptoids may act through a similar pore formation mechanism against methicillin-resistant Staphylococcus aureus (MRSA). Peptoid-induced membrane disruption is observed by scanning electron microscopy and results in a loss of membrane integrity. We demonstrate that the antimicrobial activity of the peptoids is attenuated with the addition of polyethylene glycol osmoprotectants, signifying protection from a loss of osmotic balance. This decrease in antimicrobial activity is more significant with larger osmoprotectants, indicating that peptoids form pores with initial diameters of ∼2.0-3.8 nm. The initial membrane pores formed by cyclic peptoid hexamers are comparable in diameter to those formed by larger and structurally distinct AMPs. After 24 h, the membrane pores expand to >200 nm in diameter. Together, these results indicate that cyclic peptoids exhibit a mechanism of action that includes effects manifested at the cell membrane of MRSA.


Assuntos
Peptoides/farmacologia , Polímeros/farmacologia , Antibacterianos/química , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Staphylococcus aureus Resistente à Meticilina , Peptoides/química , Polímeros/química
12.
Proc Natl Acad Sci U S A ; 109(49): 19922-7, 2012 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-23169638

RESUMO

The ability of natural peptides and proteins to influence the formation of inorganic crystalline materials has prompted the design of synthetic compounds for the regulation of crystal growth, including the freezing of water and growth of ice crystals. Despite their versatility and ease of structural modification, peptidomimetic oligomers have not yet been explored extensively as crystallization modulators. This report describes a library of synthetic N-substituted glycine peptoid oligomers that possess "dual-action" antifreeze activity as exemplified by ice crystal growth inhibition concomitant with melting temperature reduction. We investigated the structural features responsible for these phenomena and observed that peptoid antifreeze activities depend both on oligomer backbone structure and side chain chemical composition. These studies reveal the capability of peptoids to act as ice crystallization regulators, enabling the discovery of a unique and diverse family of synthetic oligomers with potential as antifreeze agents in food production and biomedicine.


Assuntos
Proteínas Anticongelantes/química , Biomimética/métodos , Peptoides/química , Cristalização/métodos , Gelo/análise , Estrutura Molecular , Temperatura de Transição , Difração de Raios X
13.
Proc Natl Acad Sci U S A ; 109(36): 14320-5, 2012 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-22908242

RESUMO

Peptoid molecules are biomimetic oligomers that can fold into unique three-dimensional structures. As part of an effort to advance computational design of folded oligomers, we present blind-structure predictions for three peptoid sequences using a combination of Replica Exchange Molecular Dynamics (REMD) simulation and Quantum Mechanical refinement. We correctly predicted the structure of a N-aryl peptoid trimer to within 0.2 Å rmsd-backbone and a cyclic peptoid nonamer to an accuracy of 1.0 Å rmsd-backbone. X-ray crystallographic structures are presented for a linear N-alkyl peptoid trimer and for the cyclic peptoid nonamer. The peptoid macrocycle structure features a combination of cis and trans backbone amides, significant nonplanarity of the amide bonds, and a unique "basket" arrangement of (S)-N(1-phenylethyl) side chains encompassing a bound ethanol molecule. REMD simulations of the peptoid trimers reveal that well folded peptoids can exhibit funnel-like conformational free energy landscapes similar to those for ordered polypeptides. These results indicate that physical modeling can successfully perform de novo structure prediction for small peptoid molecules.


Assuntos
Modelos Moleculares , Peptoides/química , Conformação Proteica , Dobramento de Proteína , Cristalografia por Raios X , Simulação de Dinâmica Molecular , Polímeros/química
14.
J Proteome Res ; 13(12): 5707-14, 2014 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-25271054

RESUMO

Measuring the synthesis of new proteins in the context of a much greater number of pre-existing proteins can be difficult. To overcome this obstacle, bioorthogonal noncanonical amino acid tagging (BONCAT) can be combined with stable isotope labeling by amino acid in cell culture (SILAC) for comparative proteomic analysis of de novo protein synthesis (BONLAC). In the present study, we show that alkyne resin-based isolation of l-azidohomoalanine (AHA)-labeled proteins using azide/alkyne cycloaddition minimizes contamination from pre-existing proteins. Using this approach, we isolated and identified 7414 BONCAT-labeled proteins. The nascent proteome isolated by BONCAT was very similar to the steady-state proteome, although transcription factors were highly enriched by BONCAT. About 30% of the methionine residues were replaced by AHA in our BONCAT samples, which allowed for identification of methionine-containing peptides. There was no bias against low-methionine proteins by BONCAT at the proteome level. When we applied the BONLAC approach to screen for brain-derived neurotrophic factor (BDNF)-induced protein synthesis, 53 proteins were found to be significantly changed 2 h after BDNF stimulation. Our study demonstrated that the newly synthesized proteome, even after a short period of stimulation, can be efficiently isolated by BONCAT and analyzed to a depth that is similar to that of the steady-state proteome.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Proteoma/análise , Proteômica/métodos , Alanina/análogos & derivados , Alanina/química , Cromatografia Líquida , Células HEK293 , Humanos , Espectrometria de Massas , Proteoma/química , Proteoma/metabolismo , Reprodutibilidade dos Testes
15.
J Am Chem Soc ; 136(24): 8772-82, 2014 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-24823488

RESUMO

Peptoids are a family of synthetic oligomers composed of N-substituted glycine units. Along with other "foldamer" systems, peptoid oligomer sequences can be predictably designed to form a variety of stable secondary structures. It is not yet evident if foldamer design can be extended to reliably create tertiary structure features that mimic more complex biomolecular folds and functions. Computational modeling and prediction of peptoid conformations will likely play a critical role in enabling complex biomimetic designs. We introduce a computational approach to provide accurate conformational and energetic parameters for peptoid side chains needed for successful modeling and design. We find that peptoids can be described by a "rotamer" treatment, similar to that established for proteins, in which the peptoid side chains display rotational isomerism to populate discrete regions of the conformational landscape. Because of the insufficient number of solved peptoid structures, we have calculated the relative energies of side-chain conformational states to provide a backbone-dependent (BBD) rotamer library for a set of 54 different peptoid side chains. We evaluated two rotamer library development methods that employ quantum mechanics (QM) and/or molecular mechanics (MM) energy calculations to identify side-chain rotamers. We show by comparison to experimental peptoid structures that both methods provide an accurate prediction of peptoid side chain placements in folded peptoid oligomers and at protein interfaces. We have incorporated our peptoid rotamer libraries into ROSETTA, a molecular design package previously validated in the context of protein design and structure prediction.


Assuntos
Modelos Moleculares , Biblioteca de Peptídeos , Peptoides/química , Dobramento de Proteína , Estrutura Molecular , Teoria Quântica
18.
Biopolymers ; 102(5): 407-15, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25059748

RESUMO

N-Substituted glycine peptoid oligomers have recently attracted attention for their metal binding capabilities. Due to their efficient synthesis on solid phase, peptoids are well suited for generation of compound libraries, followed by screening for molecular recognition and other diverse functional attributes. Ideally, peptoids could be simultaneously screened for binding to a number of metal species. Here, we demonstrate the use of bench-top X-ray fluorescence (XRF) instrumentation to screen rapidly, on solid support, a library of peptoid oligomers incorporating metal-binding functionalities. A subset of the peptoid sequences exhibited significant metal binding capabilities, including a peptoid pentamer and a nonamer that were shown to selectively bind nickel. The binding capabilities were validated by colorimetric assay and by depletion of Ni(2+) ion concentration from solution, establishing bench-top XRF as a rapid, practicable high-throughput screening technique for peptoid oligomers. This protocol will facilitate discovery of metallopeptoids with unique material properties.


Assuntos
Metais/metabolismo , Peptoides/metabolismo , Resinas Sintéticas/química , Espectrometria por Raios X , Colorimetria , Níquel , Oximas/química , Biblioteca de Peptídeos , Soluções
19.
Biopolymers ; 102(5): 369-78, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24919990

RESUMO

Peptomers are oligomeric molecules composed of both α-amino acids and N-substituted glycine monomers, thus creating a hybrid of peptide and peptoid units. Peptomers have been used in several applications such as antimicrobials, protease inhibitors, and antibody mimics. Despite the considerable promise of peptomers as chemically diverse molecular scaffolds, we know little about their conformational tendencies. This lack of knowledge limits the ability to implement computational approaches for peptomer design. Here we computationally evaluate the local structural propensities of the peptide-peptoid linkage. We find some general similarities between the peptide residue conformational preferences and the Ramachandran distribution of residues that precede proline in folded protein structures. However, there are notable differences. For example, several ß-turn motifs are disallowed when the i+2 residue is also a peptoid monomer. Significantly, the lowest energy geometry, when dispersion forces are accounted for, corresponds to a "cis-Pro touch-turn" conformation, an unusual turn motif that has been observed at protein catalytic centers and binding sites. The peptomer touch-turn thus represents a useful design element for the construction of folded oligomers capable of molecular recognition and as modules in the assembly of structurally complex peptoid-protein hybrid macromolecules.


Assuntos
Conformação Molecular , Peptídeos/química , Peptoides/química , Alanina/química , Motivos de Aminoácidos , Modelos Moleculares , Teoria Quântica , Termodinâmica
20.
Org Biomol Chem ; 11(25): 4142-6, 2013 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-23715215

RESUMO

We report the first example of chemoselective fragment condensation, through native amide bond formation, between peptoid and peptide oligomers. Peptoid oligomers bearing C-terminal salicylaldehyde esters were synthesized and ligated to peptides containing N-terminal serine or threonine residues. We investigate the ligation efficiency of peptoid oligomers varying in length, sequence, and C-terminal steric bulk. These protocols enhance accessibility of structurally complex peptoid-peptide hybrids and will facilitate the design new semi-synthetic proteins with unique attributes.


Assuntos
Peptídeos/química , Peptidomiméticos/química , Peptoides/química , Aldeídos/química , Amidas/química , Peptídeos/síntese química , Peptidomiméticos/síntese química , Peptoides/síntese química , Serina/química , Treonina/química
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