RESUMO
Alanyl-tRNA synthetase retains a conserved prototype structure throughout its biology. Nevertheless, its C-terminal domain (C-Ala) is highly diverged and has been shown to play a role in either tRNA or DNA binding. Interestingly, we discovered that Caenorhabditis elegans cytoplasmic C-Ala (Ce-C-Alac) robustly binds both ligands. How Ce-C-Alac targets its cognate tRNA and whether a similar feature is conserved in its mitochondrial counterpart remain elusive. We show that the N- and C-terminal subdomains of Ce-C-Alac are responsible for DNA and tRNA binding, respectively. Ce-C-Alac specifically recognized the conserved invariant base G18 in the D-loop of tRNAAla through a highly conserved lysine residue, K934. Despite bearing little resemblance to other C-Ala domains, C. elegans mitochondrial C-Ala robustly bound both tRNAAla and DNA and maintained targeting specificity for the D-loop of its cognate tRNA. This study uncovers the underlying mechanism of how C. elegans C-Ala specifically targets the D-loop of tRNAAla.
Assuntos
Alanina-tRNA Ligase , Caenorhabditis elegans , Motivos de Nucleotídeos , RNA de Transferência de Alanina , Animais , Alanina-tRNA Ligase/química , Alanina-tRNA Ligase/metabolismo , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Sequência Conservada , Citoplasma/enzimologia , DNA/química , DNA/metabolismo , Ligantes , Lisina/metabolismo , Mitocôndrias/enzimologia , Domínios Proteicos , RNA de Transferência de Alanina/química , RNA de Transferência de Alanina/metabolismo , Especificidade por Substrato , Conformação de Ácido NucleicoRESUMO
The unique interaction between fluorine atoms has been exploited to alter protein structures and to develop synthetic and analytical applications. To expand such fluorous interaction for novel applications, polyproline peptides represent an excellent molecular nanoscaffold for controlling the presentation of perfluoroalkyl groups on their unique secondary structure. We develop approaches to synthesis fluorinated peptides to systematically investigate how the number, location and types of the fluorous groups on polyproline affect the conformation by monitoring the transition between the two major polyproline structures PPI and PPII. This work provides valuable information on how fluorous interaction affects the peptide structure and also benefits the design of functional fluorous molecules.
Assuntos
Desenho de Fármacos , Peptídeos/síntese química , Halogenação , Estrutura Molecular , Peptídeos/químicaRESUMO
A polyproline fragment containing a catalytic dyad of His-His or Ser-His was coupled with a self-assembling peptide MAX1 to design new hydrolases (H2H5 and H2S5) for catalyzing ester hydrolysis. Circular dichroism measurements indicated that the peptides change their conformation from random coils to ß-sheets when pH increases from 5 to 10. IR spectra also displayed the vibration modes corresponding to their ß-structures at pH 9.0. Transmission electron microscopy (TEM) and atomic force microscopy (AFM) measurements showed that in solution, the designed peptides self-assemble into network fibrils having a significantly increased catalytic efficiency on ester hydrolysis. On p-nitrophenyl acetate (p-NPA) substrate, the designed peptides exhibit high catalytic efficiency at pH 9.0 (kcat/KM = 12.1 M-1 s-1 for H2H5, 13.3 M-1 s-1 for H2S5), and their efficiency is even better at pH 10.0 (kcat/KM = 24.3 M-1 s-1 for H2H5, 99.4 M-1 s-1 for H2S5). Additionally, H2H5 and H2S5 also display good activity on catalyzing the hydrolysis of p-nitrophenyl-(2-phenyl)-propanoate (p-NPP) and p-nitrophenyl methoxyacetate (p-NPMA). Combining the polyproline-based catalytic scaffold with a self-assembling peptide generates an efficient hydrolase, providing a new design for effective artificial enzymes.
Assuntos
Hidrolases , Peptídeos , Catálise , Dicroísmo Circular , Fragmentos de PeptídeosRESUMO
Leucettamine B is a natural product found in marine sponge Leucetta microraphis. Several of analogs of its family, such as aplysinopsine and clathridine, are medicinally active molecules which have applications in many pharmaceuticals and healthcare products; however, thus far, leucettamine B has not been studied. In this report, we describe the synthesis of a new class of analogs of leucettamine B obtained by Knoevenagel condensation using a microwave reactor. The 25 newly synthesized compounds were tested against MDA-MB-468, SW480, and Mahlavu cell lines for anticancer activity. Among them, the carborane-based compound (Z)-5-(benzo[d][1,3]dioxol-5-ylmethylene)-3-(1-closo-carboranyl)-2-thioxo -thiazolidin-4-one (49) and (Z)-5-(benzo[d][1,3]dioxol-5-ylmethylene)-3-(2-(pyrrolidin-1-yl)ethyl)-2-thioxothiazolidin-4-one (31) derivatives were found to have the most potential for use against tumor cells. The carborane derivative 49 had the lowest IC50 value against the SW480 cell line (4.7 µM) and the Mahlavu (6.6 µM) cell line. Furthermore, compound 31 also had a low IC50 value against SW480 (7.5 µM). Our research shows that leucettamine B analogs might have potential for use in cancer chemotherapy.
Assuntos
Antineoplásicos/farmacologia , Boranos/farmacologia , Desenho de Fármacos , Imidazóis/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Boranos/síntese química , Boranos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Imidazóis/síntese química , Imidazóis/química , Estrutura Molecular , Relação Estrutura-Atividade , Células VeroRESUMO
Polyproline is a unique type of peptide that has a stable, robust, and well-defined helical structure in an aqueous environment. These features have allowed polyproline to be used as a nanosized scaffold for applications in chemical biology and related fields. To understand its structural properties and to expand the applications, this secondary structure was tested systematically by stapling the peptide at different locations with staples of various lengths. Using the efficient copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC), we successfully prepared stapled polyproline and investigated the impact of this peptide macrocyclization through circular dichroism analysis. Whereas the stapling seems to have no significant effect on polyproline helixâ II (PPII) conformation in water, the location and the length of the staple affect the transformation of conformation in n-propanol. These results provide valuable information for future research using peptide stapling to manipulate polyproline conformation for various applications.
Assuntos
Alcinos/química , Azidas/química , Cobre/química , Peptídeos/química , Catálise , Reação de Cicloadição , Estrutura MolecularRESUMO
Numerous reports have shown that conjugated benzimidazole derivatives possess various kinds of biological activities, including anticancer properties. In this report, we designed and synthesized 24 new molecules comprising a benzimidazole ring, arene, and alkyl chain-bearing cyclic moieties. The results showed that the N-substituted benzimidazole derivatives bearing an alkyl chain and a nitrogen-containing 5- or 6-membered ring enhanced the cytotoxic effects on human breast adenocarcinoma (MCF-7) and human ovarian carcinoma (OVCAR-3) cell lines. Among the 24 synthesized compounds, (2E)-1-(1-(3-morpholinopropyl)-1H-benzimidazol-2 -yl)-3-phenyl-2-propen-1-one) (23a) reduced the proliferation of MCF-7 and OVCAR-3 cell lines demonstrating superior outcomes to those of cisplatin.
Assuntos
Benzimidazóis/farmacologia , Neoplasias da Mama/tratamento farmacológico , Chalconas/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Apoptose/efeitos dos fármacos , Benzimidazóis/síntese química , Benzimidazóis/química , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Chalconas/síntese química , Chalconas/química , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Células MCF-7 , Estrutura Molecular , Neoplasias Ovarianas/patologia , Relação Estrutura-AtividadeRESUMO
Collagen-related materials have many potential biomedical applications because of their high biocompatibility and biodegradability. Designed collagen-mimetic peptides (CMPs) could self-assemble into supramolecular structures via a variety of interactions. In particular, metal-ligand interactions can induce microscale sizes of collagen assemblies. Our previous study also successfully applied metal-histidine coordination method to promote the self-assembly of CMPs into micrometers of constructs. In an effort to broaden the metal-induced strategies on assembling designed CMPs and explore the new insights into their assembly process, herein we designed and synthesized a series of short CMPs with one or more histidine residues incorporated into the peptides and used Zn(II) to induce the formation of collagen assembled microstructures. By altering the location and the number of histidine residues, we found that the assembly rate was significantly affected as well as the morphology of the assembled architectures. The CMPs containing terminal histidine residues were found to assemble into less ordered granulated and spherical microstructures while that with only one single histidine in its middle site could form pinwheel or floret-like constructs, showing that we could modulate the morphology of collagen assemblies by changing the location and number of Zn(II)-His coordination sites. Additionally, these CMPs also exhibited catalytic activities on ester hydrolysis in the presence of Zn(II) ions, which suggested that Zn(II)-CMP assemblies could be potentially applied to the development of artificial enzymes.
Assuntos
Colágeno/química , Histidina/química , Compostos Organometálicos/química , Multimerização Proteica , Materiais Biomiméticos/química , Catálise , Hidrólise , Peptídeos/química , PolimerizaçãoRESUMO
Most of natural collagens are heterotrimers composed of two (AAB) or three (ABC) different peptide chains, and thus heterotrimeric constructs are preferable to mimic natural collagens. Exploring the forces to assemble synthetic collagen-mimetic peptides (CMPs) into heterotrimers has been an attractive topic in preparing collagen-related biomaterials. Here we designed and synthesized two cationic CMPs (CR and CK) in which multiple Arg or Lys residues are installed in their C-terminal region, and one aromatic CMP (CF) whose C-terminal end contains multiple Phe residues. Circular dichroism and NMR spectroscopy showed that AAB-type heterotrimers could form in both CR-CF and CK-CF mixtures, suggesting that the C-terminal cation-π interactions between cationic and aromatic residues could serve as a nucleation force and substantially promote the folding of heterotrimers. In particular, only one major heterotrimeric fold was found in each mixture. For CR-CF mixtures, either the heterotrimer with two CR chains and one CF chain or that with one CR chain and two CF chains could form, depending on the molar ratios of CR to CF in solution. By contrast, in CK-CF mixtures only the heterotrimer consisting of two CK chains and one CF chain was found in solution even increasing the ratio of CF, implying that the heterotrimer composed of one CK chain and two CF chains is highly unstable. Additionally, differential scanning calorimetry analysis showed that the folding of these heterotrimers is governed by entropic effects. Together, our results provide a new design to prepare AAB-type collagen heterotrimers and reveal new insights into their folding thermodynamics.
Assuntos
Materiais Biomiméticos/química , Cátions/química , Colágeno/química , Peptídeos/química , Varredura Diferencial de Calorimetria , Dicroísmo Circular , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Multimerização Proteica , Estrutura Secundária de Proteína , TermodinâmicaRESUMO
Functionalized collagen-mimetic peptides (CMPs) have been widely used in the preparation of collagen-related biomaterials. Among the reported results, the induced noncovalent interactions between the implanted functional groups or moieties were frequently the key elements to promote the self-assembly of small CMPs. In this work, we designed and synthesized a series of glycosylated CMPs in which 4-O-[ß-D-galactopyranosyl]-(2S,4R)-4-hydroxyproline (Hyp(Gal)) was incorporated to explore the effects of glycosylation on the stability and assembly of collagen triple helices. Circular dichroism measurements showed that glycosylation of hydroxyproline slightly destabilized the collagen triple helices, but did not reduce their refolding rate. Compared to non-glycosylated CMPs, the incorporation of Hyp(Gal) speeded up the assembly of CMPs, indicating that this modification could assist the self-assembly of CMPs into higher-order structures, such as fibrils. O-Galactosylation of hydroxyproline imposes contrary effects on the triple helix stability and the self-assembly rate of collagen triple helices, exhibiting a piece of important and useful information for designing collagen-related biomaterials. Our finding also suggests that instead of stabilizing the triple helical conformation of CMPs, installing additional forces between CMPs could be a crucial factor to promote the assembly of CMPs into large-scale constructs.
Assuntos
Colágeno/química , Peptídeos/química , Biomimética , Dicroísmo Circular , Colágenos Fibrilares/química , Colágenos Fibrilares/efeitos dos fármacos , Glicosilação/efeitos dos fármacos , Hidroxiprolina/química , Hidroxiprolina/farmacologia , Conformação Proteica em alfa-Hélice/efeitos dos fármacosRESUMO
A series of new conjugated compounds with a -SCH2- linkage were synthesized by chemical methods from imidazole and coumarin derivatives. The experimental results indicate that of the twenty newly synthesized imidazole-coumarin conjugates, three of them exhibited appealing EC50 values (5.1-8.4 µM) and selective indices >20 against hepatitis C virus. Their potency and selectivity were increased substantially by modification of their structure with two factors: imidazole nucleus with a hydrogen atom at the N(1) position and coumarin nucleus with a substituent, such as Cl, F, Br, Me, and OMe. These guidelines provide valuable information for further development of conjugated compounds as anti-viral agents.
Assuntos
Cumarínicos/síntese química , Hepacivirus/efeitos dos fármacos , Imidazóis/síntese química , Antivirais/síntese química , Antivirais/química , Antivirais/farmacologia , Cumarínicos/química , Cumarínicos/farmacologia , Hepacivirus/fisiologia , Imidazóis/química , Imidazóis/farmacologia , Estrutura Molecular , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacosRESUMO
Human Pin1 is involved in cancer developments and has been a pharmaceutical target. Thus, finding a high-affinity inhibitor of Pin1 has become an attractive topic. The WW domain of human Pin1 can recognize the phosphoserine/phosphothreonine-proline (pS/pT-P) motifs, while its PPIase domain catalyzes the cis/trans isomerization of prolyl bonds to regulate the cell cycle. Here we incorporated a series of 4-substituted proline derivatives into the phosphopeptides and investigated their affinities for the WW domain of Pin1 to develop better inhibitors of Pin1. On the basis of the ligand Myt1-T412 [PPA(pT)PP], we synthesized several phosphopeptides in which the proline residue in the pT-P motif was replaced with various 4-substituted proline derivatives. Isothermal titration calorimetry and fluorescence anisotropy analyses show that the replacement of proline with (2S,4R)-4-fluoroproline increases the binding affinity of the peptide. Circular dichroism measurements suggest that a more PPII-like structure of phosphopeptides makes them bind to the WW domain more tightly. Chemical shift perturbation experiments also indicate that (2S,4R)-4-fluoroproline interacts with Trp34 of the WW domain in the binding site. Results of molecular modeling further suggested that a strong C-H···π interaction induced by (2S,4R)-4-fluoroproline is important in enhancing the affinity of the peptide for the WW domain. The results of this study provide new valuable information for designing and developing effective inhibitors of human Pin1.
Assuntos
Peptidilprolil Isomerase/metabolismo , Fosfopeptídeos/química , Fosfopeptídeos/metabolismo , Prolina/análogos & derivados , Sequência de Aminoácidos , Sítios de Ligação , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Peptidilprolil Isomerase de Interação com NIMA , Peptidilprolil Isomerase/química , Prolina/química , Prolina/metabolismo , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Especificidade por SubstratoRESUMO
HP36, the helical subdomain of villin headpiece, contains a hydrophobic core composed of three phenylalanine residues (Phe47, Phe51, and Phe58). Hydrophobic effects and electrostatic interactions were shown to be the critical factors in stabilizing this core and the global structure. To assess the interactions among Phe47, Phe51, and Phe58 residues and investigate how they affect the folding stability, we implanted 4-fluorophenylalanine (Z) and 4-methylphenylalanine (X) into the hydrophobic core of HP36. We chemically synthesized HP36 and its seven variants including four single mutants whose Phe51 or Phe58 was replaced with Z or X, and three double mutants whose Phe51 and Phe58 were both substituted. Circular dichroism and nuclear magnetic resonance measurements show that the variants exhibit a native HP36 like fold, of which F51Z and three double mutants are more stable than the wild type. Molecular modeling provided detailed interaction energy within the phenylalanine residues, revealing that electrostatic interactions dominate the stability modulation upon the introduction of 4-fluorophenylalanine and 4-methylphenylalanine. Our results show that these two non-natural amino acids can successfully tune the interactions in a relatively compact hydrophobic core and the folding stability without inducing dramatic steric effects. Such an approach may be applied to other folded motifs or proteins.
Assuntos
Proteínas dos Microfilamentos/química , Fenilalanina/análogos & derivados , p-Fluorfenilalanina/química , Interações Hidrofóbicas e Hidrofílicas , Fenilalanina/química , Dobramento de Proteína , Estrutura Terciária de Proteína , Eletricidade Estática , TermodinâmicaRESUMO
A new "single-flask" method was developed for the synthesis of imidazolidines and pyrrolidines with high stereoselectivity. First, a Schiff base was arylated with an aryne. Second, an intramolecular proton transfer took place from the methylene position to the anionic aryne ring. Third, the resultant ylide reacted with a second equivalent of the same Schiff base inâ situ or an electron-deficient alkene through a (3+2) cycloaddition. These sequential tandem 1,2-addition/(3+2) cycloaddition reactions led to the desired heterocycles in 63-88% yields.
RESUMO
The pyrrolidine side chain makes proline play a unique role in protein structure and function. The C(γ) ring pucker preference and the cis-trans peptidyl bond ratio can be mediated via stereoelectronic effects. Here we used a compact triple-stranded antiparallel ß-sheet protein, the human Pin1 WW domain, to study the consequences of implanting a preorganized C(γ) ring pucker on protein structure and function. The conserved Pro37 is a key residue involved in one hydrophobic core, plays an important role in the WW domain, and adopts a C(γ) -endo ring pucker in the native structure. Pro37 was replaced with C(γ) -exo biased pucker derivatives: (2S,4R)-4-hydroxyproline (4R-Hyp), (2S,4R)-4-fluoroproline (4R-Flp), (2S,4R)-4-methoxyproline (4R-Mop), and C(γ) -endo biased pucker derivatives: (2S,4S)-4-hydroxyproline (4S-hyp), (2S,4S)-4-fluoroproline (4S-flp), (2S,4S)-4-methoxyproline (4S-mop) to examine how a preorganized pucker affects the folding stability and ligand-binding affinity. Circular dichroism measurements indicate that among the variants, only the one with 4S-flp substitution (P37flp) is more stable than the wild type, suggesting that the stabilization effects originated from preorganization of the backbone conformation and the hydrophobicity of C - F group. Analysis of ligand-binding affinity using isothermal titration calorimetry revealed that only P37flp has a stronger ligand affinity than the wild type, showing that 4S-flp can stabilize the WW domain and increase its ligand affinity. Together we have used 4-substituted proline derivatives and the WW domain to demonstrate that proline ring puckering can be a key factor in determining the folding stability of a protein but the choice of the derivative groups is also critical.
Assuntos
Modelos Moleculares , Peptidilprolil Isomerase/química , Prolina/química , Dobramento de Proteína , Sequência de Aminoácidos/genética , Calorimetria , Dicroísmo Circular , Humanos , Espectroscopia de Ressonância Magnética , Peptidilprolil Isomerase de Interação com NIMA , Peptidilprolil Isomerase/metabolismo , Ligação Proteica , Conformação Proteica , Estabilidade Proteica , Espectrometria de FluorescênciaRESUMO
Many interests have been focused on prolyl cis-trans isomerization which is related to protein folding and isomer-specific biochemical recognition. Since polyproline can adopt either type I (PPI) helices with all cis amide bonds or type II (PPII) helices with all trans amide bonds, it has been a valuable model to study the prolyl isomerization. Recent studies have shown that stereoelectronic effects govern the stability of PPII structure and the rate of PPII â PPI conversion. To further explore the terminal stereoelectronic effects on polyproline conformation, herein we synthesized a series of host-guest peptides in which (2S,4S)-4-fluoroproline (flp) or (2S,4R)-4-fluoroproline (Flp) residues are incorporated into the C- or N-terminal end of a peptide and studied the thermodynamic and kinetic consequences on polyproline conformation. Circular dichroism measurements revealed that inserting 4-fluoroproline residues into the C terminus of a polyproline peptide induces a great stereoelectronic effect on PPII stability and PPII â PPI conversion rates. From the C terminus, a (Flp)3 triplet stabilizes PPII structure and increases the transition barrier of PPII â PPI conversion by 1.53 kJ mol⻹ while a (flp)3 triplet destabilizes PPII conformation and reduce the PPII â PPI transition barrier by 4.61 kJ mol⻹. In contrast, the 4-fluoroproline substitutions at the N terminus do not exhibit distinct stereoelectronic effects on PPII stability and PPII â PPI conversion rates. Our data demonstrate that the C-terminal stereoelectronic effects have a more dramatic impact on PPII stability and PPII â PPI conversion kinetics.
Assuntos
Modelos Moleculares , Oligopeptídeos/química , Peptídeos/química , Prolina/análogos & derivados , Sequência de Aminoácidos , Dicroísmo Circular , Flúor/química , Halogenação , Cinética , Prolina/química , Conformação Proteica , Dobramento de Proteína , Estabilidade Proteica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Estereoisomerismo , TermodinâmicaRESUMO
(2R)-4-thiaproline (Thp) is an analog of proline, replacing Cγ in the pyrrolidine ring with sulfur. Its thiazolidine ring easily interconverts between endo and exo puckers due to a small energy barrier, which leads to destabilize polyproline helices. Collagen, composed of three polyproline II helices, mainly consists of X-Y-Gly triplets, where X is often proline and Y is frequently (2S,4R)-hydroxyproline. In this study, we incorporated Thp into either position-X or position-Y to investigate the consequences of such a replacement on the triple helix. Circular dichroism and differential scanning calorimetry analyses showed that the Thp-containing collagen-mimetic peptides (CMPs) can fold into stable triple helices, in which the substitution at position-Y exhibits a larger destabilization effect. Additionally, we also prepared the derivative peptides by oxidizing Thp in the peptide to N-formyl-cysteine or S,S-dioxide Thp. The results showed that the oxidized derivatives at position-X only slightly affect collagen stability, but those at position-Y induce a large destabilization effect. The consequences of incorporating Thp and its oxidized derivatives into CMPs are position dependent. Computational results suggested that the ease of interconversion between exo and endo puckers for Thp and the twist conformation of S,S-dioxide Thp may cause the destabilization effect at position-Y. We have revealed new insights into the impacts of Thp and its oxidized derivatives on collagen and demonstrated that Thp can be used to design collagen-related biomaterials.
Assuntos
Biomimética , Colágeno , Tiazolidinas , Tiazolidinas/química , Colágeno/síntese química , Colágeno/química , Estabilidade Proteica , Termodinâmica , CinéticaRESUMO
Collagen is an important and widely used biomaterial and therapeutic. The construction of large-scale collagen structures via the self-assembly of small collagen-related peptides has been extensively studied in the past decade. Here, we report a highly effective and simple means to assemble small synthetic collagen-related peptides into various higher-order structures by utilizing metal-histidine coordination. In this work, two short collagen-related peptides in which histidine residues were incorporated as metal binding sites were designed and chemically synthesized: HG(PPG)(9)GH (X9) and HG(PPG)(4)(PHG)(PPG)(4)GH (PHG). Circular dichroism measurements indicated that these two peptides form only marginally stable collagen triple helices but that their stability can be increased upon the addition of metal ions. Dynamic light scattering analyses, turbidity measurements, TEM, and SEM results demonstrated the metal ion-dependent self-assembly of X9 and PHG into supramolecular structures ranging from various nanofibrils to microscale spherical, laminated, and granulated assemblies. The topology and size of these higher-order structures depends both on the metal ion identity and the location of the binding sites. Most intriguingly, the assembled fibrils show similar D-periodicity to that of natural collagen. Our results demonstrate that metal-histidine coordination can serve as an effective force to induce the self-assembly of unstable collagen-related peptides into higher-order structures.
Assuntos
Colágeno/química , Histidina/química , Metais/química , Peptídeos/química , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Nanofibras/química , Nanofibras/ultraestruturaRESUMO
Introduction of a cationic residue at the N-terminus and an aromatic residue at the C-terminus of a collagen-related peptide can generate favorable cation-π interactions between the termini of collagen triple helices. The experimental results indicate that such cation-π interactions can promote the self-assembly of collagen triple helices into a higher-order structure in a head-to-tail manner. Our current work shows that cation-π interactions can serve as an effective force in preparing collagen-related biomaterials.
Assuntos
Materiais Biomiméticos/química , Colágenos Fibrilares/química , Peptídeos/química , Animais , Humanos , Cinética , Oligopeptídeos/química , Concentração Osmolar , Conformação Proteica , Estrutura Secundária de Proteína , Temperatura de TransiçãoRESUMO
Cation-π interactions are found to be an important noncovalent force in proteins. Collagen is a right-handed triple helix composed of three left-handed PPII helices, in which (X-Y-Gly) repeats dominate in the sequence. Molecular modeling indicates that cation-π interactions could be formed between the X and Y positions in adjacent collagen strands. Here, we used a host-guest peptide system: (Pro-Hyp-Gly)(3)-(Pro-Y-Gly-X-Hyp-Gly)-(Pro-Hyp-Gly)(3), where X is an aromatic residue and Y is a cationic residue, to study the cation-π interaction in the collagen triple helix. Circular dichroism (CD) measurements and Tm data analysis show that the cation-π interactions involving Arg have a larger contribution to the conformational stability than do those involving Lys, and Trp forms a weaker cation-π interaction with cationic residues than expected as a result of steric effects. The results also show that the formation of cation-π interactions between Arg and Phe depends on their relative positions in the strand. Moreover, the fluorinated and methylated Phe substitutions show that an electron-withdrawing or electron-donating substituent on the aromatic ring can modulate its π-electron density and the cation-π interaction in collagen. Our data demonstrate that the cation-π interaction could play an important role in stabilizing the collagen triple helix.
Assuntos
Colágeno/química , Sequência de Aminoácidos , Aminoácidos Aromáticos/química , Aminoácidos Aromáticos/metabolismo , Cátions/química , Cátions/metabolismo , Colágeno/metabolismo , Transporte de Elétrons , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Estabilidade Proteica , Estrutura Secundária de ProteínaRESUMO
This manuscript describes the interaction of low-molecular-weight DNICs with short peptides designed to explore the stability and structure of DNIC-peptide/RRE-peptide constructs. Although characterization of protein-bound and low-molecular-weight DNICs is possible via EPR, XAS, and NRVS, this study demonstrates that the combination of aqueous IR ν(NO) and UV-vis spectra can serve as an efficient tool to characterize and discriminate peptide-bound DNICs and RREs. The de novo chelate-cysteine-containing peptides KC(A)(n)CK-bound (n = 1-4) dinitrosyliron complexes KC(A)(n)CK-DNIC (CnA-DNIC) and monodentate-cysteine-containing peptides KCAAK-/KCAAHK-bound Roussin's red esters (RREs) KCAAK-RRE/KCAAHK-RRE were synthesized and characterized by aqueous IR, UV-vis, EPR, CD, XAS, and ESI-MS. In contrast to the inertness of chelate-cysteine-containing peptide-bound DNICs toward KCAAK/KCAAHK, transformation of KCAAK-RRE/KCAAHK-RRE into CnA-DNIC triggered by CnA and reversible transformation between CnA-DNIC and CnA-RRE via {Fe(NO)(2)}(9)-{Fe(NO)(2)}(10) reduced-form peptide-bound RREs demonstrate that the {Fe(NO)(2)}(9) motif displays a preference for chelate-cysteine-containing peptides over monodentate-cysteine-containing peptides. Also, this study may signify that nitrosylation of [Fe-S] proteins generating protein-bound RREs, reduced protein-bound RREs, or protein-bound DNICs are modulated by both the oxidation state of iron and the chelating effect of the bound proteins of [Fe-S] clusters.