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PURPOSE OF REVIEW: MicroRNAs (miRNAs) are emerging rapidly as a novel class of biomarkers of major organ disorders, including kidney diseases. However, current PCR-based detection methods are not amenable to development for high-throughput, cost-effective miRNA biomarker quantification. RECENT FINDINGS: MiRNA biomarkers show significant promise for diagnosis and prognosis of kidney diseases, including diabetic kidney disease, acute kidney injury, IgA nephropathy and delayed graft function following kidney transplantation. A variety of novel methods to detect miRNAs in liquid biopsies including urine, plasma and serum are being developed. As miRNAs are functional transcripts that regulate the expression of many protein coding genes, differences in miRNA profiles in disease also offer clues to underlying disease mechanisms. SUMMARY: Recent findings highlight the potential of miRNAs as biomarkers to detect and predict progression of kidney diseases. Developing in parallel, novel methods for miRNA detection will facilitate the integration of these biomarkers into rapid routine clinical testing and existing care pathways. Validated kidney disease biomarkers also hold promise to identify novel therapeutic tools and targets. VIDEO ABSTRACT: http://links.lww.com/CONH/A43.
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Nefropatias Diabéticas , MicroRNAs , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Rim/metabolismo , Nefropatias Diabéticas/metabolismo , Biomarcadores/metabolismo , Biópsia LíquidaRESUMO
The human CD8+ T cell clone 6C5 has previously been shown to recognize the tert-butyl-modified Bax161-170 peptide LLSY(3-tBu)FGTPT presented by HLA-A*02:01. This nonnatural epitope was likely created as a by-product of fluorenylmethoxycarbonyl protecting group peptide synthesis and bound poorly to HLA-A*02:01. In this study, we used a systematic approach to identify and characterize natural ligands for the 6C5 TCR. Functional analyses revealed that 6C5 T cells only recognized the LLSYFGTPT peptide when tBu was added to the tyrosine residue and did not recognize the LLSYFGTPT peptide modified with larger (di-tBu) or smaller chemical groups (Me). Combinatorial peptide library screening further showed that 6C5 T cells recognized a series of self-derived peptides with dissimilar amino acid sequences to LLSY(3-tBu)FGTPT. Structural studies of LLSY(3-tBu)FGTPT and two other activating nonamers (IIGWMWIPV and LLGWVFAQV) in complex with HLA-A*02:01 demonstrated similar overall peptide conformations and highlighted the importance of the position (P) 4 residue for T cell recognition, particularly the capacity of the bulky amino acid tryptophan to substitute for the tBu-modified tyrosine residue in conjunction with other changes at P5 and P6. Collectively, these results indicated that chemical modifications directly altered the immunogenicity of a synthetic peptide via molecular mimicry, leading to the inadvertent activation of a T cell clone with unexpected and potentially autoreactive specificities.
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Linfócitos T CD8-Positivos/imunologia , Ativação Linfocitária/imunologia , Fragmentos de Peptídeos/imunologia , Peptídeos/imunologia , Sequência de Aminoácidos , Apresentação de Antígeno/imunologia , Células Cultivadas , Epitopos de Linfócito T/imunologia , Humanos , Ligantes , Biblioteca de PeptídeosRESUMO
Altered serum and plasma microRNA (miRNA) expression profiles have been observed in numerous human diseases, with a number of studies describing circulating miRNA biomarkers for cancer diagnosis, prognosis and response to treatment, and recruitment to clinical trials for miRNA-based drug therapy already underway. Electrochemical detection of biomarkers in urine has several significant advantages over circulating biomarker analysis including safety, cost, speed and ease of conversion to the point of care environment. Consequently, much current research is underway to identify urinary miRNA biomarkers for a variety of pathologies including prostate and bladder malignancies, and renal disorders. We describe here a robust method capable of electrochemical detection of human urinary miRNAs at femtomolar concentrations using a complementary DNA-modified glassy carbon electrode. A miR-21-specific DNA hybridisation probe was immobilised onto a glassy carbon electrode modified by sulfonic acid deposition and subsequent chlorination. In our pilot system, the presence of synthetic mature miR-21 oligonucleotides increased resistance at the probe surface to electron transfer from the ferricyanide/ferrocyanide electrolyte. Response was linear for 10 nM-10 fM miR-21, with a limit of detection of 20 fM, and detection discriminated between miR-21, three point-mutated miR-21 sequences, and miR-16. We then demonstrated similar sensitivity and reproducibility of miR-21 detection in urine samples from 5 human control subjects. Our protocol provides a platform for future high-throughput screening of miRNA biomarkers in liquid biopsies.
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We set out to clone Bax-specific CD8+ T-cells from peripheral blood samples of primary chronic lymphocytic leukemia patients. A number of clones were generated using a Bax peptide pool and their T-cell epitope was mapped to two peptides sharing a common 9-aa sequence (LLSYFGTPT), restricted by HLA-A*0201. However, when these T-cell clones were tested against highly purified syntheses (>95%) of the same peptide sequence, there was no functional response. Subsequent mass spectrometric analysis and HPLC fractionation suggested that the active component in the original crude peptide preparations (77% pure) was a peptide with a tert-butyl (tBu) modification of the tyrosine residue. This was confirmed by modification of the inactive wild type (wt) sequence to generate functionally active peptides. Computer modeling of peptide:HLA-A*0201 structures predicted that the tBu modification would not affect interactions between peptide residues and the HLA binding site. However these models did predict that the tBu modification of tyrosine would result in an extension of the side chain out of the peptide-binding groove up towards the TCR. This modified product formed <1% of the original P603 crude peptide preparation and <0.05% of the original 23 peptide mixture used for T-cell stimulation. The data presented here, illustrates the potential for chemical modifications to change the immunogenicity of synthetic peptides, and highlights the exquisite capacity of TCR to discriminate between structurally similar peptide sequences. Furthermore this study highlights potential pitfalls associated with the use of synthetic peptides for the monitoring and modulating of human immune responses. This article is protected by copyright. All rights reserved.
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Two compounds, (S)-8-{[(tert-butyl-dimethyl-sil-yl)-oxy]meth-yl}-1-[(2,2,4,6,7-penta-methyl-2,3-di-hydro-benzo-furan-5-yl)sulfon-yl]-1,3,4,6,7,8-hexa-hydro-2H-pyrimido[1,2-a]pyrimidin-1-ium tri-fluoro-methane-sulfonate, C27H46N3O4SSi+·CF3O3S-, (1) and (S)-8-(iodo-meth-yl)-1-tosyl-1,3,4,6,7,8-hexa-hydro-2H-pyrimido[1,2-a]pyrimidin-1-ium iodide, C15H21IN3O2S+·I-, (2), have been synthesized and characterized. They are bicyclic guanidinium salts and were synthesized from N-(tert-but-oxy-carbon-yl)-l-me-thio-nine (Boc-l-Met-OH). The guanidine is protected by a 2,2,4,6,7-penta-methyl-dihydro-benzo-furan-5-sulfonyl (Pbf, 1) or a tosyl (2) group. In the crystals of both compounds, the guanidinium group is almost planar and the N-H forms an intra-molecular hydrogen bond in a six-membered ring to the oxygen atom of the sulfonamide protecting group.
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Severe bacterial or viral infections can induce a state of immune hyperactivation that can culminate in a potentially lethal cytokine storm. The classic example is toxic shock syndrome, a life-threatening complication of Staphylococcus aureus or Streptococcus pyogenes infection, which is driven by potent toxins known as superantigens (SAgs). SAgs are thought to promote immune evasion via the promiscuous activation of T cells, which subsequently become hyporesponsive, and act by cross-linking major histocompatibility complex class II molecules on antigen-presenting cells to particular ß-chain variable (TRBV) regions of αß T cell receptors (TCRs). Although some of these interactions have been defined previously, our knowledge of SAg-responsive TRBV regions is incomplete. In this study, we found that CD4+ and CD8+ T cells expressing TRBV12-3/12-4+ TCRs were highly responsive to streptococcal pyrogenic exotoxin C (SpeC) and toxic shock syndrome toxin-1 (TSST-1). In particular, SpeC and TSST-1 specifically induced effector cytokine production and the upregulation of multiple coinhibitory receptors among TRBV12-3/12-4+ CD4+ and CD8+ memory T cells, and importantly, these biological responses were dependent on human leukocyte antigen (HLA)-DR. Collectively, these data provided evidence of functionally determinative and therapeutically relevant interactions between SpeC and TSST-1 and CD4+ and CD8+ memory T cells expressing TRBV12-3/12-4+ TCRs, mediated via HLA-DR.
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Ativação Linfocitária , Células T de Memória , Superantígenos , Humanos , Linfócitos T CD8-Positivos/imunologia , Células T de Memória/imunologia , Receptores de Antígenos de Linfócitos T , Superantígenos/imunologiaRESUMO
Aberrant expression of the human hyaluronan synthase 2 (HAS2) gene has been implicated in the pathology of malignancy, pulmonary arterial hypertension, osteoarthritis, asthma, thyroid dysfunction, and large organ fibrosis. Renal fibrosis is associated with increased cortical synthesis of hyaluronan (HA), an extracellular matrix glycosaminoglycan, and we have shown that HA is a correlate of interstitial fibrosis in vivo. Our previous in vitro data have suggested that both HAS2 transcriptional induction and subsequent HAS2-driven HA synthesis may contribute to kidney fibrosis via phenotypic modulation of the renal proximal tubular epithelial cell (PTC). Post-transcriptional regulation of HAS2 mRNA synthesis by the natural antisense RNA HAS2-AS1 has recently been described in osteosarcoma cells, but the antisense transcript was not detected in kidney. In this study, PTC stimulation with IL-1ß or TGF-ß1 induced coordinated temporal profiles of HAS2-AS1 and HAS2 transcription. Constitutive activity of the putative HAS2-AS1 promoter was demonstrated, and transcription factor-binding sequence motifs were identified. Knockdown of Sp1/Sp3 expression by siRNA blunted IL-1ß induction of both HAS2-AS1 and HAS2, and Smad2/Smad3 knockdown similarly attenuated TGF-ß1 stimulation. Inhibition of IL-1ß-stimulated HAS2-AS1 RNA induction using HAS2-AS1-specific siRNAs also suppressed up-regulation of HAS2 mRNA transcription. The thermodynamic feasibility of HAS2-AS1/HAS2 heterodimer formation was demonstrated in silico, and locus-specific cytoplasmic double-stranded RNA was detected in vitro. In summary, our data show that transcriptional induction of HAS2-AS1 and HAS2 occurs simultaneously in PTCs and suggest that transcription of the antisense RNA stabilizes or augments HAS2 mRNA expression in these cells via RNA/mRNA heteroduplex formation.
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Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Glucuronosiltransferase/biossíntese , Túbulos Renais Proximais/metabolismo , RNA Antissenso/biossíntese , Transcrição Gênica , Linhagem Celular Tumoral , Células Epiteliais/patologia , Fibrose , Técnicas de Silenciamento de Genes , Glucuronosiltransferase/genética , Humanos , Hialuronan Sintases , Ácido Hialurônico/biossíntese , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/patologia , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Nefropatias/genética , Nefropatias/metabolismo , Nefropatias/patologia , Túbulos Renais Proximais/patologia , Ácidos Nucleicos Heteroduplexes/biossíntese , Ácidos Nucleicos Heteroduplexes/genética , RNA Antissenso/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Elementos de Resposta/genética , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína Smad3/genética , Proteína Smad3/metabolismo , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição Sp3/genética , Fator de Transcrição Sp3/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Peptide nucleic acids (PNAs, nucleic acid analogues with a peptide backbone rather than a phosphoribosyl backbone) have emerged as promising chemical agents in antigene or antisense therapeutics, as splicing modulators or in gene editing. Their main benefits, compared to DNA or RNA agents, are their biochemical stability and the lack of negative charges throughout the backbone, leading to negligible electrostatic interaction with the strand with which they are hybridizing. As a result, hybridization of PNA strands with DNA or RNA strands leads to higher binding energies and melting temperatures. A lack of natural transporters, however, necessitates the formation of PNA-containing chimeras or the formulation of nanoparticular cell delivery methods. Here, we set out to explore the progress made in using imaging agents based on PNAs in diagnostic applications and highlight selected developments and challenges.
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This paper describes a straightforward electrochemical method for rapid and robust urinary microRNA (miRNA) quantification using disposable biosensors that can discriminate between urine from diabetic kidney disease (DKD) patients and control subjects. Aberrant miRNA expression has been observed in several major human disorders, and we have identified a urinary miRNA signature for DKD. MiRNAs therefore have considerable promise as disease biomarkers, and techniques to quantify these transcripts from clinical samples have significant clinical and commercial potential. Current RT-qPCR-based methods require technical expertise, and more straightforward methods such as electrochemical detection offer attractive alternatives. We describe a method to detect urinary miRNAs using diazo sulfonamide-modified screen printed carbon electrode-based biosensors that is amenable to parallel analysis. These sensors showed a linear response to buffered miR-21, with a 17 fM limit of detection, and successfully discriminated between urine samples (n = 6) from DKD patients and unaffected control subjects (n = 6) by differential miR-192 detection. Our technique for quantitative miRNA detection in liquid biopsies has potential for development as a platform for non-invasive high-throughput screening and/or to complement existing diagnostic procedures in disorders such as DKD.
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We have explored a series of trisubstituted acridine-peptide conjugates for their ability to recognize and discriminate between DNA quadruplexes derived from the human telomere, and the c-kit and N-ras proto-oncogenes. Quadruplex affinity was measured as the peptide sequences were varied, together with their substitution position on the acridine, and the identity of the C-terminus (acid or amide). Surface plasmon resonance measurements revealed that all compounds bound to the human telomeric quadruplex with sub-micromolar affinity. Docking calculations from molecular modelling studies were used to model the effects of substituent orientation and peptide sequence. Modelling and experiment were in agreement that placement of the peptide over the face of the acridine is detrimental to binding affinity. The highest degrees of selectivity were observed towards the N-ras quadruplex by compounds capable of forming simultaneous contacts with their acridine and peptide moieties. The ligands that bound best displayed quadruplex affinities in the 1-5 nM range and at least 10-fold discrimination between the quadruplexes studied.
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Acridinas/química , Peptídeos/química , Biotinilação , DNA/química , Transferência Ressonante de Energia de Fluorescência , Quadruplex G , Humanos , Cinética , Ligantes , Modelos Químicos , Modelos Moleculares , Estrutura Terciária de Proteína , Ressonância de Plasmônio de Superfície , Telômero/ultraestrutura , Proteínas ras/metabolismoRESUMO
The 5-year survival rate of esophageal cancer is less than 10% in developing countries, where more than 90% of these cancers are esophageal squamous cell carcinomas (ESCC). Endoscopic screening is undertaken in high incidence areas. Biomarker analysis could reduce the subjectivity associated with histologic assessment of dysplasia and thus improve diagnostic accuracy. The aims of this study were therefore to identify biomarkers for esophageal squamous dysplasia and carcinoma. A publicly available dataset was used to identify genes with differential expression in ESCC compared with normal esophagus. Each gene was ranked by a support vector machine separation score. Expression profiles were examined, before validation by qPCR and IHC. We found that 800 genes were overexpressed in ESCC compared with normal esophagus (P < 10(-5)). Of the top 50 genes, 33 were expressed in ESCC epithelium and not in normal esophagus epithelium or stroma using the Protein Atlas website. These were taken to qPCR validation, and 20 genes were significantly overexpressed in ESCC compared with normal esophagus (P < 0.05). TNFAIP3 and CHN1 showed differential expression with IHC. TNFAIP3 expression increased gradually through normal esophagus, mild, moderate and severe dysplasia, and SCC (P < 0.0001). CHN1 staining was rarely present in the top third of normal esophagus epithelium and extended progressively towards the surface in mild, moderate, and severe dysplasia, and SCC (P < 0.0001). Two novel promising biomarkers for ESCC were identified, TNFAIP3 and CHN1. CHN1 and TNFAIP3 may improve diagnostic accuracy of screening methods for ESCC. Cancer Prev Res; 9(7); 558-66. ©2016 AACR.
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Biomarcadores Tumorais/análise , Carcinoma de Células Escamosas/diagnóstico , Quimerina 1/biossíntese , Neoplasias Esofágicas/diagnóstico , Lesões Pré-Cancerosas/diagnóstico , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/biossíntese , Carcinoma de Células Escamosas do Esôfago , Perfilação da Expressão Gênica , Humanos , TranscriptomaRESUMO
[structure: see text] N-Fmoc-L-p-azidotetrafluorophenylalanine was prepared from achiral starting materials using an acetamidomalonate synthesis and enzymatic resolution. A photoactive peptide containing this fluorinated residue could be assembled using solid-phase Fmoc chemistry.
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Azidas/síntese química , Peptídeos/química , Peptídeos/síntese química , Fenilalanina/análogos & derivados , Fenilalanina/síntese química , Sequência de Aminoácidos , Azidas/química , Espectrometria de Massas , Dados de Sequência Molecular , Fenilalanina/química , FotoquímicaRESUMO
A cyclic pseudo-peptide receptor for acetylcholine has been amplified and isolated from a dynamic combinatorial library by virtue of templated stabilization under thermodynamic control (see scheme, TFA=trifluoroacetic acid). This is a demonstration of significant molecular amplification in dynamic systems to evolve a novel receptor.
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Transforming growth factor-ß(1) (TGF-ß(1)) regulates cellular proliferation, differentiation, migration, and survival. The human TGF-ß(1) transcript is inherently poorly translated, and translational activation has been documented in relation to several stimuli. In this paper, we have sought to identify in cis regulatory elements within the TGF-ß(1) 5'Untranslated Region (5'UTR). In silico analysis predicted formation of stable secondary structure in a G/C-rich element between nucleotides +77 to +106, and demonstrated that this element is highly conserved across species. Circular dichroism spectroscopy confirmed the presence of secondary structure in this region. The proximal 5'UTR was inhibitory to translation in reporter gene experiments, and mutation of the secondary structure motif increased translational efficiency. Translational regulation of TGF-ß(1) mRNA is linked to altered binding of YB-1 protein to its 5'UTR. Immunoprecipitation-RT-qPCR demonstrated a high basal association of YB-1 with TGF-ß(1) mRNA. However, mutation of the secondary structure motif did not prevent interaction of YB-1 with the 5'UTR, suggesting that YB-1 binds to this region due to its G/C-rich composition, rather than a specific, sequence-dependent, binding site. These data identify a highly conserved element within the TGF-ß(1) 5'UTR that forms stable secondary structure, and is responsible for the inherent low translation efficiency of this cytokine.
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Regiões 5' não Traduzidas , Regulação da Expressão Gênica , Sequências Repetidas Invertidas , Biossíntese de Proteínas , Fator de Crescimento Transformador beta1/química , Fator de Crescimento Transformador beta1/genética , Sequência de Bases , Linhagem Celular , Sequência Conservada , Humanos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Surface plasmon resonance is a technique for detecting binding events at the surface of a thin metal film. Through the commercial availability of instrumentation and sensor chips, the technique has found widespread application for determining the affinity and kinetics of macromolecular interactions. A variety of quadruplex forming oligonucleotides have been immobilized to sensor chips to permit analysis of their binding interactions with both small molecule and protein analytes. The fold of the quadruplex must be maintained through an appropriate choice of buffer, and care must be taken to ensure that data interpretation is not hampered by non-specific binding and adsorption of the analyte to the sensor surface and instrument. Affinity constants determined by surface plasmon resonance for interactions with quadruplexes correlate meaningfully with other methods, such as UV-visible and fluorescence titrations, enzyme linked immunosorbent assay, thermal melting studies and telomerase inhibition. Kinetic measurements of the association and dissociation of duplexes of quadruplex forming oligonucleotides and their complementary strands have enabled calculation of the folding and unfolding rates of the quadruplex itself, and determination of its stability as a function of buffer composition.
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Quadruplex G , Guanina/química , Proteínas/química , Ressonância de Plasmônio de Superfície/métodos , Conformação de Ácido NucleicoRESUMO
We describe the identification of small-molecule G-quadruplex ligands using a direct ELISA screen of a one-bead-one-compound library of unnatural polyamides displayed on a branched linker with a biotin tag. This general purpose parallel screen for small molecule-oligonucleotide interactions was validated by surface plasmon resonance and ELISA of resynthesized compounds. Linear polyamides displayed similar rankings in their affinity for quadruplex as their branched counterparts. Quadruplex affinity as judged by these surface based techniques was a useful predictor of the ability of the ligands to stabilize the quadruplex to thermal unfolding in solution.
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DNA/química , Ensaio de Imunoadsorção Enzimática , Ligantes , Técnicas de Química Combinatória , Transferência Ressonante de Energia de Fluorescência , Quadruplex G , Humanos , Ressonância Magnética Nuclear Biomolecular , Oligopeptídeos/química , Peptídeos/química , Peptídeos/imunologia , Ressonância de Plasmônio de SuperfícieRESUMO
We have recently described an engineered zinc finger protein (Gq1) that binds with high specificity to the intramolecular G-quadruplex formed by the human telomeric sequence 5'-(GGTTAG)(5)-3', and that inhibits the activity of the enzyme telomerase in vitro. Here we report site-directed mutagenesis, biophysical, and molecular modeling studies that provide new insights into quadruplex recognition by the zinc finger scaffold. We show that any one finger of Gq1 can be replaced with the corresponding finger of Zif268, without significant loss of quadruplex affinity or quadruplex versus duplex discrimination. Replacement of two fingers, with one being finger 2, of Gq1 by Zif268 results in significant impairment of quadruplex recognition and loss of discrimination. Molecular modeling suggests that the zinc fingers of Gq1 can bind to the human parallel-stranded quadruplex structure in a stable arrangement, whereas Zif268-quadruplex models show significantly weaker binding energy. Modeling also suggests that an important role of the key protein finger residues in the Gq1-quadruplex complex is to maintain Gq1 in an optimum conformation for quadruplex recognition.
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DNA/química , Proteína 1 de Resposta de Crescimento Precoce/química , Conformação de Ácido Nucleico , Proteínas Recombinantes/química , Dedos de Zinco/fisiologia , Cisteína/química , DNA/genética , DNA/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/genética , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Ensaio de Imunoadsorção Enzimática , Quadruplex G , Histidina/química , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , Engenharia de Proteínas , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ressonância de Plasmônio de Superfície , Dedos de Zinco/genéticaRESUMO
Cavities and clefts are frequently important sites of interaction between natural enzymes or receptors and their corresponding substrate or ligand molecules and exemplify the types of molecular surfaces that would facilitate engineering of artificial catalysts and receptors. Even so, structural characterizations of designed cavities are rare. To address this issue, we performed a systematic study of the structural effects of single-amino acid substitutions within the hydrophobic cores of tetrameric coiled-coil peptides. Peptides containing single glycine, serine, alanine, or threonine amino acid substitutions at the buried L9, L16, L23, and I26 hydrophobic core positions of a GCN4-based sequence were synthesized and studied by solution-phase and crystallographic techniques. All peptides adopt the expected tetrameric state and contain tunnels or internal cavities ranging in size from 80 to 370 A(3). Two closely related sequences containing an L16G substitution, one of which adopts an antiparallel configuration and one of which adopts a parallel configuration, illustrate that cavities of different volumes and shapes can be engineered from identical core substitutions. Finally, we demonstrate that two of the peptides (L9G and L9A) bind the small molecule iodobenzene when present during crystallization, leaving the general peptide quaternary structure intact but altering the local peptide conformation and certain superhelical parameters. These high-resolution descriptions of varied molecular surfaces within solvent-occluded internal cavities illustrate the breadth of design space available in even closely related peptides and offer valuable models for the engineering of de novo helical proteins.