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Transient receptor potential (TRP) channels are a large and diverse family of transmembrane ion channels that are widely expressed, have important physiological roles, and are associated with many human diseases. These proteins are actively pursued as promising drug targets, benefitting greatly from advances in structural and mechanistic studies of TRP channels. At the same time, the complex, polymodal activation and regulation of TRP channels have presented formidable challenges. In this short review, we summarize recent progresses toward understanding the structural basis of TRP channel function, as well as potential ligand binding sites that could be targeted for therapeutics. A particular focus is on the current understanding of the molecular mechanisms of TRP channel activation and regulation, where many fundamental questions remain unanswered. We believe that a deeper understanding of the functional mechanisms of TRP channels will be critical and likely transformative toward developing successful therapeutic strategies targeting these exciting proteins. This endeavor will require concerted efforts from computation, structural biology, medicinal chemistry, electrophysiology, pharmacology, drug safety and clinical studies.
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Water is at the heart of almost all biological phenomena, without which no life that we know of would have been possible. It is a misleadingly complex liquid that exists in near coexistence with the vapor phase under ambient conditions. Confinement within a hydrophobic cavity can tip this balance enough to drive a cooperative dewetting transition. For a nanometer-scale pore, the dewetting transition leads to a stable dry state that is physically open but impermeable to ions. This phenomenon is often referred to as hydrophobic gating. Numerous transmembrane protein ion channels have now been observed to utilize hydrophobic gating in their activation and regulation. Here, we review recent theoretical, simulation, and experimental studies that together have started to establish the principles of hydrophobic gating and discuss how channels of various sizes, topologies, and biological functions can utilize these principles to control the thermodynamic properties of water within their interior pores for gating and regulation. Exciting opportunities remain in multiple areas, particularly on direct experimental detection of hydrophobic dewetting in biological channels and on understanding how the cell may control the hydrophobic gating in regulation of ion channels.
Assuntos
Ativação do Canal Iônico , Canais Iônicos/química , Canais Iônicos/metabolismo , Água/química , Água/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Humanos , Hidrodinâmica , Canais de Potássio Ativados por Cálcio de Condutância Alta/química , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Modelos Moleculares , Nanoporos , Receptores 5-HT3 de Serotonina/química , Receptores 5-HT3 de Serotonina/metabolismo , MolhabilidadeRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Large-conductance potassium (BK) channels are transmembrane (TM) proteins that can be synergistically and independently activated by membrane voltage and intracellular Ca2+. The only covalent connection between the cytosolic Ca2+ sensing domain and the TM pore and voltage sensing domains is a 15-residue 'C-linker'. To determine the linker's role in human BK activation, we designed a series of linker sequence scrambling mutants to suppress potential complex interplay of specific interactions with the rest of the protein. The results revealed a surprising sensitivity of BK activation to the linker sequence. Combining atomistic simulations and further mutagenesis experiments, we demonstrated that nonspecific interactions of the linker with membrane alone could directly modulate BK activation. The C-linker thus plays more direct roles in mediating allosteric coupling between BK domains than previously assumed. Our results suggest that covalent linkers could directly modulate TM protein function and should be considered an integral component of the sensing apparatus.
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Membrana Celular/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Animais , Cálcio/metabolismo , Membrana Celular/fisiologia , Fenômenos Eletrofisiológicos , Humanos , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/fisiologia , Canais de Potássio Ativados por Cálcio de Condutância Alta/química , Canais de Potássio Ativados por Cálcio de Condutância Alta/fisiologia , Estrutura Terciária de Proteína , Xenopus laevisRESUMO
Monoclonal antibodies are among the fastest growing therapeutics in the pharmaceutical industry. Detecting higher-order structure changes of antibodies upon storage or mishandling, however, is a challenging problem. In this study, we describe the use of diethylpyrocarbonate (DEPC)-based covalent labeling (CL) - mass spectrometry (MS) to detect conformational changes caused by heat stress, using rituximab as a model system. The structural resolution obtained from DEPC CL-MS is high enough to probe subtle conformation changes that are not detectable by common biophysical techniques. Results demonstrate that DEPC CL-MS can detect and identify sites of conformational changes at the temperatures below the antibody melting temperature (e.g., 55 á´¼C). The observed labeling changes at lower temperatures are validated by activity assays that indicate changes in the Fab region. At higher temperatures (e.g., 65 á´¼C), conformational changes and aggregation sites are identified from changes in CL levels, and these results are confirmed by complementary biophysical and activity measurements. Given the sensitivity and simplicity of DEPC CL-MS, this method should be amenable to the structural investigations of other antibody therapeutics.
Assuntos
Dietil Pirocarbonato/química , Fragmentos Fab das Imunoglobulinas/química , Modelos Moleculares , Rituximab/química , Espectrometria de Massas , Estrutura Quaternária de ProteínaRESUMO
The rapid emergence of antibiotic-resistant bacterial "superbugs" with concomitant treatment failure and high mortality rates presents a severe threat to global health. The superbug risk is further exacerbated by chronic infections generated from antibiotic-resistant biofilms that render them refractory to available treatments. We hypothesized that efficient antimicrobial agents could be generated through careful engineering of hydrophobic and cationic domains in a synthetic semirigid polymer scaffold, mirroring and amplifying attributes of antimicrobial peptides. We report the creation of polymeric nanoparticles with highly efficient antimicrobial properties. These nanoparticles eradicate biofilms with low toxicity to mammalian cells and feature unprecedented therapeutic indices against red blood cells. Most notably, bacterial resistance toward these nanoparticles was not observed after 20 serial passages, in stark contrast to clinically relevant antibiotics where significant resistance occurred after only a few passages.
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Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Nanopartículas/química , Polímeros/farmacologia , Compostos de Amônio Quaternário/farmacologia , Animais , Antibacterianos/síntese química , Antibacterianos/química , Antibacterianos/toxicidade , Enterobacter cloacae/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Humanos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Camundongos , Testes de Sensibilidade Microbiana , Células NIH 3T3 , Nanopartículas/toxicidade , Polímeros/síntese química , Polímeros/química , Polímeros/toxicidade , Pseudomonas aeruginosa/efeitos dos fármacos , Compostos de Amônio Quaternário/síntese química , Compostos de Amônio Quaternário/química , Compostos de Amônio Quaternário/toxicidadeRESUMO
The gating mechanism of transmembrane ion channels is crucial for understanding how these proteins control ion flow across membranes in various physiological processes. Big potassium (BK) channels are particularly interesting with large single-channel conductance and dual regulation by membrane voltage and intracellular Ca2+. Recent atomistic structures of BK channels failed to identify structural features that could physically block the ion flow in the closed state. Here, we show that gating of BK channels does not seem to require a physical gate. Instead, changes in the pore shape and surface hydrophobicity in the Ca2+-free state allow the channel to readily undergo hydrophobic dewetting transitions, giving rise to a large free energy barrier for K+ permeation. Importantly, the dry pore remains physically open and is readily accessible to quaternary ammonium channel blockers. The hydrophobic gating mechanism is also consistent with scanning mutagenesis studies showing that modulation of pore hydrophobicity is correlated with activation properties.
Assuntos
Ativação do Canal Iônico/fisiologia , Canais de Potássio Ativados por Cálcio de Condutância Alta/química , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Animais , Humanos , Interações Hidrofóbicas e Hidrofílicas , Potássio/metabolismo , Estrutura Secundária de ProteínaRESUMO
Additives play an important role in modifying the morphology and phase separation of donor and acceptor molecules in bulk heterojunction (BHJ) solar cells. Here, we report triptycene (TPC) as a small-molecule additive for supramolecular control of phase separation and concomitant improvement of the power conversion efficiency (PCE) of PTB7 donor and fullerene acceptor-based BHJ polymer solar cells. An overall 60% improvement in PCE is observed for both PTB7:PC61BM and PTB7:PC71BM blends. The improved photovoltaic (PV) performance can be attributed to three factors: (a) TPC-induced supramolecular interactions with donor:acceptor components in the blends to realize a nanoscale phase-separated morphology; (b) an increase in the charge transfer state energy that lowers the driving force for electron transfer from donor to acceptor molecules; and (c) an increase in the charge carrier mobility. An improvement in efficiency using TPC as a supramolecular additive has also been demonstrated for other BHJ blends such as PBDB-T:PC71BM and P3HT:PCBM, implying the wide applicability of this new additive molecule. A comparison of the photostability of TPC as an additive for PTB7:PCBM solar cells to that of the widely used 1,8-diiodooctane additive shows â¼30% higher retention of PV performance for the TPC-added solar cells after 34 h of AM 1.5G illumination. The results obtained suggest that the approach of using additives that can promote supramolecular interactions to modify the length scale of phase separation between donor and acceptor is very promising and can lead to the development of highly efficient and stable organic photovoltaics.
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Infections caused by multidrug-resistant (MDR) bacteria are a rapidly growing threat to human health, in many cases exacerbated by their presence in biofilms. We report here a biocompatible oil-in-water cross-linked polymeric nanocomposite that degrades in the presence of physiologically relevant biomolecules. These degradable nanocomposites demonstrated broad-spectrum penetration and elimination of MDR bacteria, eliminating biofilms with no toxicity to cocultured mammalian fibroblast cells. Notably, serial passaging revealed that bacteria were unable to develop resistance toward these nanocomposites, highlighting the therapeutic promise of this platform.
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Antibacterianos/farmacologia , Materiais Biocompatíveis/farmacologia , Biofilmes/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Nanocompostos/química , Antibacterianos/química , Antibacterianos/metabolismo , Materiais Biocompatíveis/química , Materiais Biocompatíveis/metabolismo , Reagentes de Ligações Cruzadas/química , Reagentes de Ligações Cruzadas/metabolismo , Reagentes de Ligações Cruzadas/farmacologia , Testes de Sensibilidade Microbiana , Estrutura MolecularRESUMO
Liver disease is the fifth most common cause of premature death in the Western world, with the irreversible damage caused by fibrosis, and ultimately cirrhosis, a primary driver of mortality. Early detection of fibrosis would facilitate treatment of the underlying liver disease to limit progression. Unfortunately, most cases of liver disease are diagnosed late, with current strategies reliant on invasive biopsy or fragile lab-based antibody technologies. A robust, fully synthetic fluorescent-polymer sensor array is reported, which, rapidly (in 45 minutes), detects liver fibrosis from low-volume serum samples with clinically relevant specificity and accuracy, using an easily readable diagnostic output. The simplicity, rapidity, and robustness of this method make it a promising platform for point-of-care diagnostics for detecting and monitoring liver disease.
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Cirrose Hepática/diagnóstico , Biomarcadores , Biópsia , Técnicas de Imagem por Elasticidade , Humanos , Fígado , Polímeros , Sensibilidade e EspecificidadeRESUMO
We outline an evolution process for tongue elements composed of poly(p-aryleneethynylene)s (PAE) and detergents, resulting in a chemical tongue (24 elements) that discerns antibiotics. Cross-breeding of this new tongue with tongue elements that consist of simple poly(p-phenyleneethynylene)s (PPE) at different pH-values leads to an enlarged sensor array, composed of 30 elements. This tongue was pruned, employing principal component analysis. We find that a filial tongue featuring three elements from each original array (i.e. a six element tongue) is superior to either of the prior tongues and the composite tongue in the discrimination of structurally different antibiotics. Such a selection process should be general and give an idea how to successfully generate powerful low-selectivity sensor elements and configure them into discriminative chemical tongues.
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We report a nanosensor that uses cell lysates to rapidly profile the tumorigenicity of cancer cells. This sensing platform uses host-guest interactions between cucurbit[7]uril and the cationic headgroup of a gold nanoparticle to non-covalently modify the binding of three fluorescent proteins of a multi-channel sensor in situ. This approach doubles the number of output channels to six, providing single-well identification of cell lysates with 100% accuracy. Significantly, this classification could be extended beyond the training set, determining the invasiveness of novel cell lines. The unique fingerprint of these cell lysates required minimal sample quantity (200 ng, â¼1000 cells), making the methodology compatible with microbiopsy technology.
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Técnicas Biossensoriais , Hidrocarbonetos Aromáticos com Pontes/química , Imidazóis/química , Proteínas Luminescentes/química , Nanopartículas Metálicas/química , Nanotecnologia , Neoplasias/patologia , Sítios de Ligação , Linhagem Celular Tumoral , Ouro/química , Humanos , Estrutura Molecular , Neoplasias/diagnóstico por imagemRESUMO
We present here a highly efficient sensor for bacteria that provides an olfactory output, allowing detection without the use of instrumentation and with a modality that does not require visual identification. The sensor platform uses nanoparticles to reversibly complex and inhibits lipase. These complexes are disrupted in the presence of bacteria, restoring enzyme activity and generating scent from odorless pro-fragrance substrate molecules. This system provides rapid (15 min) sensing and very high sensitivity (102 cfu/mL) detection of bacteria using the human sense of smell as an output.
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Bactérias/isolamento & purificação , Técnicas Biossensoriais/métodos , Candida/enzimologia , Lipase/metabolismo , Nanopartículas/metabolismo , Bactérias/metabolismo , Infecções Bacterianas/microbiologia , Técnicas Biossensoriais/economia , Humanos , Lipase/antagonistas & inibidores , Nanopartículas/química , Olfato , Fatores de TempoRESUMO
Using a family of cationic gold nanoparticles (NPs) with similar size and charge, we demonstrate that proper surface engineering can control the nature and identity of protein corona in physiological serum conditions. The protein coronas were highly dependent on the hydrophobicity and arrangement of chemical motifs on NP surface. The NPs were uptaken in macrophages in a corona-dependent manner, predominantly through recognition of specific complement proteins in the NP corona. Taken together, this study shows that surface functionality can be used to tune the protein corona formed on NP surface, dictating the interaction of NPs with macrophages.
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Ouro/química , Macrófagos/metabolismo , Nanopartículas Metálicas/química , Coroa de Proteína/metabolismo , Animais , Cátions , Humanos , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Tamanho da Partícula , Ligação Proteica , Células RAW 264.7 , Propriedades de SuperfícieRESUMO
Supramolecular complexes of a family of positively charged conjugated polymers (CPs) and green fluorescent protein (GFP) create a fluorescence resonance energy transfer (FRET)-based ratiometric biosensor array. Selective multivalent interactions of the CPs with mammalian cell surfaces caused differential change in FRET signals, providing a fingerprint signature for each cell type. The resulting fluorescence signatures allowed the identification of 16 different cell types and discrimination between healthy, cancerous, and metastatic cells, with the same genetic background. While the CP-GFP sensor array completely differentiated between the cell types, only partial classification was achieved for the CPs alone, validating the effectiveness of the ratiometric sensor. The utility of the biosensor was further demonstrated in the detection of blinded unknown samples, where 121 of 128 samples were correctly identified. Notably, this selectivity-based sensor stratified diverse cell types in minutes, using only 2000 cells, without requiring specific biomarkers or cell labeling.
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Fluorescência , Proteínas de Fluorescência Verde/química , Polímeros/química , Animais , Técnicas Biossensoriais/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Humanos , Camundongos , Modelos QuímicosRESUMO
Sensing using specific and selective receptors provides two very different but complementary strategies. This Sensor Issues article will discuss the merits and challenges of specific sensors, and selective sensors based on synthetic arrays. We will examine where each has been successfully applied to a sensing challenge, and then look at how a combined approach could take elements of both to provide new sensor platforms.
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Bacteria attach to the surfaces of medical devices and implants, resulting in life-threatening infections. Nonfouling coatings can be used to prevent adhesion of bacteria on the surface, while biocidal coatings kill the microbes. Combining nonfouling and biocidal properties can yield highly effective antimicrobial coatings. We demonstrate here a nanoimprint lithography (NIL)-based method to generate antibacterial coatings that both resist bacterial attachment and kill bacteria. In this strategy nanoimprint lithography was used to create water-stable films of bovine serum albumin (BSA) that are nonadhesive toward bacteria because of their negative/zwitterionic surface potential. Biocidal activity was then imparted through chlorination of cysteine sulfurs, providing slow release of chlorine and potent antimicrobial activity against pathogenic bacteria.
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Traditional plating and culturing methods used to quantify bacteria commonly require hours to days from sampling to results. We present here a simple, sensitive and rapid electrochemical method for bacterial detection in drinking water based on gold nanoparticle-enzyme complexes. The gold nanoparticles were functionalized with positively charged quaternary amine headgroups that could bind to enzymes through electrostatic interactions, resulting in inhibition of enzymatic activity. In the presence of bacteria, the nanoparticles were released from the enzymes and preferentially bound to the bacteria, resulting in an increase in enzyme activity, releasing a redox-active phenol from the substrate. We employed this strategy for the electrochemical sensing of Escherichia coli and Staphylococcus aureus, resulting in a rapid detection (<1 h) with high sensitivity (10(2) CFU mL(-1)).
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Técnicas Biossensoriais/métodos , Água Potável/microbiologia , Escherichia coli/isolamento & purificação , Ouro/química , Nanopartículas Metálicas/química , Staphylococcus aureus/isolamento & purificação , beta-Galactosidase/química , Técnicas Biossensoriais/economia , Técnicas Eletroquímicas/economia , Técnicas Eletroquímicas/métodos , Enzimas Imobilizadas/química , Limite de Detecção , Nitrofenilgalactosídeos/químicaRESUMO
Zwitterionic materials are useful tools in material science and biology as they provide high water solubility while preventing non-specific interactions. Quantum dots (QDs) functionalized with zwitterionic and quaternary ammonium ligands were synthesized to investigate their interactions with the outer membrane of HeLa cells. Quaternary ammonium functionalized quantum dots adhered strongly to the cell surface while zwitterionic QDs had no cell adhesion. These results demonstrate that future non-interacting nanoparticles based on this design are possible.
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Array-based sensing using nanoparticles (NPs) provides an attractive alternative to specific biomarker-focused strategies for cancer diagnosis. The physical and chemical properties of NPs provide both the recognition and transduction capabilities required for biosensing. Array-based sensors utilize a combined response from the interactions between sensors and analytes to generate a distinct pattern (fingerprint) for each analyte. These interactions can be the result of either the combination of multiple specific biomarker recognition (specific binding) or multiple selective binding responses, known as chemical nose sensing. The versatility of the latter array-based sensing using NPs can facilitate the development of new personalized diagnostic methodologies in cancer diagnostics, a necessary evolution in the current healthcare system to better provide personalized treatments. This review will describe the basic principle of array-based sensors, along with providing examples of both invasive and noninvasive samples used in cancer diagnosis.