RESUMO
Dementia is a brain disease which results in irreversible and progressive loss of cognition and motor activity. Despite global efforts, there is no simple and reliable diagnosis or treatment option. Current diagnosis involves indirect testing of commonly inaccessible biofluids and low-resolution brain imaging. We have developed a portable, wireless readout-based Graphene field-effect transistor (GFET) biosensor platform that can detect viruses, proteins, and small molecules with single-molecule sensitivity and specificity. We report the detection of three important amyloids, namely, Amyloid beta (Aß), Tau (τ), and α-Synuclein (αS) using DNA aptamer nanoprobes. These amyloids were isolated, purified, and characterized from the autopsied brain tissues of Alzheimer's Disease (AD) and Parkinson's Disease (PD) patients. The limit of detection (LoD) of the sensor is 10 fM, 1-10 pM, 10-100 fM for Aß, τ, and αS, respectively. Synthetic as well as autopsied brain-derived amyloids showed a statistically significant sensor response with respect to derived thresholds, confirming the ability to define diseased vs. nondiseased states. The detection of each amyloid was specific to their aptamers; Aß, τ, and αS peptides when tested, respectively, with aptamers nonspecific to them showed statistically insignificant cross-reactivity. Thus, the aptamer-based GFET biosensor has high sensitivity and precision across a range of epidemiologically significant AD and PD variants. This portable diagnostic system would allow at-home and POC testing for neurodegenerative diseases globally.
Assuntos
Doença de Alzheimer , Aptâmeros de Nucleotídeos , Grafite , Doença de Parkinson , Humanos , Peptídeos beta-Amiloides/metabolismo , Doença de Alzheimer/diagnóstico , Doença de Alzheimer/metabolismo , Doença de Parkinson/diagnóstico , Biomarcadores , Proteínas tauRESUMO
Aging, tau pathology, and chronic inflammation in the brain play crucial roles in synaptic loss, neurodegeneration, and cognitive decline in tauopathies, including Alzheimer's disease. Senescent cells accumulate in the aging brain, accelerate the aging process, and promote tauopathy progression through their abnormal inflammatory secretome known as the senescence-associated secretory phenotype (SASP). Tau oligomers (TauO)-the most neurotoxic tau species-are known to induce senescence and the SASP, which subsequently promote neuropathology, inflammation, oxidative stress, synaptic dysfunction, neuronal death, and cognitive dysfunction. TauO, brain inflammation, and senescence are associated with heterogeneity in tauopathy progression and cognitive decline. However, the underlying mechanisms driving the disease heterogeneity remain largely unknown, impeding the development of therapies for tauopathies. Based on clinical and preclinical evidence, this review highlights the critical role of TauO and senescence in neurodegeneration. We discuss key knowledge gaps and potential strategies for targeting senescence and TauO to treat tauopathies. HIGHLIGHTS: Senescence, oligomeric Tau (TauO), and brain inflammation accelerate the aging process and promote the progression of tauopathies, including Alzheimer's disease. We discuss their role in contributing to heterogeneity in tauopathy and cognitive decline. We highlight strategies to target senescence and TauO to treat tauopathies while addressing key knowledge gaps.
Assuntos
Doença de Alzheimer , Disfunção Cognitiva , Encefalite , Tauopatias , Humanos , Doença de Alzheimer/patologia , Proteínas tau/metabolismo , Tauopatias/patologia , Encéfalo/patologia , Encefalite/complicações , Encefalite/patologia , Disfunção Cognitiva/patologia , InflamaçãoRESUMO
Before the controversial approval of humanized monoclonal antibody lecanemab, which binds to the soluble amyloid-ß protofibrils, all the treatments available earlier, for Alzheimer's disease (AD) were symptomatic. The researchers are still struggling to find a breakthrough in AD therapeutic medicine, which is partially attributable to lack in understanding of the structural information associated with the intrinsically disordered proteins and amyloids. One of the major challenges in this area of research is to understand the structural diversity of intrinsically disordered proteins under in vitro conditions. Therefore, in this review, we have summarized the in vitro applications of biophysical methods, which are aimed to shed some light on the heterogeneity, pathogenicity, structures and mechanisms of the intrinsically disordered protein aggregates associated with proteinopathies including AD. This review will also rationalize some of the strategies in modulating disease-relevant pathogenic protein entities by small molecules using structural biology approaches and biophysical characterization. We have also highlighted tools and techniques to simulate the in vivo conditions for native and cytotoxic tau/amyloids assemblies, urge new chemical approaches to replicate tau/amyloids assemblies similar to those in vivo conditions, in addition to designing novel potential drugs.
RESUMO
The increase of antibiotic-resistant bacterial pathogens has created challenges in treatment and warranted the design of antibiotics against comparatively less exploited targets. The peptidoglycan (PG) biosynthesis delineates unique pathways for the design and development of a novel class of drugs. Mur ligases are an essential component of bacterial cell wall synthesis that play a pivotal role in PG biosynthesis to maintain internal osmotic pressure and cell shape. Inhibition of these enzymes can interrupt bacterial replication and hence, form attractive targets for drug discovery. In the present work, we focused on the PG biosynthesis pathway enzyme, UDP-N-acetylpyruvylglucosamine reductase, from Salmonella enterica serovar Typhi (stMurB). Biophysical characterization of purified StMurB was performed to gauge the molecular interactions and estimate thermodynamic stability for determination of attributes for possible therapeutic intervention. The thermal melting profile of MurB was monitored by circular dichroism and validated through differential scanning calorimetry experiment. Frequently used chemical denaturants, GdmCl and urea, were employed to study the chemical-induced denaturation of stMurB. In the search for natural compound-based inhibitors, against this important drug target, an in silico virtual screening based investigation was conducted with modeled stMurB structure. The three top hits (quercetin, berberine, and scopoletin) returned were validated for complex stability through molecular dynamics simulation. Further, fluorescence binding studies were undertaken for the selected natural compounds with stMurB alone and with NADPH bound form. The compounds scopoletin and berberine, displayed lesser binding to stMurB whereas quercetin exhibited stronger binding affinity than NADPH. This study suggests that quercetin can be evolved as an inhibitor of stMurB enzyme.
Assuntos
Berberina , Salmonella typhi , NADP , Quercetina , Escopoletina , Antibacterianos/farmacologiaRESUMO
DNA Gyrase is a type II topoisomerase that utilizes the energy of ATP hydrolysis for introducing negative supercoils in DNA. The protein comprises two subunits GyrA and GyrB that form a GyrA2GyrB2 heterotetramer. GyrB subunit contains the N-terminal domain (GBNTD) for ATPase activity and the C-terminal domain (GBCTD) for interaction with GyrA and DNA. Earlier structural studies have revealed three different conformational states for GBNTD during ATP hydrolysis defined as open, semi-open, and closed. Here we report, the three-dimensional structure of a new transient closed conformation of GBNTD from Salmonella Typhi (StGBNTD) at 1.94 Å resolution. Based on the structural analysis of this transient closed conformation, we propose the role of protein in the mechanism of ATP hydrolysis. We further explored the effect of pH on ATPase activity and structural stability of the GBNTD using CD and fluorescence spectroscopy at varying pH environment. Kinetic parameters obtained from the ATPase assay were correlated with its secondary and tertiary structure at their respective pH environment. The protein possessed maximum ATPase activity and structural stability at optimum pH 8. At acidic pH, a remarkable decrease in both enzymatic activity and structural stability was observed whereas at alkaline pH there was no significant change. The structural analysis of StGBNTD reveals the role of polar interactions in stabilizing the overall dimeric conformation of the protein.
Assuntos
Adenosina Trifosfatases/química , DNA Girase/química , Salmonella typhi/enzimologia , Adenosina Trifosfatases/genética , Cristalografia por Raios X , DNA Girase/genética , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Cinética , Domínios Proteicos , Salmonella typhi/genéticaRESUMO
The pathological misfolding and aggregation of the microtubule associated protein tau (MAPT), a full length Tau2N4R with 441aa, is considered the principal disease relevant constituent in tauopathies including Alzheimer's disease (AD) with an imbalanced ratio in 3R/4R isoforms. The exact cellular fluid composition, properties, and changes that coincide with tau misfolding, seed formation, and propagation events remain obscure. The proteostasis network, along with the associated osmolytes, is responsible for maintaining the presence of tau in its native structure or dealing with misfolding. In this study, for the first time, the roles of natural brain osmolytes are being investigated for their potential effects on regulating the conformational stability of the tau monomer (tauM) and its propensity to aggregate or disaggregate. Herein, the effects of physiological osmolytes myo-inositol, taurine, trimethyl amine oxide (TMAO), betaine, sorbitol, glycerophosphocholine (GPC), and citrulline on tau's aggregation state were investigated. The overall results indicate the ability of sorbitol and GPC to maintain the monomeric form and prevent aggregation of tau, whereas myo-inositol, taurine, TMAO, betaine, and citrulline promote tau aggregation to different degrees, as revealed by protein morphology in atomic force microscopy images. Biochemical and biophysical methods also revealed that tau proteins adopt different conformations under the influence of these osmolytes. TauM in the presence of all osmolytes expressed no toxicity when tested by a lactate dehydrogenase assay. Investigating the conformational stability of tau in the presence of osmolytes may provide a better understanding of the complex nature of tau aggregation in AD and the protective and/or chaotropic nature of osmolytes.
Assuntos
Doença de Alzheimer , Metilaminas , Proteínas tau , Humanos , Proteínas tau/metabolismo , Betaína , Citrulina , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Taurina/farmacologia , Inositol/metabolismo , Sorbitol/metabolismoRESUMO
The microtubule associated protein, tau, is implicated in a multitude of neurodegenerative disorders that are collectively termed as tauopathies. These disorders are characterized by the presence of tau aggregates within the brain of afflicted individuals. Mutations within the MAPT gene that encodes the tau protein form the genetic backdrop for familial forms of tauopathies, such as frontotemporal dementia (FTD), but the molecular consequences of such alterations and their pathological effects are unclear. We sought to investigate the conformational properties of the aggregates of three tau mutants: A152T, P301L, and R406W, all implicated within FTD, and compare them to those of the native form (WT-Tau 2N4R). Our immunochemical analysis reveals that mutants and WT tau oligomers exhibit similar affinity for conformation-specific antibodies but have distinct morphology and secondary structure. Additionally, these oligomers possess different dye-binding properties and varying sensitivity to proteolytic processing. These results point to conformational variety among them. We then tested the ability of the mutant oligomers to cross-seed the aggregation of WT tau monomer. Using similar array of experiments, we found that cross-seeding with mutant aggregates leads to the formation of conformationally unique WT oligomers. The results discussed in this paper provide a novel perspective on the structural properties of oligomeric forms of WT tau 2N4R and its mutant, along with shedding some light on their cross-seeding behavior.
Assuntos
Tauopatias , Proteínas tau , Proteínas tau/química , Proteínas tau/genética , Proteínas tau/metabolismo , Humanos , Tauopatias/genética , Tauopatias/metabolismo , Mutação , Conformação Proteica , Multimerização Proteica , Demência Frontotemporal/genética , Demência Frontotemporal/metabolismoRESUMO
Structural dynamics of human immunodeficiency virus 1 (HIV-1) envelope (Env) glycoprotein mediate cell entry and facilitate immune evasion. Single-molecule FRET using peptides for Env labeling revealed structural dynamics of Env, but peptide use risks potential effects on structural integrity/dynamics. While incorporating noncanonical amino acids (ncAAs) into Env by amber stop-codon suppression, followed by click chemistry, offers a minimally invasive approach, this has proved to be technically challenging for HIV-1. Here, we develope an intact amber-free HIV-1 system that overcomes hurdles of preexisting viral amber codons. We achieved dual-ncAA incorporation into Env on amber-free virions, enabling single-molecule Förster resonance energy transfer (smFRET) studies of click-labeled Env that validated the previous peptide-based labeling approaches by confirming the intrinsic propensity of Env to dynamically sample multiple conformational states. Amber-free click-labeled Env also enabled real-time tracking of single virion internalization and trafficking in cells. Our system thus permits in-virus bioorthogonal labeling of proteins, compatible with studies of virus entry, trafficking, and egress from cells.
Assuntos
HIV-1 , Provírus , Humanos , Imagem Individual de Molécula , Proteínas/metabolismo , Peptídeos/metabolismoRESUMO
Human Aurora kinase A (AurA) has recently garnered the attention of researchers worldwide as a promising effective mitotic drug target for its involvement in cancer and related inflammatory anomalies. This study has explored the binding affinity of newly identified heteroarene-fused anthraquinone derivatives against AurA. Molecular docking analyses showed that all the heteroanthraquinone compounds bind to AurA with different affinities. Molecular dynamics simulation studies revealed that the compounds maintained relatively stable binding modes in the active site pocket while inducing minimal conformational changes in the AurA structure, interacting with key residues through several noncovalent interactions, including hydrogen bonds. Fluorescence spectroscopy and biolayer interferometry binding assays with synthesized compounds against recombinantly expressed AurA further verified their binding efficacy. Naphthoisatine 3 proved to be the best binder, with compounds anthraimidazole 5 and anthrathiophene 2 showing comparable results. Overall, this study indicates decent binding of heterocyclic derivatives of anthraquinone with the target AurA, which can further be assessed by performing enzymatic assays and cellular studies. The studies also highlight the applicability of the heteroarene-fused anthraquinone scaffold to construct selective and potent inhibitors of Aurora kinases after necessary structural modifications for the development of new anticancer drugs.
RESUMO
The quest for effective anticancer therapeutics continues to be extensively pursued. Over the past century, several drugs have been developed, however, a majority of these drugs have a poor therapeutic index and increased toxicity profile. Hence, there still exists ample opportunity to discover safe and effective anticancer drugs. Aurora Kinase B (AurB), a member of the Aurora kinase family and a key regulator of mitotic cell division, is found to be frequently overexpressed in a variety of human cancers and has thus emerged as an attractive target for the design of anticancer therapeutics. In the present study, a structure-based scaffold hopping approach was utilized to modify the heterocyclic moiety of (S)-3-(3-aminopyrrolidine-1-carbonyl)-4,11-dihydroxy-2-methylanthra [2,3-b]furan-5,10-dione (anthrafuran 1) to generate a series of heteroarene-fused anthraquinone derivatives, which were then subjected to virtual screening for the identification of potential AurB inhibitors. The obtained hits were subsequently synthesized and evaluated by using a combination of in silico and biophysical techniques for elucidating their in vitro binding and inhibition activity with recombinantly expressed AurB. Four identified hits presented an improved binding profile as compared to their parent analog anthrafuran 1. One derivative, anthrathiophene 2 demonstrated excellent in vitro inhibition of AurB (7.3 µM).
Assuntos
Antraquinonas , Aurora Quinase B , Inibidores de Proteínas Quinases , Antraquinonas/síntese química , Antraquinonas/química , Antraquinonas/farmacologia , Aurora Quinase B/antagonistas & inibidores , Aurora Quinase B/química , Aurora Quinase B/metabolismo , Linhagem Celular Tumoral , Humanos , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologiaRESUMO
MurE ligase is known to play a significant role in peptidoglycan biosynthesis. It catalyzes the addition of meso-diaminopimelic acid to nucleotide precursor. The protein can adopt different conformations for its proper functioning. Different environmental conditions can alter the stability and function of enzyme due to their ability to disrupt interactions between different domains. We have explored the pH and temperature dependent conformational changes in MurE ligase from Salmonella Typhi and estimated the protein stability. The study enabled us to decipher the effect of different milieu condition in the enzyme activity. At acidic pH 3.0, StMurE ligase forms molten globule (MG) state and at alkaline pH it is in unfolded state. The different states of StMurE ligase were characterized using various spectroscopic techniques. These techniques including near-UV CD, far-UV CD, ANS fluorescence, differential scanning calorimetry and fluorescence spectroscopy helped to determine the secondary structural changes and detect local conformational modifications. The structural analysis using StMurE ligase homology model revealed the variations in ionization states of catalytic amino acid residues involved in substrate binding. This study provides an insight into the dynamics states of StMurE ligase at different environmental conditions during bacterial pathogenesis.
Assuntos
Concentração de Íons de Hidrogênio , Modelos Moleculares , Peptídeo Sintases/química , Conformação Proteica , Salmonella typhi/enzimologia , Temperatura , Varredura Diferencial de Calorimetria , Dicroísmo Circular , Cinética , Peptídeo Sintases/metabolismo , Desnaturação Proteica , Espectrometria de Fluorescência , TermodinâmicaRESUMO
Drug repurposing is an efficient alternative approach to counter the increasing drug-resistant pathogens to treat infectious diseases. FtsZ is an essential bacterial cytokinesis protein involved in the formation of cell-division complex and targeting FtsZ using FDA approved drugs is a promising strategy to identify and develop a new antibacterial drug. Using in silico pharmacophore-based screening of drug bank, molecular docking and molecular dynamics simulations, we identified six drugs inhibiting the function of stFtsZ from Salmonella Typhi. The selected drugs target stFtsZ at the hydrophobic cleft formed between the C-terminal domain and helix α7 with binding energy better than -8 kcal/mol. Out of these six drugs, benzethonium chloride showed promising results at 8 µM concentration where it inhibits stFtsZ GTPase activity by 80% and prevents polymerization. Benzethonium chloride also possesses an excellent antibacterial activity against the bacterial culture of Salmonella Typhi (ATCC 19430), Staphylococcus aureus (ATCC 43300) and Escherichia coli (ATCC 25922) with the MIC values of 8 µg/mL, 1 µg/mL and 12 µg/mL, respectively. Based on our current study, the scaffold of benzethonium chloride can be used for the development of broad-spectrum antibacterial agents against drug-resistant pathogens.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Benzetônio/farmacologia , Proteínas do Citoesqueleto/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Salmonella typhi/efeitos dos fármacos , Antibacterianos/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Benzetônio/química , Sítios de Ligação , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , Inibidores Enzimáticos/química , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Salmonella typhi/enzimologiaRESUMO
Pyruvate dehydrogenase kinase-3 (PDK3) plays important role in the glucose metabolism and is associated with cancer progression, and thus being considered as an attractive target for cancer therapy. In this study, we employed spectroscopic techniques to study the structural and conformational changes in the PDK3 at varying pH conditions ranging from pH 2.0 to 12.0. UV/Vis, fluorescence and circular dichroism spectroscopic measurements revealed that PDK3 maintains its native-like structure (both secondary and tertiary) in the alkaline conditions (pH 7.0-12.0). However, a significant loss in the structure was observed under acidic conditions (pH 2.0-6.0). The propensity of aggregate formation at pH 4.0 was estimated by thioflavin T fluorescence measurements. To further complement structural data, kinase activity assay was performed, and maximum activity of PDK3 was observed at pH 7.0-8.0 range; whereas, its activity was lost under acidic pH. To further see conformational changes at atomistic level we have performed all-atom molecular dynamics at different pH conditions for 150 ns. A well defined correlation was observed between experimental and computational studies. This work highlights the significance of structural dependence of pH for wide implications in protein-protein interaction, biological function and drug design procedures.
Assuntos
Neoplasias/metabolismo , Conformação Proteica , Piruvato Desidrogenase Quinase de Transferência de Acetil/ultraestrutura , Relação Estrutura-Atividade , Dicroísmo Circular , Glucose/química , Glucose/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Simulação de Dinâmica Molecular , Neoplasias/terapia , Estrutura Secundária de Proteína/genética , Estrutura Terciária de Proteína/genética , Piruvato Desidrogenase Quinase de Transferência de Acetil/química , Piruvato Desidrogenase Quinase de Transferência de Acetil/genética , Espectrometria de FluorescênciaRESUMO
MurE ligase catalyzes the assembly of peptide moiety, an essential component of bacterial cell wall. We have explored the conformational stability and unfolding equilibrium behaviour of the protein MurE ligase by determining the conformational free energy, entropy and enthalpy parameters under stress conditions. MurE from Salmonella enterica Serovar Typhi was cloned, expressed and purified. Conformational changes associated with increasing concentration of GdmCl- and urea-induced denaturation of MurE were monitored using Circular Dichroism (CD) and fluorescence spectroscopies. The secondary structural content of protein estimated by CD experiment is in close agreement with the predicted MurE ligase structure by homology modeling. Denaturant-induced transition curve was analyzed for thermodynamic parameters. Average values for MurE ligase of ΔGD0â¯=â¯3.13â¯kcalâ¯mol-1, mâ¯=â¯1.52â¯kcalâ¯mol-1â¯M-1 and Cm (=ΔGD0/m)â¯=â¯2.05â¯M were calculated in the presence of GdmCl whereas in the case of urea these were ΔGD0â¯=â¯3.04â¯kcalâ¯mol-1, mâ¯=â¯1.20â¯kcalâ¯mol-1â¯M-1 and Cm (=ΔGD0/m)â¯=â¯2.53â¯M. The observed superposition of normalized transition curve of two independent optical properties suggested that GdmCl- and urea-induced denaturation follow a two-state process.
Assuntos
Estrutura Molecular , Peptídeo Sintases/química , Peptídeo Sintases/metabolismo , Salmonella typhi/enzimologia , Dicroísmo Circular , Ativação Enzimática , Estabilidade Enzimática , Modelos Moleculares , Peptídeo Sintases/genética , Peptídeo Sintases/isolamento & purificação , Conformação Proteica , Desnaturação Proteica , Salmonella typhi/genética , Análise Espectral , Relação Estrutura-AtividadeRESUMO
In this study, we have analyzed the structural and functional changes in the nature of Allium sativum Protease Inhibitor (ASPI) on undergoing various denaturation with variable range of pH, temperature and urea (at pH 8.2). ASPI being anti-tryptic in nature has native molecular mass of â¼15kDa. The conformational stability, functional parameters and their correlation were estimated under different conditions using circular dichroism, fluorescence and activity measurements. ASPI was found to fall in belongs to α+ß protein. It demonstrated structural and functional stability in the pH range 5.0-12.0 and up to70°C temperature. Further decrease in pH and increase in temperature induces unfolding followed by aggregation. Chemical induced denaturation was found to be cooperative and transitions were reversible and sigmoid. Tm (midpoint of denaturation), ΔCp (constant pressure heat capacity change) and ΔHm (van't Hoff enthalpy change at Tm were calculated to be 41.25±0.2°C, 1.3±0.07kcalmol-1K-1 and 61±2kcalmol-1 respectively for thermally denatured ASPI earlier. The reversibility of the protein was confirmed for both thermally and chemically denatured ASPI. The results obtained from trypsin inhibitory activity assay and structural studies are found to be in a significant correlation and hence established structure-function relationship of ASPI.
Assuntos
Alho/química , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Proteínas Secretadas Inibidoras de Proteinases/química , Proteínas Secretadas Inibidoras de Proteinases/metabolismo , Temperatura , Relação Dose-Resposta a Droga , Concentração de Íons de Hidrogênio , Proteínas de Plantas/farmacologia , Desnaturação Proteica/efeitos dos fármacos , Estabilidade Proteica , Análise Espectral , Relação Estrutura-Atividade , Inibidores da Tripsina/química , Inibidores da Tripsina/metabolismo , Inibidores da Tripsina/farmacologia , Ureia/farmacologiaRESUMO
Carbonic anhydrase IX (CAIX) is a transmembrane glycoprotein, associated with tumor, acidification which leads to the cancer, and is considered as a potential biomarker for hypoxia-induced cancers. The overexpression of CAIX is linked with hypoxia condition which is mediated by the transcription of hypoxia-induced factor (HIF-1). To understand the biophysical properties of CAIX, we have carried out a reversible isothermal denaturation of CAIX-induced by GdmCl at pH 8.0 and 25°C. Three different spectroscopic probes, the far-UV CD at 222 nm ([θ]222), Trp fluorescence emission at 342 nm (F342) and difference molar absorption coefficient at 287 nm (Δε287) were used to estimate stability parameters, [Formula: see text] (Gibbs free energy change in the absence of GdmCl; Cm (midpoint of the denaturation curve), i.e. molar GdmCl concentration ([GdmCl]) at which ΔGD = 0; and m, the slope (=∂ΔGD/∂[GdmCl])). GdmCl induces a reversible denaturation of CAIX. Coincidence of the normalized transition curves of all optical properties suggests that unfolding/refolding of CAIX is a two-state process. We further performed molecular dynamics simulation of CAIX for 40 ns to see the dynamics of protein structure in different GdmCl concentrations. An excellent agreement was observed between in silico and in vitro studies.
Assuntos
Anidrase Carbônica IX/química , Simulação de Dinâmica Molecular , Desdobramento de Proteína , Análise Espectral , Humanos , Conformação Molecular , Ligação Proteica , Desnaturação Proteica , Desdobramento de Proteína/efeitos dos fármacos , Análise Espectral/métodosRESUMO
Calcium/calmodulin-dependent protein kinase IV (CaMKIV) is a multifunctional enzyme which belongs to the Ser/Thr kinase family. CaMKIV plays important role in varieties of biological processes such as gene expression regulation, memory consolidation, bone growth, T-cell maturation, sperm motility, regulation of microtubule dynamics, cell-cycle progression, and apoptosis. To measure stability parameters, urea-induced denaturation of CaMKIV was carried out at pH 7.4 and 25°C, using three different probes, namely far-UV CD, near-UV absorption, and tryptophan fluorescence. A coincidence of normalized denaturation curves of these optical properties suggests that urea-induced denaturation is a two-state process. Analysis of these denaturation curves gave values of 4.20 ± 0.12 kcal mol-1, 2.95 ± 0.15 M, and 1.42 ± 0.06 kcal mol-1 M-1 for [Formula: see text] (Gibbs free energy change (ΔGD) in the absence of urea), Cm (molar urea concentration ([urea]) at the midpoint of the denaturation curve), and m (=∂ΔGD/∂[urea]), respectively. All these experimental observations have been fully supported by 30 ns molecular dynamics simulation studies.
Assuntos
Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/química , Simulação de Dinâmica Molecular , Conformação Proteica , Desnaturação Proteica , Análise Espectral , Ureia/química , Dicroísmo Circular , Humanos , Desnaturação Proteica/efeitos dos fármacos , Relação Estrutura-Atividade , Ureia/farmacologiaRESUMO
Knowledge of folding/unfolding pathway is fundamental basis to study protein structure and stability. Human carbonic anhydrase II (HCAII) is a â¼29kDa, ß-sheet dominated monomeric protein of 259 amino acid residues. In the present study, the urea-induced denaturation of HCAII was carried out which was a tri-phasic process, i.e., N (native) â XI â XII â D (denatured) with stable intermediates XI and XII populated around 2 and 4M urea, respectively. The far-UV CD was used to characterize the intermediate states (XI and XII) for secondary structural content, near-UV CD for tertiary structure, dynamic light scattering for hydrodynamic radius and ANS fluorescence spectroscopy for the presence of exposed hydrophobic patches. Based on these experiments, we concluded that urea-induced XI state has characteristics of molten globule state while XII state bears characteristics features of pre-molten globule state. Characterization of the intermediates on the folding pathway will contribute to a deeper understanding of the structure-function relationship of HCAII. Furthermore, this system may provide an excellent model to study urea stress and the strategies adopted by the organisms to combat such a stress.
Assuntos
Anidrase Carbônica II/química , Desnaturação Proteica/efeitos dos fármacos , Ureia/farmacologia , Humanos , Hidrodinâmica , Interações Hidrofóbicas e Hidrofílicas , Estrutura Terciária de Proteína/efeitos dos fármacosRESUMO
Carbonic anhydrase IX (CAIX) is a transmembrane glycoprotein, overexpressed in cancer cells under hypoxia condition. In cancerous cells, CAIX plays an important role to combat the deleterious effects of a high rate of glycolytic metabolism. In order to favor tumor survival, CAIX maintains intracellular pH neutral or slightly alkaline and extracellular acidic pH. The equilibrium unfolding and conformational stability of CAIX were measured in the presence of increasing urea concentrations to understand it's structural features under stressed conditions. Two different spectroscopic techniques were used to follow urea-induced denaturation and observed that urea induces a reversible denaturation of CAIX. Coincidence of the normalized transition curves of both optical properties suggesting that denaturation of CAIX is a two-state process, i.e., native state â denatured state. Each denaturation curve was analyzed to estimate thermodynamic parameters, ΔGD0,value of Gibbs free energy change (ΔGD) associated with the urea-induced denaturation, Cm (midpoint of denaturation) and m (=δΔGD/δ[urea]). We further performed molecular dynamics simulation of CAIX for 50ns to see the dynamics of protein structure in the presence of different urea concentrations. An excellent agreement was observed between in silico and in vitro studies.
Assuntos
Anidrase Carbônica IX/química , Simulação de Dinâmica Molecular , Desnaturação Proteica/efeitos dos fármacos , Ureia/farmacologia , Relação Dose-Resposta a Droga , Estabilidade Enzimática/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Conformação Proteica , Termodinâmica , Água/químicaRESUMO
A sequence alignment of mammalian cytochromes c with yeast iso-1-cytochrome c (y-cyt-c) shows that the yeast protein contains five extra N-terminal residues. We have been interested in understanding the question: What is the role of these five extra N-terminal residues in folding and stability of the protein? To answer this question we have prepared five deletants of y-cyt-c by sequentially removing these extra residues. During our studies on the wild type (WT) protein and its deletants, we observed that the amount of secondary structure in the guanidinium chloride (GdmCl)-induced denatured (D) state of each protein is different from that of the heat-induced denatured (H) state. This finding is confirmed by the observation of an additional cooperative transition curve of optical properties between H and D states on the addition of different concentrations of GdmCl to the already heat denatured WT y-cyt-c and its deletants at pH 6.0 and 68°C. For each protein, analysis of transition curves representing processes, native (N) state â D state, N state â H state, and H state â D state, was done to obtain Gibbs free energy changes associated with all the three processes. This analysis showed that, for each protein, thermodynamic cycle accommodates Gibbs free energies associated with transitions between N and D states, N and H states, and H and D states, the characteristics required for a thermodynamic function. All these experimental observations have been supported by our molecular dynamics simulation studies.