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1.
Cell ; 168(5): 878-889.e29, 2017 02 23.
Artículo en Inglés | MEDLINE | ID: mdl-28235199

RESUMEN

Design of small molecules that disrupt protein-protein interactions, including the interaction of RAS proteins and their effectors, may provide chemical probes and therapeutic agents. We describe here the synthesis and testing of potential small-molecule pan-RAS ligands, which were designed to interact with adjacent sites on the surface of oncogenic KRAS. One compound, termed 3144, was found to bind to RAS proteins using microscale thermophoresis, nuclear magnetic resonance spectroscopy, and isothermal titration calorimetry and to exhibit lethality in cells partially dependent on expression of RAS proteins. This compound was metabolically stable in liver microsomes and displayed anti-tumor activity in xenograft mouse cancer models. These findings suggest that pan-RAS inhibition may be an effective therapeutic strategy for some cancers and that structure-based design of small molecules targeting multiple adjacent sites to create multivalent inhibitors may be effective for some proteins.


Asunto(s)
Antineoplásicos/farmacología , Terapia Molecular Dirigida , Proteínas Proto-Oncogénicas p21(ras)/antagonistas & inhibidores , Proteínas Proto-Oncogénicas p21(ras)/química , Animales , Antineoplásicos/química , Calorimetría , Línea Celular , Fibroblastos/metabolismo , Xenoinjertos , Humanos , Ratones , Trasplante de Neoplasias , Neoplasias/tratamiento farmacológico , Neoplasias Pancreáticas/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras , Transducción de Señal , Bibliotecas de Moléculas Pequeñas
2.
Immunity ; 48(1): 45-58.e6, 2018 01 16.
Artículo en Inglés | MEDLINE | ID: mdl-29287995

RESUMEN

Interleukin-23 (IL-23), an IL-12 family cytokine, plays pivotal roles in pro-inflammatory T helper 17 cell responses linked to autoimmune and inflammatory diseases. Despite intense therapeutic targeting, structural and mechanistic insights into receptor complexes mediated by IL-23, and by IL-12 family members in general, have remained elusive. We determined a crystal structure of human IL-23 in complex with its cognate receptor, IL-23R, and revealed that IL-23R bound to IL-23 exclusively via its N-terminal immunoglobulin domain. The structural and functional hotspot of this interaction partially restructured the helical IL-23p19 subunit of IL-23 and restrained its IL-12p40 subunit to cooperatively bind the shared receptor IL-12Rß1 with high affinity. Together with structural insights from the interaction of IL-23 with the inhibitory antibody briakinumab and by leveraging additional IL-23:antibody complexes, we propose a mechanistic paradigm for IL-23 and IL-12 whereby cognate receptor binding to the helical cytokine subunits primes recruitment of the shared receptors via the IL-12p40 subunit.


Asunto(s)
Sudunidad beta 1 del Receptor de Interleucina-12/metabolismo , Interleucina-23/metabolismo , Receptores de Interleucina/metabolismo , Animales , Calorimetría/métodos , Línea Celular , Humanos , Interferometría/métodos , Subunidad p40 de la Interleucina-12/metabolismo , Masculino , Ratones , Unión Proteica/fisiología , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología
3.
J Biol Chem ; 300(1): 105582, 2024 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-38141762

RESUMEN

The intracellular parasite, Toxoplasma gondii, has developed sophisticated molecular strategies to subvert host processes and promote growth and survival. During infection, T. gondii replicates in a parasitophorous vacuole (PV) and modulates host functions through a network of secreted proteins. Of these, Mitochondrial Association Factor 1b (MAF1b) recruits host mitochondria to the PV, a process that confers an in vivo growth advantage, though the precise mechanisms remain enigmatic. To address this knowledge gap, we mapped the MAF1b interactome in human fibroblasts using a commercial Yeast-2-hybrid (Y2H) screen, which revealed several previously unidentified binding partners including the GAP domain of Ral GTPase Accelerating Protein α1 (RalGAPα1(GAP)). Recombinantly produced MAF1b and RalGAPα1(GAP) formed as a stable binary complex as shown by size exclusion chromatography with a Kd of 334 nM as measured by isothermal titration calorimetry (ITC). Notably, no binding was detected between RalGAPα1(GAP) and the structurally conserved MAF1b homolog, MAF1a, which does not recruit host mitochondria. Next, we used hydrogen deuterium exchange mass spectrometry (HDX-MS) to map the RalGAPα1(GAP)-MAF1b interface, which led to identification of the "GAP-binding loop" on MAF1b that was confirmed by mutagenesis and ITC to be necessary for complex formation. A high-confidence Alphafold model predicts the GAP-binding loop to lie at the RalGAPα1(GAP)-MAF1b interface further supporting the HDX-MS data. Mechanistic implications of a RalGAPα1(GAP)-MAF1b complex are discussed in the context of T. gondii infection and indicates that MAF1b may have evolved multiple independent functions to increase T. gondii fitness.


Asunto(s)
Proteínas Activadoras de GTPasa , Mitocondrias , Mapas de Interacción de Proteínas , Proteínas Protozoarias , Toxoplasma , Humanos , Sitios de Unión , Calorimetría , Cromatografía en Gel , Fibroblastos/metabolismo , Fibroblastos/parasitología , Proteínas Activadoras de GTPasa/química , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Espectrometría de Masas de Intercambio de Hidrógeno-Deuterio , Mitocondrias/metabolismo , Mitocondrias/parasitología , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Toxoplasma/química , Toxoplasma/genética , Toxoplasma/metabolismo , Técnicas del Sistema de Dos Híbridos
4.
Methods ; 225: 52-61, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38492901

RESUMEN

Isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) are two commonly used methods to probe biomolecular interactions. ITC can provide information about the binding affinity, stoichiometry, changes in Gibbs free energy, enthalpy, entropy, and heat capacity upon binding. SPR can provide information about the association and dissociation kinetics, binding affinity, and stoichiometry. Both methods can determine the nature of protein-protein interactions and help understand the physicochemical principles underlying complex biochemical pathways and communication networks. This methods article discusses the practical knowledge of how to set up and troubleshoot these two experiments with some examples.


Asunto(s)
Calorimetría , Unión Proteica , Resonancia por Plasmón de Superficie , Termodinámica , Resonancia por Plasmón de Superficie/métodos , Calorimetría/métodos , Cinética , Proteínas/química , Proteínas/metabolismo , Mapeo de Interacción de Proteínas/métodos , Entropía
5.
Nature ; 571(7764): 257-260, 2019 07.
Artículo en Inglés | MEDLINE | ID: mdl-31217589

RESUMEN

Increasing global food demand, low grain reserves and climate change threaten the stability of food systems on national to global scales1-5. Policies to increase yields, irrigation and tolerance of crops to drought have been proposed as stability-enhancing solutions1,6,7. Here we evaluate a complementary possibility-that greater diversity of crops at the national level may increase the year-to-year stability of the total national harvest of all crops combined. We test this crop diversity-stability hypothesis using 5 decades of data on annual yields of 176 crop species in 91 nations. We find that greater effective diversity of crops at the national level is associated with increased temporal stability of total national harvest. Crop diversity has stabilizing effects that are similar in magnitude to the observed destabilizing effects of variability in precipitation. This greater stability reflects markedly lower frequencies of years with sharp harvest losses. Diversity effects remained robust after statistically controlling for irrigation, fertilization, precipitation, temperature and other variables, and are consistent with the variance-scaling characteristics of individual crops required by theory8,9 for diversity to lead to stability. Ensuring stable food supplies is a challenge that will probably require multiple solutions. Our results suggest that increasing national effective crop diversity may be an additional way to address this challenge.


Asunto(s)
Productos Agrícolas/clasificación , Productos Agrícolas/provisión & distribución , Abastecimiento de Alimentos/métodos , Abastecimiento de Alimentos/estadística & datos numéricos , Geografía , Riego Agrícola/estadística & datos numéricos , Biodiversidad , Calorimetría , Productos Agrícolas/crecimiento & desarrollo , Sequías/estadística & datos numéricos , Fertilizantes/provisión & distribución , Modelos Teóricos , Probabilidad , Lluvia , Temperatura
6.
Proc Natl Acad Sci U S A ; 119(45): e2205322119, 2022 11 08.
Artículo en Inglés | MEDLINE | ID: mdl-36367955

RESUMEN

We present in situ calorimetry, thermal conductivity, and thermal diffusivity measurements of materials using temperature-sensing optical wireless integrated circuits (OWiCs). These microscopic and untethered optical sensors eliminate input wires and reduce parasitic effects. Each OWiC has a mass of ∼100 ng, a 100-µm-scale footprint, and a thermal response time of microseconds. We demonstrate that they can measure the thermal properties of nearly any material, from aerogels to metals, on samples as small as 100 ng and over thermal diffusivities covering four orders of magnitude. They also function over a broad temperature range, and we present proof-of-concept measurements of the thermodynamic phase transitions in both liquid crystal 5CB and gadolinium.


Asunto(s)
Cristales Líquidos , Conductividad Térmica , Temperatura , Calorimetría , Termodinámica
7.
Proc Natl Acad Sci U S A ; 119(11): e2112382119, 2022 03 15.
Artículo en Inglés | MEDLINE | ID: mdl-35271392

RESUMEN

SignificanceUnderstanding the molecular forces driving the unfolded polypeptide chain to self-assemble into a functional native structure remains an open question. However, identifying the states visited during protein folding (e.g., the transition state between the unfolded and native states) is tricky due to their transient nature. Here, we introduce calorimetric force spectroscopy in a temperature jump optical trap to determine the enthalpy, entropy, and heat capacity of the transition state of protein barnase. We find that the transition state has the properties of a dry molten globule, that is, high free energy and low configurational entropy, being structurally similar to the native state. This experimental single-molecule study characterizes the thermodynamic properties of the transition state in funneled energy landscapes.


Asunto(s)
Proteínas Bacterianas , Pinzas Ópticas , Pliegue de Proteína , Ribonucleasas , Imagen Individual de Molécula , Proteínas Bacterianas/química , Calorimetría/métodos , Conformación Proteica , Desnaturalización Proteica , Ribonucleasas/química , Imagen Individual de Molécula/métodos , Termodinámica
8.
Biochemistry ; 63(1): 116-127, 2024 01 02.
Artículo en Inglés | MEDLINE | ID: mdl-38127721

RESUMEN

FixL is an oxygen-sensing heme-PAS protein that regulates nitrogen fixation in the root nodules of plants. In this paper, we present the first photothermal studies of the full-length wild-type FixL protein from Sinorhizobium meliloti and the first thermodynamic profile of a full-length heme-PAS protein. Photoacoustic calorimetry studies reveal a quadriphasic relaxation for SmFixL*WT and the five variant proteins (SmFixL*R200H, SmFixL*R200Q, SmFixL*R200E, SmFixL*R200A, and SmFixL*I209M) with four intermediates from <20 ns to ∼1.5 µs associated with the photodissociation of CO from the heme. The altered thermodynamic profiles of the full-length SmFixL* variant proteins confirm that the conserved heme domain residues R200 and I209 are important for signal transduction. In contrast, the truncated heme domain, SmFixLH128-264, shows only a single, fast monophasic relaxation at <50 ns associated with the fast disruption of a salt bridge and release of CO to the solvent, suggesting that the full-length protein is necessary to observe the conformational changes that propagate the signal from the heme domain to the kinase domain.


Asunto(s)
Hemoproteínas , Sinorhizobium meliloti , Proteínas Quinasas/metabolismo , Histidina Quinasa/genética , Histidina Quinasa/metabolismo , Sinorhizobium meliloti/química , Hemo/química , Ligandos , Hemoproteínas/metabolismo , Oxígeno/metabolismo , Calorimetría , Proteínas Bacterianas/química
9.
J Biol Chem ; 299(2): 102854, 2023 02.
Artículo en Inglés | MEDLINE | ID: mdl-36592931

RESUMEN

Carbohydrate-binding modules (CBMs) are the noncatalytic modules that assist functions of the catalytic modules in carbohydrate-active enzymes, and they are usually discrete structural domains in larger multimodular enzymes. CBMs often occur in tandem in different alginate lyases belonging to the CBM families 13, 16, and 32. However, none of the currently known CBMs in alginate lyases specifically bind to an internal alginate chain. In our investigation of the multidomain alginate lyase Dp0100 carrying several ancillary domains, we identified an alginate-binding domain denoted TM6-N4 using protein truncation analysis. The structure of this CBM domain was determined at 1.35 Å resolution. TM6-N4 exhibited an overall ß-sandwich fold architecture with two antiparallel ß-sheets. We identified an extended binding groove in the CBM using site-directed mutagenesis, docking, and surface electrostatic potential analysis. Affinity analysis revealed that residues of Lys10, Lys22, Lys25, Lys27, Lys31, Arg36, and Tyr159 located on the bottom or the wall of the shallow groove are responsible for alginate binding, and isothermal titration calorimetry analyses indicated that the binding cleft consists of six subsites for sugar recognition. This substrate binding pattern is typical for type B CBM, and it represents the first CBM domain that specifically binds internal alginate chain. Phylogenetic analysis supports that TM6-N4 constitutes the founding member of a new CBM family denoted as CBM96. Our reported structure not only facilitates the investigation of the CBM-alginate ligand recognition mechanism but also inspires the utilization of the CBM domain in biotechnical applications.


Asunto(s)
Alginatos , Carbohidratos , Humanos , Alginatos/química , Calorimetría , Carbohidratos/química , Cristalografía por Rayos X , Mutagénesis Sitio-Dirigida , Filogenia , Unión Proteica
10.
J Biol Chem ; 299(9): 105098, 2023 09.
Artículo en Inglés | MEDLINE | ID: mdl-37507023

RESUMEN

RasGAP (p120RasGAP), the founding member of the GTPase-activating protein (GAP) family, is one of only nine human proteins to contain two SH2 domains and is essential for proper vascular development. Despite its importance, its interactions with key binding partners remains unclear. In this study we provide a detailed viewpoint of RasGAP recruitment to various binding partners and assess their impact on RasGAP activity. We reveal the RasGAP SH2 domains generate distinct binding interactions with three well-known doubly phosphorylated binding partners: p190RhoGAP, Dok1, and EphB4. Affinity measurements demonstrate a 100-fold weakened affinity for RasGAP-EphB4 binding compared to RasGAP-p190RhoGAP or RasGAP-Dok1 binding, possibly driven by single versus dual SH2 domain engagement with a dominant N-terminal SH2 interaction. Small-angle X-ray scattering reveals conformational differences between RasGAP-EphB4 binding and RasGAP-p190RhoGAP binding. Importantly, these interactions do not impact catalytic activity, implying RasGAP utilizes its SH2 domains to achieve diverse spatial-temporal regulation of Ras signaling in a previously unrecognized fashion.


Asunto(s)
Proteínas Tirosina Quinasas Receptoras , Proteína Activadora de GTPasa p120 , Humanos , Proteínas Activadoras de GTPasa/metabolismo , Proteína Activadora de GTPasa p120/química , Fosforilación , Proteínas Activadoras de ras GTPasa/química , Proteínas Activadoras de ras GTPasa/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal , Dominios Homologos src , Calorimetría , Péptidos/metabolismo , Modelos Moleculares , Estructura Terciaria de Proteína , Dispersión del Ángulo Pequeño
11.
Biochem Biophys Res Commun ; 695: 149467, 2024 02 05.
Artículo en Inglés | MEDLINE | ID: mdl-38211531

RESUMEN

Staphylococcus aureus (S. aureus), a Gram-positive bacterium, causes a wide range of infections, and diagnosis at an early stage is challenging. Targeting the maltodextrin transporter has emerged as a promising strategy for imaging bacteria and has been able to image a wide range of bacteria including S. aureus. However, little is known about the maltodextrin transporter in S. aureus, and this prevents new S. aureus specific ligands for the maltodextrin transporter from being developed. In Gram-positive bacteria, including S. aureus, the first step of maltodextrin transport is the binding of the maltodextrin-binding protein malE to maltodextrins. Thus, understanding the binding affinity and characteristics of malE from S. aureus is important to developing efficient maltodextrin-based imaging probes. We evaluated the affinity of malE of S. aureus to maltodextrins of various lengths. MalE of S. aureus (SAmalE) was expressed in E. coli BL21(DE3) and purified by Ni-NTA resin. The affinities of SAmalE to maltodextrins were evaluated with isothermal titration calorimetry. SAmalE has low affinity to maltose but binds to maltotriose and longer maltodextrins up to maltoheptaose with affinities up to Ka = 9.02 ± 0.49 × 105 M-1. SAmalE binding to maltotriose-maltoheptaose was exothermic and fit a single-binding site model. The van't Hoff enthalpy in the binding reaction of SAmalE with maltotriose was 9.9 ± 1.3 kcal/mol, and the highest affinity of SAmalE was observed with maltotetraose with Ka = 9.02 ± 0.49 × 105 M-1. In the plot of ΔH-T*ΔS, the of Enthalpy-Entropy Compensation effect was observed in binding reaction of SAmalE to maltodextrins. Acarbose and maltotetraiol bind with SAmalE indicating that SAmalE is tolerant of modifications on both the reducing and non-reducing ends of maltodextrins. Our results show that unlike ECmalE and similar to the maltodextrin binding protein of Streptococci, SAmalE primarily binds to maltodextrins via hydrogen bonds. This is distinct from the maltodextrin binding protein of Streptococci, SAmalE that binds to maltotetraiol with high affinity. Understanding the binding characteristics and tolerance to maltodextrins modifications by maltodextrin binding proteins will hopefully provide the basis for developing bacterial species-specific maltodextrin-based imaging probes.


Asunto(s)
Proteínas Portadoras , Staphylococcus aureus , Proteínas Portadoras/metabolismo , Staphylococcus aureus/metabolismo , Escherichia coli/metabolismo , Oligosacáridos/metabolismo , Proteínas Bacterianas/metabolismo , Polisacáridos/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Calorimetría , Unión Proteica
12.
Biochem Biophys Res Commun ; 711: 149908, 2024 06 04.
Artículo en Inglés | MEDLINE | ID: mdl-38613867

RESUMEN

RNA aptamers are oligonucleotides, selected through Systematic Evolution of Ligands by EXponential Enrichment (SELEX), that can bind to specific target molecules with high affinity. One such molecule is the RNA aptamer that binds to a blue-fluorescent Hoechst dye that was modified with bulky t-Bu groups to prevent non-specific binding to DNA. This aptamer has potential for biosensor applications; however, limited information is available regarding its conformation, molecular interactions with the ligand, and binding mechanism. The study presented here aims to biophysically characterize the Hoechst RNA aptamer when complexed with the t-Bu Hoechst dye and to further optimize the RNA sequence by designing and synthesizing new sequence variants. Each variant aptamer-t-Bu Hoechst complex was evaluated through a combination of fluorescence emission, native polyacrylamide gel electrophoresis, fluorescence titration, and isothermal titration calorimetry experiments. The results were used to design a minimal version of the aptamer consisting of only 21 nucleotides. The performed study also describes a more efficient method for synthesizing the t-Bu Hoechst dye derivative. Understanding the biophysical properties of the t-Bu Hoechst dye-RNA complex lays the foundation for nuclear magnetic resonance spectroscopy studies and its potential development as a building block for an aptamer-based biosensor that can be used in medical, environmental or laboratory settings.


Asunto(s)
Aptámeros de Nucleótidos , Aptámeros de Nucleótidos/química , Colorantes Fluorescentes/química , Conformación de Ácido Nucleico , Técnicas Biosensibles/métodos , Secuencia de Bases , Espectrometría de Fluorescencia/métodos , Técnica SELEX de Producción de Aptámeros/métodos , Calorimetría/métodos , ARN/química
13.
Chembiochem ; 25(12): e202400154, 2024 Jun 17.
Artículo en Inglés | MEDLINE | ID: mdl-38616168

RESUMEN

Histamine is a biogenic amine that poses a potential threat to public health due to its toxicological effects. In this study, we identified histamine-binding peptides by screening a random 12-mer peptide library, employing a novel biopanning approach that excluded histidine-binding sequences in the final round. This additional step enhanced the selectivity of the peptides and prevented interference from histidine during detection. The binding affinities of synthesized peptides to histamine were assessed using isothermal titration calorimetry (ITC). Among the identified peptides, HBF10 (SGFRDGIEDFLW) and HBF26 (IPLENQHKIYST) showed significant affinity to histamine, with Ka values of 2.56×104 (M-1) and 8.94×104 (M-1), respectively. Notably, the identified peptides did not demonstrate binding affinity towards histidine, despite its structural similarity to histamine. Subsequently, the surface plasmon resonance (SPR) sensor surface was prepared by immobilizing the peptide HBF26 to investigate the potential of the peptide as a recognition agent for histamine detection. The findings suggest that the identified peptides have an affinity to histamine specifically, showcasing their potential applications as diagnostic agents with specific targeting capabilities.


Asunto(s)
Histamina , Biblioteca de Péptidos , Péptidos , Resonancia por Plasmón de Superficie , Histamina/química , Histamina/metabolismo , Péptidos/química , Péptidos/metabolismo , Calorimetría , Secuencia de Aminoácidos , Unión Proteica , Humanos
14.
Chembiochem ; 25(14): e202400116, 2024 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-38668388

RESUMEN

The 4-aminoquinoline class of compounds includes the important antimalarial compounds amodiaquine and chloroquine. Despite their medicinal importance, the mode of action of these compounds is poorly understood. In a previous study we observed these compounds, as well as quinine and mefloquine, tightly bind the DNA cocaine-binding aptamer. Here, we further explore the range of nucleic acid structures bound by these compounds. To gauge a wide range of binding affinities, we used isothermal titration calorimetry to explore high affinity binding (nM to tens of µM) and NMR spectroscopy to assay weak binding biding in the hundreds of micromolar range. We find that amodiaquine tightly binds all double stranded DNA structures explored. Mefloquine binds double stranded DNA duplex molecules tightly and weakly associates with a three-way junction DNA construct. Quinine and chloroquine only weakly bind duplex DNA but do not tightly bind any of the DNA constructs explored. A simulation of the free energy of binding of these ligands to the Dickerson-Drew dodecamer resulted in an excellent agreement between the simulated and experimental free energy. These results provide new insight into the DNA binding of clinically important antimalarial compounds and may play a role in future development of new antimalarials.


Asunto(s)
Amodiaquina , ADN , ADN/química , ADN/metabolismo , Amodiaquina/química , Amodiaquina/metabolismo , Amodiaquina/análogos & derivados , Antimaláricos/química , Antimaláricos/metabolismo , Conformación de Ácido Nucleico , Sitios de Unión , Termodinámica , Calorimetría
15.
J Mol Recognit ; 37(2): e3075, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-38191989

RESUMEN

The binding of four alkaloids with human serum albumin (HSA) was investigated by isothermal titration calorimetry (ITC), spectroscopy and molecular docking techniques. The findings demonstrated that theophylline or caffeine can bind to HAS, respectively. The number of binding sites and binding constants are obtained. The binding mode is a static quenching process. The effects of steric hindrance, temperature, salt concentration and buffer solution on the binding indicated that theophylline and HSA have higher binding affinity than caffeine. The fluorescence and ITC results showed that the interaction between HSA and theophylline or caffeine is an entropy-driven spontaneous exothermic process. The hydrophobic force was the primary driving factor. The experimental results were consistent with the molecular docking data. Based on the molecular structures of the four alkaloids, steric hindrance might be a major factor in the binding between HSA and these four alkaloids. This study elucidates the mechanism of interactions between four alkaloids and HSA.


Asunto(s)
Alcaloides , Albúmina Sérica Humana , Humanos , Albúmina Sérica Humana/química , Simulación del Acoplamiento Molecular , Cafeína , Teofilina , Espectrometría de Fluorescencia , Termodinámica , Sitios de Unión , Calorimetría/métodos , Unión Proteica , Dicroismo Circular
16.
Arch Biochem Biophys ; 756: 109995, 2024 06.
Artículo en Inglés | MEDLINE | ID: mdl-38621448

RESUMEN

T4 polynucleotide kinase (T4 PNK) phosphorylates the 5'-terminus of DNA and RNA substrates. It is widely used in molecular biology. Single nucleotides can serve as substrates if a 3'-phosphate group is present. In this study, the T4 PNK-catalyzed conversion of adenosine 3'-monophosphate (3'-AMP) to adenosine-3',5'-bisphosphate was characterized using isothermal titration calorimetry (ITC). Although ITC is typically used to study ligand binding, in this case the instrument was used to evaluate enzyme kinetics by monitoring the heat production due to reaction enthalpy. The reaction was initiated with a single injection of 3'-AMP substrate into the sample cell containing T4 PNK and ATP at pH 7.6 and 30 °C, and Michaelis-Menten analysis was performed on the reaction rates derived from the plot of differential power versus time. The Michaelis-Menten constant, KM, was 13 µM, and the turnover number, kcat, was 8 s-1. The effect of inhibitors was investigated using pyrophosphate (PPi). PPi caused a dose-dependent decrease in the apparent kcat and increase in the apparent KM under the conditions tested. Additionally, the intrinsic reaction enthalpy and the activation energy of the T4 PNK-catalyzed phosphorylation of 3'-AMP were determined to be -25 kJ/mol and 43 kJ/mol, respectively. ITC is seldom used as a tool to study enzyme kinetics, particularly for technically-challenging enzymes such as kinases. This study demonstrates that quantitative analysis of kinase activity can be amenable to the ITC single injection approach.


Asunto(s)
Calorimetría , Polinucleótido 5'-Hidroxil-Quinasa , Cinética , Calorimetría/métodos , Polinucleótido 5'-Hidroxil-Quinasa/metabolismo , Polinucleótido 5'-Hidroxil-Quinasa/química , Adenosina Monofosfato/química , Adenosina Monofosfato/metabolismo , Termodinámica , Bacteriófago T4/enzimología , Difosfatos/química , Difosfatos/metabolismo , Fosforilación
17.
Anal Biochem ; 694: 115602, 2024 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-38977233

RESUMEN

Modern isothermal titration calorimetry instruments give great precision, but for comparable accuracy they require chemical calibration. For the heat factor, one recommended process is HCl into the weak base TRIS. In studying this reaction with a VP-ITC and two Nano-ITCs, we have encountered some problems, most importantly a titrant volume shortfall Δv ≈ 0.3 µL, which we attribute to diffusive loss of HCl in the syringe tip. This interpretation is supported by a mathematical treatment of the diffusion problem. The effect was discovered through a variable-v protocol, which thus should be used to properly allow for it in any reaction that similarly approaches completion. We also find that the effects from carbonate contamination and from OH- from weak base hydrolysis can be more significant that previously thought. To facilitate proper weighting in the least-squares fitting of data, we have estimated data variance functions from replicate data. All three instruments have low-signal precision of σ ≈ 1 µJ; titrant volume uncertainty is a factor of ∼2 larger for the Nano-ITCs than for the VP-ITC. The final heat factors remain uncertain by more than the ∼1 % precision of the instruments and are unduly sensitive to the HCl concentration.


Asunto(s)
Calorimetría , Calorimetría/métodos , Calibración , Ácido Clorhídrico/química
18.
Anal Biochem ; 687: 115446, 2024 04.
Artículo en Inglés | MEDLINE | ID: mdl-38147946

RESUMEN

Binding interactions often involve heterogeneous samples displaying a distribution of binding sites that vary in affinity and binding enthalpy. Examples include biological samples like proteins and chemically produced samples like modified cyclodextrins. Experimental studies often ignore sample heterogeneity and treat the system as an interaction of two homogeneous species, i.e. a chemically well-defined ligand binding to one type of site. The present study explores, by simulations and experiments, the impact of heterogeneity in isothermal titration calorimetry (ITC) setups where one of the binding components is heterogeneous. It is found that the standard single-site model, based on the assumption of two homogeneous binding components, provides excellent fits to simulated ITC data when the binding free energy is normally distributed and all sites have similar binding enthalpies. In such cases, heterogeneity can easily go undetected but leads to underestimated binding constants. Heterogeneity in the binding enthalpy is a bigger problem and may result in enthalpograms of increased complexity that are likely to be misinterpreted as two-site binding or other complex binding models. Finally, it is shown that heterogeneity can account for previously observed experimental anomalies. All simulations are accessible in Google Colab for readers to experiment with the simulation parameters.


Asunto(s)
Proteínas , Ligandos , Proteínas/química , Termodinámica , Entropía , Calorimetría , Unión Proteica
19.
Biomacromolecules ; 25(2): 903-923, 2024 Feb 12.
Artículo en Inglés | MEDLINE | ID: mdl-38170471

RESUMEN

Stimuli-responsive microgels with ionizable functional groups offer versatile applications, e.g., by the uptake of oppositely charged metal ions or guest molecules such as drugs, dyes, or proteins. Furthermore, the incorporation of carboxylic groups enhances mucoadhesive properties, crucial for various drug delivery applications. In this work, we successfully synthesized poly{N-vinylcaprolactam-2,2'-[(5-acrylamido-1-carboxypentyl)azanediyl]diacetic acid} [p(VCL/NTAaa)] microgels containing varying amounts of nitrilotriacetic acid (NTA) using precipitation polymerization. We performed fundamental characterization by infrared (IR) spectroscopy and dynamic and electrophoretic light scattering. Despite their potential multiresponsiveness, prior studies on NTA-functionalized microgels lack in-depth analysis of their stimuli-responsive behavior. This work addresses this gap by assessing the microgel responsiveness to temperature, ionic strength, and pH. Morphological investigations were performed via NMR relaxometry, nanoscale imaging (AFM and SEM), and reaction calorimetry. Finally, we explored the potential application of the microgels by conducting cytocompatibility experiments and demonstrating the immobilization of the model protein cytochrome c in the microgels.


Asunto(s)
Microgeles , Microgeles/química , Ácido Nitrilotriacético , Sistemas de Liberación de Medicamentos , Temperatura , Calorimetría
20.
Eur Biophys J ; 53(4): 225-238, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38613566

RESUMEN

Calibration of titration calorimeters is an ongoing problem, particularly with calorimeters with reaction vessel volumes < 10 mL in which an electrical calibration heater is positioned outside the calorimetric vessel. Consequently, a chemical reaction with a known enthalpy change must be used to accurately calibrate these calorimeters. This work proposes the use of standard solutions of potassium acid phthalate (KHP) titrated into solutions of excess sodium hydroxide (NaOH) or excess tris(hydroxymethyl)aminomethane (TRIS) as standard reactions to determine the collective accuracy of the relevant variables in a determination of the molar enthalpy change for a reaction. KHP is readily available in high purity, weighable for easy preparation of solutions with accurately known concentrations, stable in solution, not compromised by side reactions with common contaminants such as atmospheric CO2, and non-corrosive to materials used in calorimeter construction. Molar enthalpy changes for these reactions were calculated from 0 to 60 °C from reliable literature data for the pKa of KHP, the molar enthalpy change for protonation of TRIS, and the molar enthalpy change for ionization of water. The feasibility of using these reactions as enthalpic standards was tested in several calorimeters; a 50 mL CSC 4300, a 185 µL NanoITC, a 1.4 mL VP-ITC, and a TAM III with 1 mL reaction vessels. The results from the 50 mL CSC 4300, which was accurately calibrated with an electric heater, verified the accuracy of the calculated standard values for the molar enthalpy changes of the proposed reactions.


Asunto(s)
Calorimetría , Hidróxido de Sodio , Trometamina , Hidróxido de Sodio/química , Calibración , Trometamina/química , Temperatura , Estándares de Referencia , Termodinámica
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