RESUMEN

Macromolecular crowding bridges in vivo and in vitro studies by simulating cellular complexities such as high viscosity and limited space while maintaining the experimental feasibility. Over the last two decades, the impact of macromolecular crowding on protein stability and activity has been a significant topic of study and discussion, though still lacking a thorough mechanistic understanding. This article investigates the role of associated water dynamics on protein stability and activity within crowded environments, using bromelain and Ficoll-70 as the model systems. Traditional crowding theory primarily attributes protein stability to entropic effects (excluded volume) and enthalpic interactions. However, our recent findings suggest that water structure modulation plays a crucial role in a crowded environment. In this report, we strengthen the conclusion of our previous study, i.e., rigid-associated water stabilizes proteins via entropy and destabilizes them via enthalpy, while flexible water has the opposite effect. In the process, we addressed previous shortcomings with a systematic concentration-dependent study using a single-domain protein and component analysis of solvation dynamics. More importantly, we analyze bromelain's hydrolytic activity using the Michaelis-Menten model to understand kinetic parameters like maximum velocity (Vmax) achieved by the system and the Michaelis-Menten coefficient (KM). Results indicate that microviscosity (not the bulk viscosity) controls the enzyme-substrate (ES) complex formation, where an increase in the microviscosity makes the ES complex formation less favorable. On the other hand, flexible associated water dynamics were found to favor the rate of product formation significantly from the ES complex, while rigid associated water hinders it. This study improves our understanding of protein stability and activity in crowded environments, highlighting the critical role of associated water dynamics.

Asunto(s)

Bromelaínas , Agua , Agua/química , Bromelaínas/química , Bromelaínas/metabolismo , Estabilidad Proteica , Cinética , Ficoll/química , Termodinámica , Viscosidad

RESUMEN

Given the fact that the cellular interior is crowded by many different kinds of macromolecules, it is important that in vitro studies be carried out in the presence of mixed crowder systems. In this regard, we have used binary crowders formed by the combination of some of the commonly used crowding agents, namely, Ficoll 70, Dextran 70, Dextran 40, and PEG 8000 (PEG 8), to study how these affect enzyme activity, dynamics, and crowder diffusion. The enzyme chosen is AK3L1, an isoform of adenylate kinase. To investigate its dynamics, we have carried out three single point mutations (A74C, A132C, and A209C) with the cysteine residues being labeled with a coumarin-based solvatochromic probe [CPM: (7-diethylamino-3-(4-maleimido-phenyl)-4-methylcoumarin)]. Both enzyme activity and dynamics decreased in the binary mixtures as compared with the sum of the individual crowders, suggesting a reduction in excluded volume (in the mixture). To gain deeper insights into the binary mixtures, fluorescence correlation spectroscopy studies were carried out using fluorescein isothiocyanate-labeled Dextran 70 and tetramethylrhodamine-labeled AK3L1 as the diffusion probes. Diffusion in binary mixtures was observed to be much more constrained (relative to the sum of the individual crowders) for the labeled enzyme as compared to the labeled crowder showing different environments being faced by the two species. This was further confirmed during imaging of the phase-separated droplets formed in the binary mixtures having PEG as one of the crowding agents. The interior of these droplets was found to be rich in crowders and densely packed, as shown by confocal and digital holographic microscopy images, with the enzymes predominantly residing outside these droplets, that is, in the relatively less crowded regions. Taken together, our data provide important insights into various aspects of the simplest form of mixed crowding, that is, composed of just two components, and also hint at the enhanced complexity that the cellular interior presents toward having a detailed and comprehensive understanding of the same.

Asunto(s)

Adenilato Quinasa , Polietilenglicoles , Difusión , Adenilato Quinasa/metabolismo , Adenilato Quinasa/química , Adenilato Quinasa/genética , Polietilenglicoles/química , Ficoll/química , Dextranos/química , Dextranos/metabolismo , Espectrometría de Fluorescencia , Mutación Puntual , Cumarinas/química , Cumarinas/metabolismo

RESUMEN

The cellular environment is crowded with macromolecules of different shapes and sizes. The effect of this macromolecular crowding has been studied in a variety of synthetic crowding environments: two popular examples are the compact colloid-like Ficoll macromolecule and the globular protein bovine serum albumin (BSA). Recent studies have indicated that a significant component of bound or surface-associated water in these crowders reduces the available free volume. In this work, Brillouin light scattering experiments were performed on aqueous solutions of Ficoll 70 and Ficoll 400 with concentrations ranging from 1 to 35 wt % and BSA with concentrations of 1 to 27 wt %. From the dependence of spectral peak parameters on polymer concentration, we determined fundamental solution properties: hypersound velocity, adiabatic bulk modulus and compressibility, apparent viscosity, and hypersound attenuation. The existing theory that ignores intermolecular interactions can capture only the observed linear trends in the frequency shift up to a threshold concentration, beyond which a quadratic term accounting for intermolecular interactions is necessary. This likely indicates a transition from the dilute to semidilute regime. In the Ficoll solutions (but not BSA), we see evidence for a central mode, which is indicative of relaxation in the hydration shell of Ficoll.

Asunto(s)

Albúmina Sérica Bovina , Agua , Ficoll/química , Albúmina Sérica Bovina/química , Sustancias Macromoleculares , Análisis Espectral , Soluciones/química

RESUMEN

Macromolecular crowding affects many cellular processes such as diffusion, biochemical reaction kinetics, protein-protein interactions, and protein folding. Mapping the heterogeneous, dynamic crowding in living cells or tissues requires genetically encoded, site-specific, crowding sensors that are compatible with quantitative, noninvasive fluorescence micro-spectroscopy. Here, we carried out time-resolved 2P-fluorescence measurements of a new mEGFP-linker-mScarlet-I macromolecular crowding construct (GE2.3) to characterize its environmental sensitivity in biomimetic crowded solutions (Ficoll-70, 0-300 g L-1) via Förster resonance energy transfer (FRET) analysis. The 2P-fluorescence lifetime of the donor (mEGFP) was measured under magic-angle polarization, in the presence (intact) and absence (enzymatically cleaved) of the acceptor (mScarlet-I), as a function of the Ficoll-70 concentration. The FRET efficiency was used to quantify the sensitivity of GE2.3 to macromolecular crowding and to determine the environmental dependence of the mEGFP-mScarlet-I distance. We also carried out time-resolved 2P-fluorescence depolarization anisotropy to examine both macromolecular crowding and linker flexibility effects on GE2.3 rotational dynamics within the context of the Stokes-Einstein model as compared with theoretical predictions based on its molecular weight. These time-resolved 2P-fluorescence depolarization measurements and conformational population analyses of GE2.3 were also used to estimate the free energy gain upon the structural collapse in crowded environment. Our results further the development of a rational engineering design for bioenvironmental sensors without the interference of cellular autofluorescence. Additionally, these results in well-defined environments will inform our future in vivo studies of genetically encoded GE2.3 towards the mapping of the crowded intracellular environment under different physiological conditions.

Asunto(s)

Técnicas Biosensibles , Transferencia Resonante de Energía de Fluorescencia , Ficoll/química , Espectrometría de Fluorescencia , Transferencia Resonante de Energía de Fluorescencia/métodos , Sustancias Macromoleculares/metabolismo , Ambiente Controlado

RESUMEN

Because steric crowding is most effective when the crowding agent is similar in size to the molecule that it acts upon and the average macromolecule inside cells is much larger than a small protein or peptide, steric crowding is not predicted to affect their folding inside cells. On the other hand, chemical interactions should perturb in-cell structure and stability because they arise from interactions between the surface of the small protein or peptide and its environment. Indeed, previous in vitro measurements of the λ-repressor fragment, λ6-85 , in crowding matrices comprised of Ficoll or protein crowders support these predictions. Here, we directly quantify the in-cell stability of λ6-85 and distinguish the contribution of steric crowding and chemical interactions to its stability. Using a FRET-labeled λ6-85 construct, we find that the fragment is stabilized by 5°C in-cells compared to in vitro. We demonstrate that this stabilization cannot be explained by steric crowding because, as anticipated, Ficoll has no effect on λ6-85 stability. We find that the in-cell stabilization arises from chemical interactions, mimicked in vitro by mammalian protein extraction reagent (M-PER™). Comparison between FRET values in-cell and in Ficoll confirms that U-2 OS cytosolic crowding is reproduced at macromolecule concentrations of 15% w/v. Our measurements validate the cytomimetic of 15% Ficoll and 20% M-PER™ that we previously developed for protein and RNA folding studies. However, because the in-cell stability of λ6-85 is reproduced by 20% v/v M-PER™ alone, we predict that this simplified mixture could be a useful tool to predict the in-cell behaviors of other small proteins and peptides.

Asunto(s)

Mamíferos , Pliegue de Proteína , Animales , Ficoll/química , Estabilidad Proteica

RESUMEN

The cellular environment is crowded by macromolecules of various sizes, shapes, and charges, which modulate protein structure, function and dynamics. Herein, we contemplated the effect of three different macromolecular crowders: dextran-40, Ficoll-70 and PEG-35 on the structure, active-site conformational dynamics, function and relative domain movement of multi-domain human serum albumin (HSA). All the crowders used in this study have zero charges and similar sizes (at least in the dilute region) but different shapes and compositions. Some observations follow the traditional crowding theory. For example, all the crowders increased the α-helicity of HSA and hindered the conformational fluctuation dynamics. However, some observations are not in line with the expectations, such as an increase in the size of HSA with PEG-35 and uncorrelated domain movement of HSA with Ficoll-70 and PEG-35. The relative domain movement is correlated with the activity, suggesting that such moves are essential for protein function. The interaction between HSA and Ficoll-70 is proposed to be hydrophobic in nature. Overall, our results provide a somewhat systematic study of the shape-dependent macromolecular crowding effect on various protein properties and present a possible new insight into the mechanism of macromolecular crowding.

Asunto(s)

Proteínas , Albúmina Sérica Humana , Ficoll/química , Humanos , Sustancias Macromoleculares/química , Conformación Molecular , Proteínas/química

RESUMEN

PURPOSE: Vitreous humor is a complex biofluid whose composition determines its structure and function. Vitreous viscosity will affect the delivery, distribution, and half-life of intraocular drugs, and key physiological molecules. The central pig vitreous is thought to closely match human vitreous viscosity. Diffusion is inversely related to viscosity, and diffusion is of fundamental importance for all biochemical reactions. Fluorescence Recovery After Photobleaching (FRAP) may provide a novel means of measuring intravitreal diffusion that could be applied to drugs and physiological macromolecules. It would also provide information about vitreous viscosity, which is relevant to drug elimination, and delivery. METHODS: Vitreous viscosity and intravitreal macromolecular diffusion of fluorescently labelled macromolecules were investigated in porcine eyes using fluorescence recovery after photobleaching (FRAP). Fluorescein isothiocyanate conjugated (FITC) dextrans and ficolls of varying molecular weights (MWs), and FITC-bovine serum albumin (BSA) were employed using FRAP bleach areas of different diameters. RESULTS: The mean (±standard deviation) viscosity of porcine vitreous using dextran, ficoll and BSA were 3.54 ± 1.40, 2.86 ± 1.13 and 4.54 ± 0.13 cP respectively, with an average of 3.65 ± 0.60 cP. CONCLUSIONS: FRAP is a feasible and practical optical method to quantify the diffusion of macromolecules through vitreous.

Asunto(s)

Recuperación de Fluorescencia tras Fotoblanqueo/métodos , Cuerpo Vítreo/metabolismo , Animales , Bevacizumab/química , Bevacizumab/metabolismo , Dextranos/química , Difusión , Ficoll/química , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/química , Ranibizumab/química , Ranibizumab/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular/química , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Albúmina Sérica Bovina/química , Porcinos , Viscosidad

RESUMEN

Macromolecular crowding, inside the physiological interior, modulates the energy landscape of biological macromolecules in multiple ways. Amongst these, enzymes occupy a special place and hence understanding the function of the same in the crowded interior is of utmost importance. In this study, we have investigated the manner in which the multidomain enzyme, AK3L1 (PDB ID: 1ZD8), an isoform of adenylate kinase, has its features affected in presence of commonly used crowders (PEG 8, Dextran 40, Dextran 70, and Ficoll 70). Michaelis Menten plots reveal that the crowders in general enhance the activity of the enzyme, with the Km and Vmax values showing significant variations. Ficoll 70, induced the maximum activity for AK3L1 at 100 g/L, beyond which the activity reduced. Ensemble FRET studies were performed to provide insights into the relative domain (LID and CORE) displacements in presence of the crowders. Solvation studies reveal that the protein matrix surrounding the probe CPM (7-diethylamino-3-(4-maleimido-phenyl)-4-methylcoumarin) gets restricted in presence of the crowders, with Ficoll 70 providing the maximum rigidity, the same being linked to the decrease in the activity of the enzyme. Through our multipronged approach, we have observed a distinct correlation between domain displacement, enzyme activity and associated dynamics. Thus, keeping in mind the complex nature of enzyme activity and the surrounding bath of dense soup that the biological entity remains immersed in, indeed more such approaches need to be undertaken to have a better grasp of the "enzymes in the crowd".

Asunto(s)

Adenilato Quinasa/química , Simulación de Dinámica Molecular , Dominio Catalítico , Dextranos/química , Ficoll/química , Transferencia Resonante de Energía de Fluorescencia , Polietilenglicoles/química , Solventes/química

RESUMEN

Many protein misfolding diseases (e.g. type II diabetes and Alzheimer's disease) are characterised by amyloid deposition. Human islet amyloid polypeptide (hIAPP, involved in type II diabetes) spontaneously undergoes liquid-liquid phase separation (LLPS) and a kinetically complex hydrogelation, both catalysed by hydrophobic-hydrophilic interfaces (e.g. air-water interface and/or phospholipids-water interfaces). Gelation of hIAPP phase-separated liquid droplets initiates amyloid aggregation and the formation of clusters of interconnected aggregates, which grow and fuse to eventually percolate the whole system. Droplet maturation into irreversible hydrogels via amyloid aggregation is thought to be behind the pathology of several diseases. Biological fluids contain a high volume fraction of macromolecules, leading to macromolecular crowding. Despite crowding agent addition in in vitro studies playing a significant role in changing protein phase diagrams, the mechanism underlying enhanced LLPS, and the effect(s) on stages beyond LLPS remain poorly or not characterised.We investigated the effect of macromolecular crowding and increased viscosity on the kinetics of hIAPP hydrogelation using rheology and the evolution of the system beyond LLPS by microscopy. We demonstrate that increased viscosity exacerbated the kinetic variability of hydrogelation and of the phase separated-aggregated system, whereas macromolecular crowding abolished heterogeneity. Increased viscosity also strengthened the gel meshwork and accelerated aggregate cluster fusion. In contrast, crowding either delayed cluster fusion onset (dextran) or promoted it (Ficoll). Our study highlights that an in vivo crowded environment would critically influence amyloid stages beyond LLPS and pathogenesis.

Asunto(s)

Amiloide/química , Proteínas Amiloidogénicas/química , Hidrogeles/química , Polipéptido Amiloide de los Islotes Pancreáticos/química , Agua/química , Enfermedad de Alzheimer/metabolismo , Dextranos/química , Diabetes Mellitus Tipo 2/metabolismo , Ficoll/química , Glicerol/química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Fosfolípidos/química , Agregado de Proteínas , Agregación Patológica de Proteínas , Factores de Tiempo , Viscosidad

RESUMEN

Macromolecular crowding can have significant consequences on the structure and dynamics of a protein. The size and shape of a co-solute molecule and the nature of protein contribute significantly in macromolecular crowding, which results in different outcomes in similar conditions. The structure of apo-myoglobin (apo-Mb) both in the absence and presence of denaturants (GdmCl and urea) was investigated in crowded conditions at pH 7.0, with a comparable size of crowders (~70 kDa) but of different shapes (ficoll and dextran) at various concentrations using spectroscopic techniques like absorption and circular dichroism to monitor changes in secondary and tertiary structure, respectively. The crowders in the absence of denaturants showed structural stabilization of the tertiary structure while no significant change in the secondary structure was observed. The effect of crowders on the stability of the protein was also investigated using probes such as Δε291 and θ222 using chemical denaturants. The analysis of chemical-induced denaturation curves showed that both the crowders stabilize apo-Mb by increasing the values of the midpoint of transition (Cm) and change in free energy in the absence of denaturant (∆GD°), and it was observed that dextran 70 shows more stabilization than ficoll 70 under similar conditions. In this study apo-Mb showed stabilization under crowded conditions, which is a deviation from earlier work from our group where holo form of the same protein was destabilized. This study emphasizes that volume exclusion is a dominant force in a simple protein while soft interactions may play important role in the proteins that are possessing prosthetic group. Hence, the effect of crowders is protein-dependent, and excluded volume plays a great role in the stabilization of apo-Mb, which does not interact with the crowders.

Asunto(s)

Apoproteínas/química , Guanidina/farmacología , Hemo/química , Sustancias Macromoleculares/química , Mioglobina/química , Desnaturalización Proteica , Urea/farmacología , Animales , Dextranos/química , Ficoll/química , Caballos , Conformación Proteica , Desnaturalización Proteica/efectos de los fármacos , Estabilidad Proteica/efectos de los fármacos , Espectrofotometría Ultravioleta , Termodinámica

RESUMEN

We previously developed a new vitrification method (equilibrium vitrification) by which two-cell mouse embryos can be vitrified in liquid nitrogen in a highly dehydrated/concentrated state using low concentrations of cryoprotectants. In the present study, we examined whether this method is effective for mouse embryos at multiple developmental stages. Four-cell embryos, eight-cell embryos, morulae, and blastocysts were vitrified with EDFS10/10a, 10% (v/v) ethylene glycol and 10% (v/v) DMSO in FSa solution. The FSa solution was PB1 medium containing 30% (w/v) Ficoll PM-70 plus 0.5 M sucrose. The state of dehydration/concentration was assessed by examining the survival of vitrified embryos after storage at -80°C. When four-cell embryos and eight-cell embryos were vitrified with EDFS10/10a in liquid nitrogen and then stored at -80°C, the survival rate was high, even after 28 days, with relatively high developmental ability. On the other hand, the survival of morulae and blastocysts vitrified in liquid nitrogen and stored at -80°C for four days was low. Therefore, morulae and blastocysts cannot be vitrified in a highly dehydrated/concentrated state using the same method as with two-cell embryos. However, when blastocysts were shrunken artificially before vitrification, survival was high after storage at -80°C for four days with high developmental ability. In conclusion, the equilibrium vitrification method using low concentrations of cryoprotectants, which is effective for two-cell mouse embryos, is also useful for embryos at multiple stages. This method enables the convenient transportation of vitrified embryos using dry ice.

Asunto(s)

Criopreservación/instrumentación , Crioprotectores/farmacología , Vitrificación , Animales , Blastocisto/patología , Blástula/patología , Supervivencia Celular , Hielo Seco , Glicol de Etileno/química , Femenino , Ficoll/química , Técnicas In Vitro , Ratones , Ratones Endogámicos ICR , Mórula/patología , Oocitos/citología , Concentración Osmolar , Manejo de Especímenes/métodos , Sacarosa/química , Temperatura

RESUMEN

Combining broadband dielectric spectroscopy and nuclear magnetic resonance studies, we analyze the reorientation dynamics and the translational diffusion associated with the glassy slowdown of the eutectic aqueous dimethyl sulfoxide solution in nano-sized confinements, explicitly, in silica pores with different diameters and in ficoll and lysozyme matrices at different concentrations. We observe that both rotational and diffusive dynamics are slower and more heterogeneous in the confinements than in the bulk but the degree of these effects depends on the properties of the confinement and differs for the components of the solution. For the hard and the soft matrices, the slowdown and the heterogeneity become more prominent when the size of the confinement is reduced. In addition, the dynamics are more retarded for dimethyl sulfoxide than for water, implying specific guest-host interactions. Moreover, we find that the temperature dependence of the reorientation dynamics and of the translational diffusion differs in severe confinements, indicating a breakdown of the Stokes-Einstein-Debye relation. It is discussed to what extent these confinement effects can be rationalized in the framework of core-shell models, which assume bulk-like and slowed-down motions in central and interfacial confinement regions, respectively.

Asunto(s)

Dimetilsulfóxido/química , Vidrio/química , Agua/química , Espectroscopía Dieléctrica/métodos , Difusión , Ficoll/química , Hidrodinámica , Espectroscopía de Resonancia Magnética , Modelos Estadísticos , Simulación de Dinámica Molecular , Muramidasa , Dióxido de Silicio/química , Solventes , Temperatura

RESUMEN

OBJECTIVE: To investigate the effects of macromolecular crowding on the folding and aggregation of MUC5AC with different levels of glycosylation during refolding. METHODS: Part 1:An in vitro catalytic reaction comprising the ppGalNAc T2 enzyme, uridine-5'-diphospho-N-galactosamine (UDP-GalNAc) and an 11-amino acid peptide substrate, was used to assess the enzyme activity of the ppGalNAc T2 enzyme in macromolecular crowding environment respectively with bovine serum albumin (BSA), polyethylene glycol (PEG2000), Dextran70 and Ficoll70 at different concentration and temperature. Part 2: The recombinant MUC5AC was expressed in HEK293 cells and purified by nickel column chromatography. The purified protein was treated with PNGase F, and the degree of glycosylation was analyzed by SDS-PAGE. Macromolecular crowding was simulated using PEG2000 at the concentrations of 50, 100, and 200 g/L. Deglycosylated-MUC5AC (d-MUC5AC) and glycosylated MUC5AC (g-MUC5AC) were denatured by GdnHCl and renatured by dilution in a refolding buffer. Protein aggregation was monitored continuously by absorbance reading at 488 nm using a UV spectrophotometer at 25 °C. The refolded proteins were centrifuged, the protein concentration of the supernatant was measured, and refolding yield in different refolding buffers was determined. RESULTS: Enzyme activityof ppGalNAc T2 was observed to increase with increasing crowding agent concentration, with highest enzyme activity at 200 g/L. Compared with the group in the absence of crowding reagent, the refolding yield of g-MUC5AC and d-MUC5AC were reduced significantly in the presence of different concentrations of PEG2000 (200, 100, and 50 g/L). Compared with the dilute solution, aggregation increased significantly in the presence of PEG2000, especially at 200 g/L. Moreover, in the crowded reagent with the same concentration, the refolding yield of d-MUC5AC was higher than that of g-MUC5AC, whereas the degree of aggregation of d-MUC5AC was lower than that of g-MUC5AC. CONCLUSION: The crowded intracellular environment reduces the refolding rate of MUC5AC and strongly induces the misfolding and aggregation of glycosylated MUC5AC.

Asunto(s)

Dextranos/farmacología , Ficoll/farmacología , Mucina 5AC/metabolismo , Polietilenglicoles/farmacología , Procesamiento Proteico-Postraduccional , Albúmina Sérica Bovina/farmacología , Secuencia de Aminoácidos , Animales , Bovinos , Clonación Molecular , Dextranos/química , Escherichia coli/genética , Escherichia coli/metabolismo , Ficoll/química , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Glicosilación/efectos de los fármacos , Células HEK293 , Humanos , Cinética , Mucina 5AC/química , Péptidos/síntesis química , Péptidos/metabolismo , Polietilenglicoles/química , Agregado de Proteínas/efectos de los fármacos , Pliegue de Proteína/efectos de los fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Albúmina Sérica Bovina/química , Uridina Difosfato N-Acetilgalactosamina/análogos & derivados , Uridina Difosfato N-Acetilgalactosamina/química , Uridina Difosfato N-Acetilgalactosamina/metabolismo

RESUMEN

Heat shock protein 90 (Hsp90) is a key regulator of nitric oxide synthase (NOS) in vivo. Despite its functional importance, little is known about the underlying molecular mechanism. Here, purified dimeric human Hsp90α was used to investigate whether (and if so, how) Hsp90 affects the FMN-heme interdomain electron transfer (IET) step in NOS. Hsp90α increases the IET rate for rat neuronal NOS (nNOS) in a dose-saturable manner, and a single charge-neutralization mutation at conserved Hsp90 K585 abolishes the effect. The kinetic results with added Ficoll 70, a crowder, further indicate that Hsp90 enhances the FMN-heme IET through specific association with nNOS. The Hsp90-nNOS docking models provide hints on the putative role of Hsp90 in constraining the available conformational space for the FMN domain motions.

Asunto(s)

Electrones , Mononucleótido de Flavina/química , Proteínas HSP90 de Choque Térmico/química , Hemo/química , Óxido Nítrico Sintasa de Tipo I/química , Animales , Ácido Aspártico/química , Ácido Aspártico/metabolismo , Sitios de Unión , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Ficoll/química , Mononucleótido de Flavina/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismo , Hemo/metabolismo , Humanos , Lisina/química , Lisina/metabolismo , Simulación del Acoplamiento Molecular , Mutación , NADP/química , NADP/metabolismo , Óxido Nítrico Sintasa de Tipo I/genética , Óxido Nítrico Sintasa de Tipo I/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Electricidad Estática

RESUMEN

BACKGROUND: Cryopreserved human peripheral blood mononuclear cells (PBMCs) are a commonly used sample type for a variety of immunological assays. Many factors can affect the quality of PBMCs, and careful consideration and validation of an appropriate PBMC isolation and cryopreservation method is important for well-designed clinical studies. A major point of divergence in PBMC isolation protocols is the collection of blood, either directly into vacutainers pre-filled with density gradient medium or the use of conical tubes containing a porous barrier to separate the density gradient medium from blood. To address potential differences in sample outcome, we isolated, cryopreserved, and compared PBMCs using parallel protocols differing only in the use of one of two common tube types for isolation. METHODS: Whole blood was processed in parallel using both Cell Preparation Tubes™ (CPT, BD Biosciences) and Lymphoprep™ Tubes (Axis-Shield) and assessed for yield and viability prior to cryopreservation. After thawing, samples were further examined by flow cytometry for cell yield, cell viability, frequency of 10 cell subsets, and capacity for stimulation-dependent CD4+ and CD8+ T cell intracellular cytokine production. RESULTS: No significant differences in cell recovery, viability, frequency of immune cell subsets, or T cell functionality between PBMC samples isolated using CPT or Lymphoprep tubes were identified. CONCLUSION: CPT and Lymphoprep tubes are effective and comparable methods for PBMC isolation for immunological studies.

Asunto(s)

Separación Celular/métodos , Criopreservación/métodos , Ficoll/química , Leucocitos Mononucleares/citología , Ácido Metrizoico/química , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Supervivencia Celular/inmunología , Células Cultivadas , Citocinas/inmunología , Citometría de Flujo/métodos , Humanos , Leucocitos Mononucleares/inmunología

RESUMEN

Pseudo-isocyanine chloride (PIC) is a cationic dyestuff that exhibits self-assembly in aqueous solution, promoted either by increasing the PIC concentration or by decreasing the temperature. PIC-aggregates exhibit a characteristic and sharp absorption band as well as a fluorescence band at a wavelength of 573 nm making PIC an interesting candidate to analyze the self-assembly process in various environments. The present work developed PIC-based, synthetic model systems, suitable to investigate how macromolecular crowding influences self-assembly processes. Four synthetic additives were used as potential crowders: Triethylene glycol (TEG), polyethylene glycol (PEG), Ficoll 400 as a highly branched polysaccharide, and sucrose corresponding to the monomeric unit of Ficoll. Combined UV/Vis spectroscopy and time-resolved light scattering revealed a strong impact of crowding based on excluded volume effects only for Ficoll 400. Sucrose had hardly any influence on the self-assembly of PIC and PEG and TEG impeded the PIC self-assembly. Development of such a PIC based model system led over to in-cell experiments. HeLa cells were infiltrated with PIC solutions well below the aggregation threshold in the infiltrating solution. In the cellular environment, PIC was exposed to a significant crowding and immediately started to aggregate. As was demonstrated by fluorescence imaging, the extent of aggregation can be modulated by exposing the cells to salt-induced osmotic stress. The results suggest future use of such a system as a sensor for the analysis of in vitro and in vivo crowding effects on self-assembly processes.

Asunto(s)

Cianuros/química , Ficoll/química , Polietilenglicoles/química , Fluorescencia , Células HeLa , Humanos , Sustancias Macromoleculares , Temperatura

RESUMEN

Generally, in vivo function and structural changes are studied by probing proteins in a dilute solution under in vitro conditions, which is believed to be mimicking proteins in intracellular milieu. Earlier, thermal-induced denaturation of myoglobin, in the milieu of crowder molecule showed destabilization of the metal protein. Destabilization of protein by thermal-induced denaturation involves a large extrapolation, so, the reliability is questionable. This led us to measure the effects of macromolecular crowding on its stability by chemical-induced denaturation of the protein using probes like circular dichroism and absorption spectroscopy in the presence of dextran 70 and ficoll 70 at various pHs (acidic: 6.0, almost neutral:7.0 and basic: 8.0). Observations showed that the degree of destabilization of myoglobin was greater due to ficoll 70 as compared to that of dextran 70 so it can be understood that the nature of the crowder or the shape of the crowder has an important role towards the stability of proteins. Additionally, the degree of destabilization was observed as pH dependent, however the pH dependence is different for different crowders. Furthermore, isothermal titration calorimetry and molecular docking studies confirmed that both the crowders (ficoll and dextran) bind to heme moiety of myoglobin and a single binding site was observed for each.

Asunto(s)

Dextranos/química , Ficoll/química , Hemo/química , Simulación del Acoplamiento Molecular , Mioglobina/química , Desnaturalización Proteica , Animales , Caballos

RESUMEN

The five members of the family of tumor suppressors ING contain a Plant Homeodomain (PHD) that specifically recognizes histone H3 trimethylated at lysine 4 (H3K4me3) with an affinity in the low micromolar range. Here, we use NMR to show that in the presence of 15% Ficoll 70, an inert macromolecular crowding agent, the mode of binding does not change but the affinity increases by one order of magnitude. The affinity increases also for unmethylated histone H3 tail, but the difference with H3K4me3 is larger in the presence of Ficoll. These results indicate that in the cellular milieu, the affinity of the ING proteins for their chromatin target is larger than previously thought.

Asunto(s)

Proteínas de Ciclo Celular/metabolismo , Histonas/metabolismo , Proteínas de Homeodominio/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Sitios de Unión , Proteínas de Ciclo Celular/química , Ficoll/química , Histonas/química , Proteínas de Homeodominio/química , Humanos , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Dominios Proteicos , Mapas de Interacción de Proteínas , Proteínas Supresoras de Tumor/química

RESUMEN

The cell is an extremely complex environment, notably highly crowded, segmented, and confining. Overall, there is overwhelming and ever-growing evidence that to understand how biochemical reactions proceed in vivo, one cannot separate the biochemical actors from their environment. Effects such as excluded volume, obstructed diffusion, weak nonspecific interactions, and fluctuations all team up to steer biochemical reactions often very far from what is observed in ideal conditions. In this paper, we use Ficoll PM70 and PEG 6000 to build an artificial crowded milieu of controlled composition and density in order to assess how such environments influence the biocatalytic activity of lactate dehydrogenase (LDH). Our measurements show that the normalized apparent affinity and maximum velocity decrease in the same fashion, a behavior reminiscent of uncompetitive inhibition, with PEG resulting in the largest reduction. In line with previous studies on other enzymes of the same family, and in agreement with the known role of a surface loop involved in enzyme isomerization and regulation of access to the active site, we suggest that the crowding matrix interferes with the conformational ensemble of the enzyme. This likely results in both impaired enzyme-complex isomerization and thwarted product release. Molecular dynamics simulations confirm that excluded-volume effects lead to an entropic force that effectively tends to push the loop closed, thereby effectively shifting the conformational ensemble of the enzyme in favor of a more stable complex isoform. Overall, our study substantiates the idea that most biochemical kinetics cannot be fully explained without including the subtle action of the environment where they take place naturally, in particular accounting for important factors such as excluded-volume effects and also weak nonspecific interactions when present, confinement, and fluctuations.

Asunto(s)

Ficoll/química , L-Lactato Deshidrogenasa/antagonistas & inhibidores , L-Lactato Deshidrogenasa/química , Biocatálisis , Dominio Catalítico , Difusión , Entropía , Humanos , Cinética , Simulación de Dinámica Molecular , NAD/química , Polietilenglicoles/química , Ácido Pirúvico/química

RESUMEN

The loss of albumin in urine (albuminuria) predicts cardiovascular outcome. Under physiological conditions, small amounts of albumin are filtered by the glomerulus and reabsorbed in the tubular system up until the absorption limit is reached. Early increases in pathological albumin filtration may, thus, be missed by analyzing albuminuria. Therefore, the use of tracers to test glomerular permselectivity appears advantageous. Fluorescently labeled tracer fluorescein isothiocyanate (FITC)-polysucrose (i.e., FITC-Ficoll), can be used to study glomerular permselectivity. FITC-polysucrose molecules are freely filtered by the glomerulus but not reabsorbed in the tubular system. In mice and rats, FITC-polysucrose has been investigated in models of glomerular permeability by using technically complex procedures (i.e., radioactive measurements, high-performance liquid chromatography [HPLC], gel filtration). We have modified and facilitated a FITC-polysucrose tracer-based protocol to test early and small increases in glomerular permeability to FITC-polysucrose 70 (size of albumin) in mice. This method allows repetitive urine analyses with small urine volumes (5 µL). This protocol contains information on how the tracer FITC-polysucrose 70 is applied intravenously and urine is collected via a simple urinary catheter. Urine is analyzed via a fluorescence plate reader and normalized to a urine concentration marker (creatinine), thereby avoiding technically complex procedures.

Asunto(s)

Ficoll/química , Fluoresceína-5-Isotiocianato/química , Fluoresceína-5-Isotiocianato/metabolismo , Glomérulos Renales/metabolismo , Animales , Femenino , Ratones , Permeabilidad , Ratas