Biomedical researchers have become increasingly aware of the limitations of conventional 2-dimensional tissue cell culture systems, including coated Petri dishes, multi-well plates and slides, to ...fully address many critical issues in cell biology, cancer biology and neurobiology, such as the 3-D microenvironment, 3-D gradient diffusion, 3-D cell migration and 3-D cell-cell contact interactions. In order to fully understand how cells behave in the 3-D body, it is important to develop a well-controlled 3-D cell culture system where every single ingredient is known. Here we report the development of a 3-D cell culture system using a designer peptide nanofiber scaffold with mouse adult neural stem cells. We attached several functional motifs, including cell adhesion, differentiation and bone marrow homing motifs, to a self-assembling peptide RADA16 (Ac-RADARADARADARADA-COHN2). These functionalized peptides undergo self-assembly into a nanofiber structure similar to Matrigel. During cell culture, the cells were fully embedded in the 3-D environment of the scaffold. Two of the peptide scaffolds containing bone marrow homing motifs significantly enhanced the neural cell survival without extra soluble growth and neurotrophic factors to the routine cell culture media. In these designer scaffolds, the cell populations with beta-Tubulin(+), GFAP(+) and Nestin(+) markers are similar to those found in cell populations cultured on Matrigel. The gene expression profiling array experiments showed selective gene expression, possibly involved in neural stem cell adhesion and differentiation. Because the synthetic peptides are intrinsically pure and a number of desired function cellular motifs are easy to incorporate, these designer peptide nanofiber scaffolds provide a promising controlled 3-D culture system for diverse tissue cells, and are useful as well for general molecular and cell biology.
The enzyme nitrogenase catalyzes the reduction of N2 to ammonia but also that of protons to H2. These reactions compete at the mechanistically central ‘Janus’ intermediate, denoted E4(4H), which has ...accumulated 4e–/4H+ as two bridging Fe–H–Fe hydrides on the active-site cofactor. This state can lose e–/H+ by hydride protonolysis (HP) or become activated by reductive elimination (re) of the two hydrides and bind N2 with H2 loss, yielding an E4(2N2H) state that goes on to generate two NH3 molecules. Thus, E4(4H) represents the key branch point for these competing reactions. Here, we present a steady-state kinetic analysis that precisely describes this competition. The analysis demonstrates that steady-state, high-electron flux turnover overwhelmingly populates the E4 states at the expense of less reduced states, quenching HP at those states. The ratio of rate constants for E4(4H) hydride protonolysis (k HP) versus reductive elimination (k re) provides a sensitive measure of competition between these two processes and thus is a central parameter of nitrogenase catalysis. Analysis of measurements with the three nitrogenase variants (Mo-nitrogenase, V-nitrogenase, and Fe-nitrogenase) reveals that at a fixed N2 pressure their tendency to productively react with N2 to produce two NH3 molecules and an accompanying H2, rather than diverting electrons to the side reaction, HP production of H2, decreases with their ratio of rate constants, k re /k HP: Mo-nitrogenase, 5.1 atm–1; V-nitrogenase, 2 atm–1; and Fe-nitrogenase, 0.77 atm–1 (namely, in a 1:0.39:0.15 ratio). Moreover, the lower catalytic effectiveness of the alternative nitrogenases, with more H2 production side reaction, is not caused by a higher k HP but by a significantly lower k re .
How protein stability and new functions trade off Tokuriki, Nobuhiko; Stricher, Francois; Serrano, Luis ...
PLOS computational biology/PLoS computational biology,
02/2008, Letnik:
4, Številka:
2
Journal Article
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Numerous studies have noted that the evolution of new enzymatic specificities is accompanied by loss of the protein's thermodynamic stability (DeltaDeltaG), thus suggesting a tradeoff between the ...acquisition of new enzymatic functions and stability. However, since most mutations are destabilizing (DeltaDeltaG>0), one should ask how destabilizing mutations that confer new or altered enzymatic functions relative to all other mutations are. We applied DeltaDeltaG computations by FoldX to analyze the effects of 548 mutations that arose from the directed evolution of 22 different enzymes. The stability effects, location, and type of function-altering mutations were compared to DeltaDeltaG changes arising from all possible point mutations in the same enzymes. We found that mutations that modulate enzymatic functions are mostly destabilizing (average DeltaDeltaG = +0.9 kcal/mol), and are almost as destabilizing as the "average" mutation in these enzymes (+1.3 kcal/mol). Although their stability effects are not as dramatic as in key catalytic residues, mutations that modify the substrate binding pockets, and thus mediate new enzymatic specificities, place a larger stability burden than surface mutations that underline neutral, non-adaptive evolutionary changes. How are the destabilizing effects of functional mutations balanced to enable adaptation? Our analysis also indicated that many mutations that appear in directed evolution variants with no obvious role in the new function exert stabilizing effects that may compensate for the destabilizing effects of the crucial function-altering mutations. Thus, the evolution of new enzymatic activities, both in nature and in the laboratory, is dependent on the compensatory, stabilizing effect of apparently "silent" mutations in regions of the protein that are irrelevant to its function.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Green photosynthetic bacteria, one of the phototrophs, have the largest and most efficient light-harvesting antenna systems, called chlorosomes. The core part of chlorosomes consists of unique ...bacteriochlorophyll c/d/e molecules. In the biosynthetic pathway of these molecules, a BciC enzyme catalyzes the removal of the C132-methoxycarbonyl group of chlorophyllide a. Two sequential reactions have been proposed for the BciC enzymatic demethoxycarbonylation: the BciC enzyme would catalyze the hydrolysis of the C132-methoxycarbonyl group, and the resulting carboxylic acid would be rapidly decarboxylated to generate pyrochlorophyllide a. In this study, we computationally predicted the three-dimensional structure of the BciC protein. Its active site was proposed based on structural analysis using docking simulation. In vitro enzymatic reaction assays of mutated BciC supported the prediction. The BciC enzymatic hydrolysis would be an aspartic/glutamic acid hydrolase, which involves the amino residues E85 and D180. Furthermore, Y58 and H126 might depend on stabilization and/or recognition with the substrate. Most importantly, H137 would protonate 13-CO or deprotonate C132-COOH in the hydrolyzed product to promote decarboxylation. In conclusion, the BciC enzyme has the dual functions of hydrolysis and decarboxylation.
The primary quinone electron acceptor QA is a key component in the electron transfer regulation in photosystem II (PSII), and hence accurate estimation of its redox potential, E m(QA –/QA), is ...crucial in understanding the regulatory mechanism. Although fluorescence detection has been extensively used for monitoring the redox state of QA, it was recently suggested that this method tends to provide a higher E m(QA –/QA) estimate depending on the sample status due to the effect of measuring light Kato et al. (2019) Biochim. Biophys. Acta 1860, 148082. In this study, we applied the Fourier transform infrared (FTIR) spectroelectrochemistry, which uses non-reactive infrared light to monitor the redox state of QA, to investigate the effects of stromal- and lumenal-side perturbations on E m(QA –/QA) in PSII. It was shown that replacement of bicarbonate bound to the non-heme iron with formate upshifted E m(QA –/QA) by ∼55 mV, consistent with the previous fluorescence measurement. In contrast, an E m(QA –/QA) difference between binding of 3-(3,4-dichlorophenyl)-1,1-dimethylurea and bromoxynil was found to be ∼30 mV, which is much smaller than the previous estimate, ∼100 mV, by the fluorescence method. This ∼30 mV difference was verified by the decay kinetics of the S2QA – recombination. On the lumenal side, Mn depletion hardly affected the E m(QA –/QA), confirming the previous FTIR result. However, removal of the extrinsic proteins by NaCl or CaCl2 wash downshifted the E m(QA –/QA) by 14–20 mV. These results suggest that electron flow through QA is regulated by changes both on the stromal and lumenal sides of PSII.
Since the establishment of site-specific mutagenesis of single amino acids to interrogate protein function in the 1970s, biochemists have sought to tailor protein structure in the native cell ...environment. Fine-tuning the chemical properties of proteins is an indispensable way to address fundamental mechanistic questions. Unnatural amino acids (UAAs) offer the possibility to expand beyond the 20 naturally occurring amino acids in most species and install new and useful chemical functions. Here, we review the literature about advances in UAA incorporation technology from chemoenzymatic aminoacylation of modified tRNAs to in vitro translation systems to genetic encoding of UAAs in the native cell environment and whole organisms. We discuss innovative applications of the UAA technology to challenges in bioengineering and medicine.
The active site of Hyd-1, an oxygen-tolerant membrane-bound NiFe-hydrogenase from Escherichia coli, contains four highly conserved residues that form a “canopy” above the bimetallic center, closest ...to the site at which exogenous agents CO and O2 interact, substrate H2 binds, and a hydrido intermediate is stabilized. Genetic modification of the Hyd-1 canopy has allowed the first systematic and detailed kinetic and structural investigation of the influence of the immediate outer coordination shell on H2 activation. The central canopy residue, arginine 509, suspends a guanidine/guanidinium side chain at close range above the open coordination site lying between the Ni and Fe atoms (N–metal distance of 4.4 Å): its replacement with lysine lowers the H2 oxidation rate by nearly 2 orders of magnitude and markedly decreases the H2/D2 kinetic isotope effect. Importantly, this collapse in rate constant can now be ascribed to a very unfavorable activation entropy (easily overriding the more favorable activation enthalpy of the R509K variant). The second most important canopy residue for H2 oxidation is aspartate 118, which forms a salt bridge to the arginine 509 headgroup: its mutation to alanine greatly decreases the H2 oxidation efficiency, observed as a 10-fold increase in the potential-dependent Michaelis constant. Mutations of aspartate 574 (also salt-bridged to R509) to asparagine and proline 508 to alanine have much smaller effects on kinetic properties. None of the mutations significantly increase sensitivity to CO, but neutralizing the expected negative charges from D118 and D574 decreases O2 tolerance by stabilizing the oxidized resting NiIII–OH state (“Ni-B”). An extensive model of the catalytic importance of residues close to the active site now emerges, whereby a conserved gas channel culminates in the arginine headgroup suspended above the Ni and Fe.
Two 15 μs all-atom simulations of the A2A adenosine receptor were obtained in a ternary mixture of cholesterol, saturated phosphatidylcholine lipids, and unsaturated phosphatidylcholine lipids. An ...analysis of local lipid solvation is reported on the basis of a Voronoi tessellation of the upper and lower leaflets, identifying first and second solvation shells. The local environments of both the inactive state and the partially active state of the receptor are significantly enriched with unsaturated chains but depleted of cholesterol and saturated chains, relative to the bulk membrane composition. In spite of the local depletion of cholesterol, the partially active receptor binds cholesterol at three locations during the entire simulation trajectory. These long-lived interactions represent the extreme of a very broad distribution of first-solvation shell lipid lifetimes, confounding sharp distinctions between lipid interactions. The broad distributions of lifetimes also make equilibrating the local lipid environment difficult, necessitating long simulation times.
Abstract
Circular dichroism (CD) spectroscopy is a widely used method to study the protein secondary structure. However, for decades, the general opinion was that the correct estimation of β-sheet ...content is challenging because of the large spectral and structural diversity of β-sheets. Recently, we showed that the orientation and twisting of β-sheets account for the observed spectral diversity, and developed a new method to estimate accurately the secondary structure (PNAS, 112, E3095). BeStSel web server provides the Beta Structure Selection method to analyze the CD spectra recorded by conventional or synchrotron radiation CD equipment. Both normalized and measured data can be uploaded to the server either as a single spectrum or series of spectra. The originality of BeStSel is that it carries out a detailed secondary structure analysis providing information on eight secondary structure components including parallel-β structure and antiparallel β-sheets with three different groups of twist. Based on these, it predicts the protein fold down to the topology/homology level of the CATH protein fold classification. The server also provides a module to analyze the structures deposited in the PDB for BeStSel secondary structure contents in relation to Dictionary of Secondary Structure of Proteins data. The BeStSel server is freely accessible at http://bestsel.elte.hu.
Nanozymes are nanomaterials exhibiting intrinsic enzyme-like characteristics that have increasingly attracted attention, owing to their high catalytic activity, low cost and high stability. This ...combination of properties has enabled a broad spectrum of applications, ranging from biological detection assays to disease diagnosis and biomedicine development. Since the intrinsic peroxidase activity of Fe
O
nanoparticles (NPs) was first reported in 2007, >40 types of nanozymes have been reported that possess peroxidase-, oxidase-, haloperoxidase- or superoxide dismutase-like catalytic activities. Given the complex interdependence of the physicochemical properties and catalytic characteristics of nanozymes, it is important to establish a standard by which the catalytic activities and kinetics of various nanozymes can be quantitatively compared and that will benefit the development of nanozyme-based detection and diagnostic technologies. Here, we first present a protocol for measuring and defining the catalytic activity units and kinetics for peroxidase nanozymes, the most widely used type of nanozyme. In addition, we describe the detailed experimental procedures for a typical nanozyme strip-based biological detection test and demonstrate that nanozyme-based detection is repeatable and reliable when guided by the presented nanozyme catalytic standard. The catalytic activity and kinetics assays for a nanozyme can be performed within 4 h.
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