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1.
A mixture of acids with known pK′ and in a known concentration was dissolved to simulate the ionic character of hemoglobin. The titration curves of the mixture were obtained in water and 3.6 M KCl at 20°C. These curves were subjected to analysis by a reiterative curve-fitting procedure to determine if one could evaluate both the group ionization constants and the numbers of groups. This approach was successful in that the calculated parameters were within the experimental error encountered in obtaining these constants on each of the model compounds. However, analysis for the electrostatic interaction parameter w indicated that there was a possible effect on the ionization of formic acid, pyridine, imidazole, and the α-amino group of glycylglycine in water and on imidazole and the α-amino group of glycylglycine in 3.6 M KCl. 相似文献
2.
Chymotrypsinogen, nitrated chymotrypsinogen (two of the four tyrosyls nitrated), acetylated chymotrypsinogen (all amino groups blocked), and nitrated-acetylated chymotrypsinogen were titrated as f(pH) in an isoperibolic calorimeter at 20°C. After appropriate correction and reduction of both the potentiometric and thermal titration data, the parameters N (ionizable groups per group-set), pK′, and ΔHi (heat of ionization) were evaluated using the iterative curve-fitting algorithm of the MLAB computer program. The pK′ parameters so obtained for the two normally ionizing tyrosyl groups in chymotrypsinogen and the two nitrated tyrosyl groups in the nitrated proteins essentially agreed with the results of spectral titration. Excellent fits to all data could be obtained using evaluated parameter sets of N and pK′ for the potentiometric titration data (groups vs pH plots) and N, pK′, and ΔHi sets for the calorimetric data (total heat vs pH plots). The invocation of electrostatic interaction effects was not required to explain the data satisfactorily, despite the differences in charge number and type among the four proteins. Rather, the data can be represented by series expressions of the mass-action law. Using all information, viz., the consequences of functional group modification, the downscale shift in tyrosyl group pK's on nitration, and the numerical values of the evaluated N, pK′, and ΔHi parameter sets for all proteins, the chemical identity of the various classes of group sets can be assigned with reasonable assurance. 相似文献
3.
A model for the electrostatic properties of hydrated collagen fibrils, based on the concept of a “penetrable” protein, has been evaluated through studies of collagen fibrils that had been chemically modified to change their electrostatic properties,. A value of 0.28 ± 0.07 ml/g was found for the intrafibrillar space sterically inaccessible to a molecule that had an equivalent spherical radius of 4.5 Å. The net intrinsic charge on reconstituted collagen is +14 mol/mol under physiological conditions, but decreases, at constant pH, with ionic strength. A value of 7.1 for the pK of the histidine and α-amino groups in reconstituted collagen was obtained through the application of the electrostatic model to this effect. The values obtained for calcium binding parameters for collagen fibrils, under solution conditions in which the nonspecific electrostatic properties of collagen fibrils were eliminated (3–5 M tetramethyl ammonium chloride), were in agreement with values obtained in 0.16 M NaCl solutions calculated through the use of the electrostatic theory. These are 0.73 ± 0.23 and 56.2 ± 12.3 sites per molecule with intrinsic association constants of 1101 ± 386 and 21.4 ± 5.2 M?1, respectively. The model also predicts that an average 4-mV potential difference exists between the reconstituted collagen fibrils and physiological solutions, and that collagen fibrils under such conditions have piezoelectriclike properties. The pattern of interaction of ions with collagen fibrils is such that an allosteric mechanism for the catalytic step in the mineralization of collagen is a possibility. 相似文献
4.
Analysis of the ionization constants and heats of ionization of reduced and oxidized horse heart cytochrome c 总被引:1,自引:0,他引:1
Simultaneous curve fitting for the ionization parameters of oxidized and reduced horse heart cytochrome c in 0.15M KCl and 20°C yields values for the ionization constants (as pK′) and the heats of ionization (ΔHi) which can reconstruct either the potentiometric or thermal titration curves. Reduced cytochrome c requires 8 sets of groups, whereas oxidized cytochrome c requires 10 sets of groups. The additional groups in the oxidized preparation appear to involve the ferriheme (pK′, 9.25; ΔHi, 13.7 kcal/mol) and a tyrosine (pK′ ? 10.24) that is not present in the reduced form. The potentiometric and thermal difference curves (reduced – oxidized) involve the appearance of 17 kcal/mol centered at pH 9.7 and 5.8 kcal/mol centered at pH 4.9. The carboxyl groups in both species appear to be normal for the hydrogen-bonded form. Only one histidine has normal ionization properties (pK′, 6.7; ΔHi, 7.5 kcal/mol), as do 17 of the lysine residues (pK′, 10.8; ΔHi, 11.5 kcal/mol). 相似文献
5.
Roberto P. Christen Spyros I. Nomikos E. T. Smith 《Journal of biological inorganic chemistry》1996,1(6):515-522
The change in the equilibrium reduction potentials of the iron-sulfur proteins, Pyrococcus furiosus rubredoxin and P. furiosus ferredoxin, and heme protein, horse cytochrome c, has been calculated as a function of temperature using a numerical solution to the Poisson-Boltzman equation. Working curves
for different internal dielectric constants were generated to best reproduce experimental observation. Based on a comparison
of the experimental and simulated change in reduction potential with temperature, it is concluded that the dielectric constant
of proteins is temperature-dependent and varies from protein to protein. For example, the temperature-dependent reduction
potential of cytochrome c can only be simulated using a different temperature-dependent dielectric constant for each oxidation state, but this was
not the case for rubredoxin or ferredoxin. The role of changes in ionization states of cytochrome c at alkaline pHs, where the reduction potential is known to be pH-dependent at room temperature, is also discussed in terms
of electrostatic interaction energies as a function of temperature. It appears that temperature/reduction potential profiles
may provide a direct method for measuring relative changes in internal protein dielectric constants.
Received: 29 April 1996 / Accepted: 1 August 1996 相似文献
6.
Victor S. Sivozhelezov 《Molecular Engineering》1996,6(4):405-414
Ionic strength dependencies of electron transfer between Cytochrome b
5 and variants of yeast Cytochrome c were analyzed by curve fitting to the simple model of the electrostatic interaction between the two proteins assuming the process to be non-diffusion-controlled. Mutagenesis of Lys79, but not Lys72, leads to an increase of effective radius of the interacting charged species, suggesting that the mutation effects of the two residues on the electrostatic field distribution near the contact site are different, even within the crude electrostatic model used. Extrapolation of the ionic strength dependencies to infinite ionic strength resulted in similar values, around (2–3)×10-6 for all Cyt c variants considered thus showing the lysine residue mutations to primarily affect protein association rather than the electron transfer directly. Based on the ionic strength dependencies of binding constants of the two proteins into an electrostatically stabilized complex, the monomolecular electron transfer rate constant was estimated to be 1.1×104–1.6×105 s-1. The electrostatic part of the binding energy of the complex at I=0.19 was estimated to be-2.4 kcal/mol, strongly at variance with the values-13.0 and-6.4 kcal/mol reported for the two types of complexes identified using Brownian dynamics techniques. Possible reasons for this discrepancy are discussed. 相似文献
7.
8.
O. S. Knyazeva I. B. Kovalenko A. M. Abaturova G. Yu. Riznichenko E. A. Grachev A. B. Rubin 《Biophysics》2010,55(2):221-227
A multiparticle computer model of plastocyanin-cytochrome f complex formation in the thylakoid lumen has been designed, which takes into account the electrostatic interactions of proteins
and membrane. The Poisson-Boltzmann formalism was used to determine the electrostatic potentials of the electron carrier proteins
and the thylakoid membrane at different ionic strengths. The membrane electrostatic field was shown to influence plastocyanin
diffusion and interaction with cytochrome f. The rate constants for plastocyanin-cytochrome f complex formation were calculated as a function of ionic strength and membrane surface charge. 相似文献
9.
10.
Titration and fluorometric studies of the tyrosine side chain of angiotensin II and related peptides
Luiz Juliano Cecilia Laluce Maria C. F. Oliveira Antonio C. M. Paiva 《Biopolymers》1979,18(7):1793-1807
The effect of charged side chains on the ionization and fluorescence of the Tyr4 phenolic group in angiotensin (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) was investigated. Several synthetic peptides related to angiotensin were titrated spectrophotometrically and quantum yields of tyrosine fluorescence were also determined. The electrostatic interactions were interpreted according to the Kirkwood-Tanford theory, and the results were related to a recently proposed model [J. L. De Coen and E. Ralston (1977) Biopolymers 16 , 1929] for angiotensin conformation in solution. The titration and fluorescence results are in good agreement with the folded conformations of this model, with the exception that the data indicate a weaker interaction between the histidine side chain and the C-terminal carboxyl groups than that proposed in the model. 相似文献
11.
The ionization constants of the tyrosyl groups of chymotrypsinogen and of nitrated-chymotrypsinogen (two tyrosyl residues nitrated) have been determined by difference spectrophotometry. In chymotrypsinogen, two of the four tyrosyl groups ionize without any time dependence. Above pH greater than ca. 12.5, time-dependent spectral changes are seen for 0.7 group equivalent. The data can be fitted to the values of pK′1 9.75 ± 0.07, pK′2 11.55 ± 0.05, pK′3 13.30 ± 0.05. In nitrated-chymotrypsinogen, the two nitrated tyrosyl residues have pK′1 6.44 and pK′2 8.30. For both proteins, these pK′ values are in agreement with those evaluated from potentiometric titration and calorimetric data using computer-assisted curve-fitting analysis. 相似文献
12.
Lindman S Bauer MC Lund M Diehl C Mulder FA Akke M Linse S 《Biophysical journal》2010,99(10):3365-3373
Understanding the role of electrostatics in protein stability requires knowledge of these interactions in both the folded and unfolded states. Electrostatic interactions can be probed experimentally by characterizing ionization equilibria of titrating groups, parameterized as pKa values. However, pKa values of the unfolded state are rarely accessible under native conditions, where the unfolded state has a very low population. Here, we report pKa values under nondenaturing conditions for two unfolded fragments of the protein G B1 domain that mimic the unfolded state of the intact protein. pKa values were determined for carboxyl groups by monitoring their pH-dependent 13C chemical shifts. Monte Carlo simulations using a Gaussian chain model provide corrections for changes in electrostatic interactions that arise from fragmentation of the protein. Most pKa values for the unfolded state agree well with model values, but some residues show significant perturbations that can be rationalized by local electrostatic interactions. The pH-dependent stability was calculated from the experimental pKa values of the folded and unfolded states and compared to experimental stability data. The use of experimental pKa values for the unfolded state results in significantly improved agreement with experimental data, as compared to calculations based on model data alone. 相似文献
13.
The adherence of an environmental strain of Pseudomonas sp. to titanium was evaluated modifying the pH (2 to 8) and ionic strength (0.1 and 0.6 M NaCl) of the electrolyte solution.
Results were analyzed considering the participation of the different interfacial forces under the Derjaguin–Landau–Verwey–Overbeek
(DLVO) theory. At 0.1 M, maximal bacterial adhesion was at pH 6, in agreement with the point of zero charge of the titanium
surface. Similar adhesion values were observed at both sides of this point despite the opposite electrostatic condition of
the surface oxide. At 0.6 M an absence of bacterial adhesion was observed throughout the pH range tested. The changes in bacterial
adhesion are in agreement with the changes in the number of reinforced H-bond-forming sites on the titanium surface calculated
using a simple model for the ionization of OH group adsorbed to the surface. Journal of Industrial Microbiology & Biotechnology (2001) 26, 303–308.
Received 25 May 2000/ Accepted in revised form 14 February 2001 相似文献
14.
《Journal of liposome research》2013,23(2):187-197
AbstractWith the aid of a flow cell assembly the desorption of cationic liposomes prepared from mixtures of dipalmitoylphoshatidylcholine (DDPC), cholesterol, and either dimethyldioctadecylammonium bromide (DDAB) or 3,β[N-(N1,N-dimethylethylenediamine)-carbamoyl]cholesterol (DC-chol) from immoblized biofilms of Staphylococcus aureus has been studied as a function of shear stress by confocal microscopy. A shear stress theory has been adapted from fluid mechanics of laminar flow between parallel plates and used to determine the critical shear stress for liposome desorption. The critical shear stress for both DDAB and DC-chol liposomes has been determined as a function of cationic lipid content and hence surface charge as reflected in their zeta potentials. The critical shear stress has been used to obtain the potential energy of liposome–biofilm interaction which together with the electrostatic interaction energy has enabled estimates of the London-Hamaker constants to be made. The values of the London-Hamaker constants at small liposome-bacterial cell separation were found to be independent of liposome composition. 相似文献
15.
Expression of tomato reference genes using established primer sets: Stability across experimental set‐ups
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Fabio Rezzonico Philippe C. Nicot Johannes Fahrentrapp 《Journal of Phytopathology》2018,166(2):123-128
Reliable reference genes are critical for relative quantification using quantitative real‐time PCR (qPCR). Ten tomato genes (Solanum lycopersicum) and their respective primer sets, which have been used over the last 6 years as references in expression studies, were evaluated for their performance using leaf tissue samples grown under semi‐controlled conditions and infected with grey mould (Botrytis cinerea) or late blight (Phytophthora infestans). The target genes coding for U6 snRNA‐associated Sm‐like protein LSm7, calcineurin B‐like protein and V‐type proton ATPase were the most stable expressed of all the genes tested in three experimental repetitions. Evaluation of candidate reference genes with geNorm and NormFinder softwares yielded the lowest mean values for their respective primer sets LSM7, SlCBL1 and SlATPase, suggesting stable expression. However, SlATPase primer set revealed a comparably high intra‐group variation and was thus not considered further. In follow‐up experiments with P. infestans, the geNorm and NormFinder values of primer sets LSM7 and SlCBL1 were even lower, indicating the stability of their expression also under these conditions. Primer efficiency differed by ‐18 to +5 percentage points from values presented in the literature. Our findings show that a reference primer set which delivers the best results in one system may be outperformed by another under different experimental conditions, thus recommending a reassessment of both expression stability and qPCR efficiency whenever the biological or technical experimental set‐up is changed. On the basis of our results, we recommend the use of LSM7 and SlCBL1 as reference primer sets for gene expression studies on plant tissue derived from open or semi‐controlled conditions. 相似文献
16.
The types of binding of different mono- and divalent ions to sites of the
constitutive pectic acids of the Nitella cell walls
were investigated by performing ion exchanges at different pH. The
experimental results were then analysed in the framework of a model derived
from the polyelectrolyte theory in which the competitive process of
dissociation of the exchange sites and their complexation by counterions
are taken into account. Divalent ions Ca2+ and
Mn2+ interacted specifically with the exchange sites
to give rise to strong thermodynamic association constants. They also
induced conformational transitions of the pectic acids which allowed some
site-specific association with monovalent ions, although the latter, in the
absence of divalent ions, interacted only in a purely electrostatic manner
with the charged sites. The complexation phenomenon of the monovalent ions
also results in a feedback process which enhances or depletes the
site-specific interactions of the divalent counterions. Changes in the
counterion association with the wall exchange sites will take place without
modification in the wall electrostatic field, when divalent ions are
present at the usual pH. These specific interactions are supported by the
values of the residual interaction energy, calculated from the variations
of the apparent pKa of the polygalacturonic acids with
their degree of protonation. 相似文献
17.
Jean. M. Sogbedji Harold. M. van Es Jeff J. Melkonian Robert R. Schindelbeck 《Plant and Soil》2006,282(1-2):343-360
Mathematical models may be used to develop management strategies that optimize the use of nutrients from complex sources such
as manure in agriculture. The Precision Nitrogen Management (PNM) model is based on the LEACHN model and a maize N uptake/growth
and yield model and focuses on developing more precise N management recommendations. The PNM model was evaluated for simulating
drain flow nitrate-nitrogen (NO3-N) in a 3-yr study involving different times of liquid manure application on two soil textural extremes, a clay loam and
a loamy sand under maize (Zea mays, L.) production. The model was calibrated for major N transformation rate constants including mineralization, nitrification
and denitrification, and its performance was tested using two different calibration scenarios with increasing levels of generalization:
(i) separate sets of rate constants for each individual soil type and (ii) a single set of rate constants for both soil types.
When calibrated for each manure application treatment for each soil type, the model provided good simulations of monthly and
seasonal drain flow NO3-N concentrations. The correlation coefficient (r) and Willmott’s index of agreement (d) ranged from 0.63 to 0.96 and 0.72 to 0.92, respectively. The calibrated model performed reasonably well when rate constant
values averaged over manure application treatment for each soil type were used, with r and d values between 0.54 and 0.97, and 0.70 and 0.94, respectively, and greater accuracy for the clay loam soil. When rate constant
values were averaged over manure application treatments and soil types, model performance was reasonably accurate for the
fall time manure application on the clay loam (r and d of 0.60 and 0.91 and 0.72 and 0.92, respectively) and satisfactory for the spring time on the clay loam and the fall and
spring times for the loamy sand soil (r and d between 0.56 and 0.90 and 0.58 and 0.84, respectively). The use of the model for predicting N dynamics under manure-fertilized
maize cropping appears promising. 相似文献
18.
Intrinsically disordered inhibitor of glutamine synthetase is a functional protein with random‐coil‐like pKa values
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José L. Neira Francisco J. Florencio M. Isabel Muro‐Pastor Bruno Rizzuti 《Protein science : a publication of the Protein Society》2017,26(6):1105-1115
The sequential action of glutamine synthetase (GS) and glutamate synthase (GOGAT) in cyanobacteria allows the incorporation of ammonium into carbon skeletons. In the cyanobacterium Synechocystis sp. PCC 6803, the activity of GS is modulated by the interaction with proteins, which include a 65‐residue‐long intrinsically disordered protein (IDP), the inactivating factor IF7. This interaction is regulated by the presence of charged residues in both IF7 and GS. To understand how charged amino acids can affect the binding of an IDP with its target and to provide clues on electrostatic interactions in disordered states of proteins, we measured the pKa values of all IF7 acidic groups (Glu32, Glu36, Glu38, Asp40, Asp58, and Ser65, the backbone C‐terminus) at 100 mM NaCl concentration, by using NMR spectroscopy. We also obtained solution structures of IF7 through molecular dynamics simulation, validated them on the basis of previous experiments, and used them to obtain theoretical estimates of the pKa values. Titration values for the two Asp and three Glu residues of IF7 were similar to those reported for random‐coil models, suggesting the lack of electrostatic interactions around these residues. Furthermore, our results suggest the presence of helical structure at the N‐terminus of the protein and of conformational changes at acidic pH values. The overall experimental and in silico findings suggest that local interactions and conformational equilibria do not play a role in determining the electrostatic features of the acidic residues of IF7. 相似文献
19.
Microscopic mechanisms that govern the titration response and pKa values of buried residues in staphylococcal nuclease mutants
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To probe the microscopic mechanisms that govern the titration behavior of buried ionizable groups, microsecond explicit solvent molecular dynamics simulations are carried out for several mutants of Staphylococcal nuclease using both fixed charge and polarizable force fields. While the ionization of Asp 66, Glu 66, and Lys 125 lead to enhanced structural fluctuations and partial unfolding of adjacent α‐helical regions, the ionization of Lys 25 causes local unfolding of adjacent β sheets. Using the sampled conformational ensembles, good agreement with experimental pKa values is obtained with Poisson–Boltzmann calculations using a protein dielectric constant of 2–4 for V66D/E; slightly larger dielectric constants are needed for Lys mutants especially L25K, suggesting that structural responses beyond microseconds are involved in ionization of Lys 25. Overall, the set of unbiased simulations provides insights into the spatial and temporal scales of protein and solvent motions that dictate the diverse titration behaviors of buried protein residues. Proteins 2017; 85:268–281. © 2016 Wiley Periodicals, Inc. 相似文献
20.
Hamid Tanzadehpanah Hanie Mahaki Neda Hosseinpour Moghadam Sadegh Salehzadeh Omid Rajabi Rezvan Najafi 《Journal of biomolecular structure & dynamics》2019,37(4):823-836
This study was carried out to evaluate the binding interaction of gefitinib (GEF) with human serum albumin (HSA) and calf thymus DNA (ct-DNA) using fluorescence, UV–Visible, zeta potential measurements and molecular docking methods in order to understand its pharmacokinetic mechanism. By increasing the temperature, a steady decrease in Stern–Volmer quenching constants was observed for HSA binding properties; this indicates a static type of fluorescence quenching. Negative values were calculated for Gibbs free energy (ΔG), enthalpy (ΔH), and entropy (ΔS) changes, indicating that the reaction is spontaneous and enthalpy-driven. Probe competitive experimental results showed that GEF contains the same binding site as warfarin and are consistent with modeling results. The zeta potential of the HSA increased with increasing GEF, which represents the presence of electrostatic interactions in the system. DNA binding properties were investigated in the presence of three probes. The experimental results showed that by increasing GEF to DNA-AO (acridine-orange) and DNA-MB (methylene-blue) system, the fluorescence intensity and absorbance spectra had no considerable change. Furthermore, with the addition of GEF to DNA, the zeta potential decreased gradually, indicating that the hydrophobic interaction between the GEF and the bases of DNA is the major factor. Thus, GEF can bind to DNA via a groove binding mode. It was also found that GEF entered into the minor groove in the A–T rich region of DNA fragment and bind via van der-Waals forces and three H-bond with double strands of DNA. This is in good agreement with experimental results. 相似文献