A method for estimation of drug affinity constants to the open conformational state of calcium channels.

The affinity of D600 to calcium channels in the open state has been examined in isolated smooth muscle cells of the rabbit ear artery. Calcium channel currents were measured in high external barium solution by means of the patch-clamp technique. The current inhibition in various D600 concentrations (3-100 microM) on application of trains of short test pulses (20-80 ms) has been studied in nonmodified calcium channels and in cells where the calcium channels were modified by the agonist dihydropyridine (+) 202,791 (100 nM). The kinetics of the peak current decay has been analyzed with a mathematical model which is based on the experimental finding that D600 interacts primarily with calcium channels in the open conformational state. The model approach allows the estimation of drug affinity constants of D600 to the calcium channel in the open conformation. An association rate constant to the open conformational state of D600 of 6.16 x 10(4) M-1 s-1 was estimated. The association rate of the drug was not significantly changed after the calcium channels have been modified with 100 nM (+) 202,791. A method for correction of rate constants for possible drug trapping is discussed.     

A fluorescence stopped-flow study on troponin labeled with N-ethyl maleimide and N-(p-(2-benzimidazolyl)phenyl) maleimide

The kinetics of the conformational change of the troponin-C (TN-C) subunit in N-(p-(2-benzimidazolyl)phenyl) maleimide (BIPM)-N-ethyl maleimide (NEM)-labeled troponin induced by calcium binding or removal were studied with the fluorescence stopped-flow method. The kinetic process of the conformational change was biphasic, the rate constants of the two phases were determined as a function of the free calcium ion concentration of the protein solution. The kinetic behaviour of the conformational change of TN-C in BIPM-NEM-labeled troponin was explained by a simple molecular kinetic mechanism: (Formula: see text) This molecular kinetic mechanism is different from that of the isolated TN-C which we found in the previous work (1). That is, formation of a complex of TN-C with troponin-I (TN-I) and troponin-T (TN-T) modifies the molecular kinetic mechanism of the conformational change of TN-C.     

Pathophysiological condition changes the conformation of a flexible FBG-related protein, switching it from pathogen-recognition to host-interaction

Although homeostatic disturbance of the blood pH and calcium in the vicinity of tissue injury/malignancy/local infection seems subtle, it can cause substantial pathophysiological consequences, a phenomenon which has remained largely unexplored. The fibrinogen-related proteins (FREPs) containing fibrinogen-like domain (FBG) represent a conserved protein family with a common calcium-binding region, implying the presence of elements responsive to physiological perturbation. Here, we studied the molecular interaction between a representative FREP, the M-ficolin, and an acute phase blood protein, the C-reactive protein (CRP), both of which are known to trigger and control seminal pathways in infection and injury. Using hydrogen-deuterium exchange mass spectrometry, we showed that the C-terminal region of M-ficolin FBG underwent dramatic conformational change upon pH and calcium perturbations. Biochemical and biophysical assays showed that under defined pathophysiological condition (pH 6.5, 2.0 mM calcium), the FBG:CRP interaction occurred more strongly compared to that under physiological condition (pH 7.4, 2.5 mM calcium). We identified the binding interface between CRP and FBG, locating it to the pH- and calcium-sensitive C-terminal region of FBG. By site-directed mutagenesis, we determined H284 in the N-acetylglucosamine (GlcNAc)-binding pocket of the FBG, to be the critical CRP-binding residue. This conformational switch involving H284, explains how the pathophysiologically-driven FBG:CRP interaction diverts the M-ficolin away from GlcNAc/pathogen-recognition to host protein–protein interaction, thus enabling the host to regain homeostatic control. Our elucidation of the binding interface at the flexible FBG domain provides insights into the bioactive centre of the M-ficolin, and possibly other FREPs, which might aid future development of immunomodulators.     

Cooperative Binding of Stromal Interaction Molecule 1 (STIM1) to the N and C Termini of Calcium Release-activated Calcium Modulator 1 (Orai1)

Calcium flux through store-operated calcium entry is a central regulator of intracellular calcium signaling. The two key components of the store-operated calcium release-activated calcium channel are the Ca²⁺-sensing protein stromal interaction molecule 1 (STIM1) and the channel pore-forming protein Orai1. During store-operated calcium entry activation, calcium depletion from the endoplasmic reticulum triggers a series of conformational changes in STIM1 that unmask a minimal Orai1-activating domain (CRAC activation region (CAD)). To gate Orai1 channels, the exposed STIM1-activating domain binds to two sites in Orai1, one in the N terminus and one in the C terminus. Whether the two sites operate as distinct binding domains or cooperate in CAD binding is unknown. In this study, we show that the N and C-terminal domains of Orai1 synergistically contribute to the interaction with STIM1 and couple STIM1 binding with channel gating and modulation of ion selectivity.     

Does intrinsic fluorescence reflect conformational changes in the Ca2+-ATPase of sarcoplasmic reticulum?

We have investigated the kinetics of the intrinsic fluorescence drop observed when ATP is added to purified sarcoplasmic reticulum ATPase in a potassium-free medium containing magnesium and calcium, at pH 6 and 20°C. Under these conditions, analysis of the fluorescence drop is complex. Several events contributed to the rate of the fluorescence drop initiated by turnover, including phosphorylation, conformational transition of the phosphorylated complex, and dephosphorylation. On the other hand, when 75% of total fluorescence was quenched by energy transfer to the membrane-bound ionophore A23187, the observed turnoverdependent drop in residual fluorescence mainly reflected the conformational transition of the phosphorylated ATPase. Combination of fast kinetics with the quenching of selected tryptophan residues is suggested to be a promising tool for the study of proteins containing many of these residues.     

Dimethyl sulfoxide induces heme oxygenase-1 expression via JNKs and Nrf2 pathways in human umbilical vein endothelial cells

Prothrombin interacts with phosphatidylserine containing platelet membranes via its N-terminal, γ-carboxyglutamate (gla) residue-rich domain. Once bound it is cleaved to form the active protease, thrombin (factor IIa). Human prothrombin was cleaved with cathepsin G in the absence of calcium and magnesium ions. Under these conditions, the gla domain was removed. Phospholipid protected the protein from this proteolytic event, and this suggests that a conformational change may be induced by interaction with phospholipids. Binding of prothrombin to a surface containing 20% phosphatidylserine/80% phosphatidylcholine was detected by surface plasmon resonance, whereas no interaction with gla-domainless prothrombin was observed. Binding of intact prothrombin in the presence of calcium ions showed complex association kinetics, suggesting multiple modes of initial interaction with the surface. The kinetics of the dissociation phase could be fitted to a two-phase, exponential decay. This implies that there are at least two forms of the protein on the surface one of which dissociates tenfold more slowly than the other. Taken together, these data suggest that, on binding to a membrane surface, prothrombin undergoes a conformational change to a form which binds more tightly to the membrane.     

Amylase release from rat parotid glands II. Calcium kinetics

The kinetics of ⁴⁵Ca²⁺ uptake, efflux, and calcium potentiation of amylase release by slices of rat parotid glands were examined. Pretreatment of the tissue with 11.25 mM ⁴⁵Ca²⁺ medium increased the total tissue ⁴⁵calcium content. Lanthanum (1 mM) decreased tissue uptake, blocked the slow components of exchange and appeared to inhibit transcellular calcium movement. Neither dibutyryl cyclic AMP nor caffeine caused consistently significant effects on ⁴⁵Ca²⁺ kinetics, or total ⁴⁵calcium content. Carbamylcholine increased the initial rate of ⁴⁵Ca²⁺ uptake, but had no effect on total uptake.Elevation of the extracellular Ca²⁺ concentration to 11.25 mM during stimulation of amylase release resulted in an initial decrease in the rate of amylase release followed by a potentiation of release which developed slowly, requiring 40–50 min to reach the maximal response.The inability to detect release-related changes in either calcium influx or mobilization, and the lengthy times and high Ca²⁺ concentrations required to achieve calcium potentiation suggests that calcium does not couple amylase release.     

Fluorescence studies of the sarcoplasmic reticulum calcium pump.

Intrinsic Tryptophan fluorescence has been used to reveal conformational changes of the Sarcoplasmic Reticulum Calcium pump. It is shown that upon binding of calcium ions the fluorescence is enhanced. The effect being reversed after removal of dependence calcium ions by EGTA. The calcium concentration dependence of this fluorescence change and the effect of inhibitors is compared with the activation of calcium dependent ATPase. We conclude that calcium ions induces a conformational change of the calcium pump and that this change is responsible for the activation of the ATPase activity.     

G-Quadruplex conformational change driven by pH variation with potential application as a nanoswitch

Background

G-Quadruplex is a highly polymorphic structure, and its behavior in acidic condition has not been well studied.

Methods

Circular dichroism (CD) spectra were used to study the conformational change of G-quadruplex. The thermal stabilities of the G-quadruplex were measured with CD melting. Interconversion kinetics profiles were investigated by using CD kinetics. The fluorescence of the inserted 2-Aminopurine (Ap) was monitored during pH change and acrylamide quenching, indicating the status of the loop. Proton NMR was adopted to help illustrate the change of the conformation.

Results

G-Quadruplex of specific loop was found to be able to transform upon pH variation. The transformation was resulted from the loop rearrangement. After screening of a library of diverse G-quadruplex, a sequence exhibiting the best transformation property was found. A pH-driven nanoswitch with three gears was obtained based on this transition cycle.

Conclusions

Certain G-quadruplex was found to go through conformational change at low pH. Loop was the decisive factor controlling the interconversion upon pH variation. G-Quadruplex with TT central loop could be converted in a much milder condition than the one with TTA loop. It can be used to design pH-driven nanodevices such as a nanoswitch.

General significance

These results provide more insights into G-quadruplex polymorphism, and also contribute to the design of DNA-based nanomachines and logic gates.     

Electrogenic steps of the SR Ca-ATPase enzymatic cycle and the effect of curcumin

Sarcoplasmic reticulum (SR) vesicles were adsorbed on an octadecanethiol/phosphatidylcholine mixed bilayer anchored to a gold electrode, and the Ca-ATPase contained in the vesicles was activated by ATP concentration jumps in the presence of calcium ions. The resulting capacitive current transients are compared with those calculated on the basis of the enzymatic cycle of the calcium pump. This comparison provides information on the kinetics of the E(2)-E(1) conformational change and on its pH dependence. The alteration in the current transients following ATP concentration jumps in the presence of curcumin is examined. In particular, curcumin decreases the rate of slippage of the Ca-ATPase, and at concentrations above 10 microM reduces calcium transport by this pump.     

Kinetics of conformational change of troponin-C induced by binding or removal of calcium ion

The kinetics of conformational change of troponin-C (TN-C) induced by binding or removal of calcium ion were studied in the presence or absence of magnesium ion by measuring the fluorescence of tyrosyl residues by stopped-flow spectrofluorometry. The result was analyzed in terms of first-order kinetics. Two phases were observed both in pCa-up and in pCa-down experiments. The dependence of the rate constants on pCa was explained by a simple mechanism as follows; (see article). The dissociation constants of calcium bound to TN-C, K and K', calculated from the experimentally determined rate constants were K = 3.16 X 10(-7) M, K' = 1.58 X 10(-6) M in the absence of magnesium ion, and K = K' = 1 X 10(-6) M in the presence of 2 mM MgCl2.     

2-Color calcium pump reveals closure of the cytoplasmic headpiece with calcium binding

The sarco(endo)plasmic reticulum calcium ATPase (SERCA) undergoes conformational changes while transporting calcium, but the details of the domain motions are still unclear. The objective of the present study was to measure distances between the cytoplasmic domains of SERCA2a in order to reveal the magnitude and direction of conformational changes. Using fluorescence microscopy of live cells, we measured intramolecular fluorescence resonance energy transfer (FRET) from a donor fluorescent protein fused to the SERCA N-terminus to an acceptor fluorescent protein fused to either the N-, P-, or transmembrane domain. The "2-color" SERCA constructs were catalytically active as indicated by ATPase activity in vitro and Ca uptake in live cells. All constructs exhibited dynamic FRET changes in response to the pump ligands calcium and thapsigargin (Tg). These FRET changes were quantified as an index of SERCA conformational changes. Intramolecular FRET decreased with Tg for the two N-domain fusion sites (at residue 509 or 576), while the P- (residue 661) and TM-domain (C-terminus) fusions showed increased FRET with Tg. The magnitude of the Tg-dependent conformational change was not decreased by coexpression of phospholamban (PLB), nor did PLB slow the kinetics of Tg binding. FRET in ionophore-permeabilized cells was lower in EGTA than in saturating calcium for all constructs, indicating a decrease in domain separation distance with the structural transition from E2 (Ca-free) to E1 (Ca-bound). The data suggest closure of the cytoplasmic headpiece with Ca-binding. The present results provide insight into the structural dynamics of the Ca-ATPase. In addition, the 2-color SERCA constructs developed for this study may be useful for evaluating candidate small molecule regulators of Ca uptake activity.     

Chromium (VI) uptake by grape stalks wastes encapsulated in calcium alginate beads: equilibrium and kinetics studies

Abstract

The removal of hexavalent chromium from aqueous solution using grape stalks wastes encapsulated in calcium alginate (GS–CA) beads was investigated. Cr(VI) sorption kinetics were evaluated as a function of chromium initial concentration and grape stalks (GS) content in the calcium alginate (CA) beads. The process follows pseudo second-order kinetics. Transport properties of hexavalent chromium on GS–CA beads was characterised by calculating chromium diffusion coefficient using the Linear Absorption Model (LAM). Langmuir isotherms, at pH 3.0 were used to describe sorption equilibrium data as a function of GS percentage in the CAbeads. Maximum uptake obtained was 86.42 mmol of Cr(VI) per L of wet sorbent volume. Results indicated that both kinetic and equilibrium models describe adequately the adsorption process.     

Review of conformation-specific affinity purification methods for plasma vitamin K-dependent proteins

There are seven known vitamin K-dependent proteins in blood. These proteins require calcium ion for expressing their full biological activities. Calcium ion also induces conformational changes in this class of proteins. Taking advantage of the ligand induced conformational changes, a number of unique approaches of affinity chromatography have been developed. These methodologies have been very useful tools for both the purification and for understanding the structure–function relationships of this class of proteins. One method is the use of metal ion dependent immunoaffinity chromatography. The antigen can be dissociated from the antibodies with either the removal or addition of calcium ion under physiological conditions. The other method is pseudoaffinity chromatography. This method uses conventional ion-exchange or hydrophobic resin and manipulates the mobilities of the proteins on these resins by the presence or absence of calcium ions. Researchers working with other calcium binding proteins or other proteins that are known to undergo ligand induced conformational changes may benefit from the experience of these unique conformation-specific affinity chromatography approaches.     

Electrogenic steps of the SR Ca-ATPase enzymatic cycle and the effect of curcumin

Sarcoplasmic reticulum (SR) vesicles were adsorbed on an octadecanethiol/phosphatidylcholine mixed bilayer anchored to a gold electrode, and the Ca-ATPase contained in the vesicles was activated by ATP concentration jumps in the presence of calcium ions. The resulting capacitive current transients are compared with those calculated on the basis of the enzymatic cycle of the calcium pump. This comparison provides information on the kinetics of the E₂-E₁ conformational change and on its pH dependence. The alteration in the current transients following ATP concentration jumps in the presence of curcumin is examined. In particular, curcumin decreases the rate of slippage of the Ca-ATPase, and at concentrations above 10 μM reduces calcium transport by this pump.     

Correlating calcium binding, Förster resonance energy transfer, and conformational change in the biosensor TN-XXL

Genetically encoded calcium indicators have become instrumental in imaging signaling in complex tissues and neuronal circuits in vivo. Despite their importance, structure-function relationships of these sensors often remain largely uncharacterized due to their artificial and multimodular composition. Here, we describe a combination of protein engineering and kinetic, spectroscopic, and biophysical analysis of the Förster resonance energy transfer (FRET)-based calcium biosensor TN-XXL. Using fluorescence spectroscopy of engineered tyrosines, we show that two of the four calcium binding EF-hands dominate the FRET output of TN-XXL and that local conformational changes of these hands match the kinetics of FRET change. Using small-angle x-ray scattering and NMR spectroscopy, we show that TN-XXL changes from a flexible elongated to a rigid globular shape upon binding calcium, thus resulting in FRET signal output. Furthermore, we compare calcium titrations using fluorescence lifetime spectroscopy with the ratiometric approach and investigate potential non-FRET effects that may affect the fluorophores. Thus, our data characterize the biophysics of TN-XXL in detail and may form a basis for further rational engineering of FRET-based biosensors.     

Proline can have opposite effects on fast and slow protein folding phases

Proline isomerization is well known to cause additional slow phases during protein refolding. We address a new question: does the presence of prolines significantly affect the very fast kinetics that lead to the formation of folding intermediates? We examined both the very slow (10-100 min) and very fast (4 micro s-2.5 ms) folding kinetics of the two-domain enzyme yeast phosphoglycerate kinase by temperature-jump relaxation. Phosphoglycerate kinase contains a conserved cis-proline in position 204, in addition to several trans-prolines. Native cis-prolines have the largest effect on folding kinetics because the unfolded state favors trans isomerization, so we compared the kinetics of a P204H mutant with the wild-type as a proof of principle. The presence of Pro-204 causes an additional slow phase upon refolding from the cold denatured state, as reported in the literature. Contrary to this, the fast folding events are sped up in the presence of the cis-proline, probably by restriction of the conformational space accessible to the molecule. The wild-type and Pro204His mutant would be excellent models for off-lattice simulations probing the effects of conformational restriction on short timescales.     

Asymmetric Effect of Mechanical Stress on the Forward and Reverse Reaction Catalyzed by an Enzyme

The concept of modulating enzymatic activity by exerting a mechanical stress on the enzyme has been established in previous work. Mechanical perturbation is also a tool for probing conformational motion accompanying the enzymatic cycle. Here we report measurements of the forward and reverse kinetics of the enzyme Guanylate Kinase from yeast (Saccharomyces cerevisiae). The enzyme is held in a state of stress using the DNA spring method. The observation that mechanical stress has different effects on the forward and reverse reaction kinetics suggests that forward and reverse reactions follow different paths, on average, in the enzyme''s conformational space. Comparing the kinetics of the stressed and unstressed enzyme we also show that the maximum speed of the enzyme is comparable to the predictions of the relaxation model of enzyme action, where we use the independently determined dissipation coefficient for the enzyme''s conformational motion. The present experiments provide a mean to explore enzyme kinetics beyond the static energy landscape picture of transition state theory.     

Conformational Behavior and Flexibility of Terminally Blocked Cysteine and Cystine

Conformational potential energy hypersurfaces, PES, for the terminally blocked L-Cysteine, L,L-Cystine and D,L-Cystine have been analyzed by means of molecular mechanics in combination with the programs ROSE, CICADA, PANIC and COMBINE. Low energy conformations and conformational transitions, conformational channels, have been located. Global and fragmental flexibility and conformational softness have been calculated for each conformer as well as for the entire molecule. The PES analyses were used for simulation of conformational movement based on Boltzmann probability of the points obtained on the PES. Boltzmann travelling revealed interesting correlated conformational movement where three or even more dihedral angles changed simultaneously. It could be shown that conformational behavior and flexibility were strongly influenced by the absolute configurations of the amino acids in the peptides.     

Mathematical modeling of transmembrane calcium transport kinetics in smooth muscle cells

A mathematical model, which describes kinetics of transmembrane calcium transport in a smooth muscular cell, has been elaborated and investigated taking into account that the change of calcium cations concentration within a cell is determined by two mutually opposite processes: an increase of a carrying capacity of calcium channels of plasma membrane under signal substance action and calcium removal from the intracellular space by Mg2+, ATP-dependent calcium pump localized on the plasma membrane. The fundamental difference of the proposed model against the models analyzed in literature before is that the cellular system returns to the initial stationary state after enzyme-catalysed transformation of the signal substance. The results of calculations showed that this model really described the experimental kinetics of the transmembrane calcium transport. In this paper the influence of different parameters (Michaelis constant and ultimate rate of calcium pump, initial concentrations of signal substance and enzyme decomposing it, rate constants) on kinetics of calcium transport through the plasma membrane has been investigated in detail.