Molecular functions and structural changes of membrane proteins in an aqueous environment can be elucidated by reaction-induced FTIR difference spectroscopy upon photolysis of caged compounds. The achieved detection of IR band changes even due to single amino acid residues is, however, only possible in the presence of very high protein concentrations, implying that a low water content must be present. In general, the films are formed by controlled dehydration of membrane protein suspensions at reduced pressure and low temperature. For the retention of enzymatic activity of Na,K-ATPase, for example, a cosolvent such as glycerol is required. In order to interprete the results obtained by FTIR spectroscopy, it is important to know whether essential properties of the proteins such as hydration are changed upon film formation. Therefore, a differential scanning calorimetry (DSC) study has been carried out with purified Na,K-ATPase and Ca-ATPase in suspension, in form of pellets obtained by high-speed ultracentrifugation and in thin films. As relevant thermoanalytical properties, the endothermic denaturation transitions of the proteins have been studied.

Thermodynamics and kinetics of the interaction between T cell receptor specific for cytomegalovirus peptide (TCR CMV ) and its specific ligand, pp65–HLA-A*0201 complex, were studied by surface plasmon resonance and stopped-flow methods. In the latter measurements, fluorescence resonance energy transfer (FRET) between fluorescently labeled reactants was used. Thermodynamic data derived from surface plasmon resonance measurements suggest that the complex formation is driven by both favorable enthalpy and entropy. Two reaction phases were resolved by the stopped-flow measurements. The rate constant of the first step was calculated to be close to the diffusion-controlled limit rate (3·10 5 to 10 6 M −1 ·s −1 ), whereas the second step's reaction rate was found to be concentration independent and relatively slow (2–4 s −1 at 25°C). These findings strongly suggest that the interactions between the TCR and its ligand, the peptide–MHC complex, proceed by a two-step mechanism, in which the second step is an induced-fit process, rate determining for antigen recognition by TCR.

Microcalorimetric titrations allow to recognize and investigate high-affinity ligand binding to Na,K-ATPase. Titrations with the cardiac glycoside Ouabain, which acts as a specific inhibitor of the enzyme, have provided not only the thermodynamic parameters of high-affinity binding with a stoichiometric coefficient of about 0.6 but also evidence for low-affinity binding to the lipid. The marked enthalpic contribution of -95 kJ mol-1 at 298.2 K is partially compensated by a large negative entropy change, attributed to an increased interaction between water and the protein. The calorimetric ADP and ATP titrations at 298.2 K are indicative of high-affinity nucleotide binding either in 3 mM NaCl, 3 mM MgCl2 or at high ionic strength such as 120 mM choline chloride. However, no binding is detected in the buffer solution alone at low ionic strength. The affinities for ADP and ATP are similar, around 106 M-1 and the stoichiometric coefficients are close to that of Ouabain binding. The exothermic binding of ADP is characterized by a ΔH and ΔS value of -65 kJ mol-1 and -100 J mol-1 K-1, respectively. TheΔH value for ATP binding is larger than for ADP and is compensated by a larger, unfavorable ΔS value. This leads to an enthalpy/entropy compensation, which could express that H-bond formation represents the major type of interaction. As for Ouabain, the negative ΔS values that are also characteristic of nucleotide binding can indicate an increase of solvate interaction with the protein due to a conformational transition occurring subsequent to the binding process. The resulting binding constants are discussed with regard to the results of other studies employing different techniques. A molecular interaction model for nucleotide binding is suggested.

Differential scanning calorimetry (DSC) studies of micellar, 60 mM solutions of the octaethyleneglycol alkylethers C 14 E 8 and C 16 E 8 provide evidence for a narrow endothermic transition at 41 and 32°C,respectively, characterized by an enthalpy change of 2 kJ mol −1 for both detergents. The observed thermal transition is indicative of a concerted transition of the surfactant molecules, as illustrated on the basis of a simple molecular model. The effect of co-solvents such as different alcohols on the thermal transition is investigated. Glycerol markedly lowers the transition temperature whereas the transition is absent in the presence of at least 10% ethanol. The calorimetric transition correlates with the temperature dependent increase of viscosity and static light scattering as well as with changes observed by small-angle neutron scattering (SANS). The SANS results provide clear evidence for a distinct structural change occurring at the transition temperature, which is interpreted as a sphere-to-rod transition of the detergent micelles. Moreover, the rod length increases with increasing temperature. We suggest that the process causing the thermal transition acts as the prerequisite of the growth process.

The receptor–ligand interaction between the cardiac glycoside Ouabain and purified, membrane-bound as well as micellar Na,K-ATPase is investigated. Calorimetric titrations are carried out with micromolar concentrations of the phosphorylated protein in the presence of Mg2+. The measured heat changes provide evidence for an exothermic, high affinity and specific receptor binding process as well as for a low affinity, nonspecific binding to the lipid part of the nanoparticulate membrane fragments. The degree of lipid binding markedly depends on the lipid composition of the tissue. The measured time course of the heat change resulting from specific binding to the receptor site is unusually slow and is limited by the binding kinetics of the ligand. A course estimation of the Ouabain binding kinetics leads to a rate constant around 104mol−1ls−1. Receptor binding is characterized by affinities ranging between 107 and 108mol−1l, ΔH values around −95kJmol−1 and ΔS values of about −130JK−1mol−1 at 25°C. The enthalpic contribution is assumed to be mainly due to hydrogen bond formations between the ligand and the receptor site whereas the large, negative entropy change may be attributed to an increased interaction between water and the protein as a consequence of a conformational transition. The evaluation of the titrations provides stoichiometric coefficients around 0.55, which implies that only about 50–60% of the Na,K-ATPase protomers are capable to bind the cardiotonic steroid. This result is consistent with radioactive phosphorylation studies and appears to be a typical feature of kidney-type Na,K-ATPase preparations. Possible implications of this finding are discussed. As a general result, this study demonstrates how simple and suitable calorimetric titrations with micromolar protein concentrations can be for the purpose of a quantitative characterization of a receptor in nanoparticulate membrane systems.

The kinetics of Na+-dependent phosphorylation of the Na+,K+-ATPase by ATP were investigated via the stopped-flow technique using the fluorescent label RH421 (saturating [ATP], [Na+], and [Mg2+], pH 7.4, and 24°C). The well-established effect of buffer composition on the E2-E1 equilibrium was used as a tool to investigate the effect of the initial enzyme conformation on the rate of phosphorylation of the enzyme. Preincubation of pig kidney enzyme in 25 mM histidine and 0.1 mM EDTA solution (conditions favoring E2) yielded a 1/τ value of 59 s−1. Addition of MgCl2 (5 mM), NaCl (2 mM), or ATP (2 mM) to the preincubation solution resulted in increases in 1/τ to values of 129, 167, and 143 s−1, respectively. The increases can be attributed to a shift in the enzyme conformational equilibrium before phosphorylation from the E2 state to an E1 or E1-like state. The results thus demonstrate conclusively that the E2 → E1 transition does in fact limit the rate of subsequent reactions of the pump cycle. Based on the experimental results, the rate constant of the E2 → E1 transition under physiological conditions could be estimated to be ∼65 s−1 for pig kidney enzyme and 90 s−1 for enzyme from rabbit kidney. Taking into account the rates of other partial reactions, computer simulations show these values to be consistent with the turnover number of the enzyme cycle (∼48 s−1 and ∼43 s−1 for pig and rabbit, respectively) calculated from steady-state measurements. For enzyme of the α1 isoform the E2 → E1 conformational change is thus shown to be the major rate-determining step of the entire enzyme cycle.

Low dissociation or reverse rate constants of single-step or multistep complex formation equilibria are usually obtained with reduced precision from standard stopped-flow binding experiments by determination of the intercept of the concentration dependence of k(obs). Large and fast concentration jumps, based on two different step-motor-driven mixing setups, are performed with 60-300-fold dilutions that allow the precise, convenient, and independent determination of dissociation rate constants in the range of approximately 0.1-100 s(-1) in a single stopped-flow dissociation experiment. A theoretical basis is developed for the design and for the evaluation of such dilution experiments by considering the rebinding occurring during dissociation. The kinetics of three chemical systems are investigated, the binding of Mg2+ to 8-hydroxyquinoline as well as of Ca2+ and K+ to the cryptand [2.2.2], by carrying out standard stopped-flow binding as well as dissociation experiments employing various dilution factors. The advantage of the dilution method for investigating chemical and biological systems is emphasized.

The coupling between the fluorescence properties of the (trifluoromethyl)coumarino fluorophore and the protolytic state of the ion binding moiety of two fluorescent cryptands, F221 and F222, is investigated experimentally by carrying out steady-state and time-resolved fluorescence measurements. The high-intensity fluorescence emission of the diprotonated state of the these alkali ion-selective indicators, characterized by quantum yields of 0.6 and 0.83 as well as by lifetimes of 5.3 and 5.6 ns, are markedly quenched upon deprotonation, which leads to the monoprotonated state with quantum yields of 0.07 and 0.02 as well as lifetimes of 1.0 and 0.19 ns, respectively. The corresponding pK(a1) values are 7.07 for F221 and 5.85 for F222. The formation of the fully deprotonated state of the fluorescent cryptands, characterized by pK(a2) values of 10.6 and 9.3, respectively, is accompanied by a comparatively small additional reduction of the fluorescence quantum yield and lifetime. As a framework for the understanding of the pH-dependent fluorescence parameters, we suggest the concept of fluorescence quenching via photoinduced electron transfer (PeT), where the quenching process is assumed to be controlled by the pH-dependent availability of nonprotonated bridgehead N-atoms of the cryptand. These N-atoms act as electron donors with respect to the excited fluorophore, which functions as electron acceptor. In order to quantify the PeT energetics in the case of F221 and its monoprotonated state, one-electron oxidation and reduction potentials are determined by cyclic voltammetry for the parent cryptand [2.2.1] and the fluorophore derivative I as suitable model compounds, respectively. Experimental redox data are supplemented by simple estimations of the electrostatic energy contributions for the intramolecular radical ion pair produced through photoinduced charge separation and by corrections for the hydration energies. The resulting thermodynamic driving forces for PeT in the deprotonated F221 and its monoprotonated form show that PeT is favored for both of these species in water, where only a minor endergonic shift is observed for the monoprotonated as compared to the fully deprotonated compound. Therefore, the fully de- and the monoprotonated state of these fluorescent cryptands are regarded as being responsible for the reduction of the fluorescence quantum yield and the appearance of the second lifetime tau(2) at high pH. In contrast, PeT is expected to be completely blocked for the diprotonated state of these (trifluoromethyl)coumarino cryptands.

A narrow, reversible endothermic main transition is found in the aqueous micellar phase of octaethylene glycol tetradecyl ether (C 14 E 8 ) by DSC, characterized by a transition temperature of 41°C and a Δ H value of 0.5 kcal mol −1 , which is not observed by light scattering. This transition is assigned to a cooperative conformational rearrangement of the assembled amphiphilic detergent molecules and not to a micelle aggregation process. It is suggested that the detergent’s polar head group is primarily involved in this rearrangement.

The effects of lyotropic anions, particularly perchlorate, on the kinetics of partial reactions of the Na+,K+-ATPase from pig kidney were investigated by two different kinetic techniques: stopped flow in combination with the fluorescent label RH421 and a stationary electrical relaxation technique. It was found that 130 mM NaClO4 caused an increase in the Kd values of both the high- and low-affinity ATP-binding sites, from values of 7.0 (± 0.6) μM and 143 (± 17) μM in 130 mM NaCl solution to values of 42 (± 3) μM and 660 (± 100) μM in 130 mM NaClO4 (pH 7.4, 24°C). The half-saturating concentration of the Na+-binding sites on the E1 conformation was found to decrease from 8–10 mM in NaCl to 2.5–3.5 mM in NaClO4 solution. The rate of equilibration of the reaction, E1P(Na+)3 ↔ E2P + 3Na+, decreased from 393 (± 51) s−1 in NaCl solution to 114 (± 15) s−1 in NaClO4. This decrease is attributed predominantly to an inhibition of the E1P(Na+)3 → E2P(Na+)3 transition. The effects can be explained in terms of electrostatic interactions due to perchlorate binding within the membrane and/or protein matrix of the Na+,K+-ATPase membrane fragments and alteration of the local electric field strength experienced by the protein. The kinetic results obtained support the conclusion that the conformational transition E1P(Na+)3 → E2P(Na+)3 is a major charge translocating step of the pump cycle.

Stationary and time-resolved fluorescence of FITC–Na,K-ATPase is investigated as a function of pH in the presence of different ligands, cations, and the monoclonal anti-FITC antibody 4-4-20. The binding of K+ and of the antibody leads to the same decreased fluorescence intensity level. Antibody binding is observed only under conditions where the enzyme exists in the conformational state F1, and not in the form of the Na+ or K+ complex or when it is phosphorylated with inorganic phosphate in the presence of Mg2+. For the interpretation of the results it is shown that the fluorophore is not essentially affected by an acidity change of the bound dye, so that pK variations responsible for the observed intensity changes can be excluded in favor of a static quenching process

The kinetics of K+-stimulated dephosphorylation of the Na+,K+-ATPase were investigated at pH 7.4, 24 degrees C, and an ATP concentration of 1.0 mM via the stopped-flow technique using the fluorescent label RH421. Two different mixing procedures were used: (a) premixing with ATP to allow phosphorylation to go to completion, followed by mixing with KCl; and (b) simultaneous mixing with ATP and KCl. Using mixing procedure (a), the dephosphorylation rate constant of enzyme complexed with K+ ions could be determined directly to be =366 s-1 and the rate constant for spontaneous dephosphorylation (without K+) =60 s-1. The K+ concentration dependence of the observed reciprocal time constant showed half-saturation at a K+ concentration of 2.4-2.6 mM with positive cooperativity involved in the occupation of the K+ binding sites on the E2P conformation of the enzyme. Using mixing procedure (b), it was found that at saturating K+ concentrations the dephosphorylation of the enzyme is rate-limited by its phosphorylation, which occurs with a rate constant of approximately 190 s-1 (1). These results show that all reactions occurring after phosphorylation and prior to dephosphorylation, i.e., the E1P to E2P conformational transition as well as Na+ release and K+ binding steps, must be fast (>190 s-1).

The kinetics of Na+-dependent partial reactions of the Na+,K+-ATPase were investigated via the stopped-flow technique using the fluorescent labels RH421 and BIPM. After the enzyme is mixed with MgATP, both labels give almost identical kinetic responses. Under the chosen experimental conditions two exponential time functions are necessary to fit the data. The dominant fast phase, 1/tau1 approximately 180 s-1 (saturating [ATP] and [Na+], pH 7.4 and 24 degrees C), is attributed to phosphorylation of the enzyme and a subsequent conformational change (E1ATP(Na+)3 --> E2P(Na+)3 + ADP). The rate of the phosphorylation reaction measured by the acid quenched-flow technique was 190 s-1 at 100 microM ATP, suggesting that phosphorylation controls the kinetics of the RH421 signal and that the conformational change is very fast (>/=600 s-1). The rate of the RH421 signal was optimal at pH 7.5. The Na+ concentration dependence of 1/tau1 showed half-saturation at a Na+ concentration of 8-10 mM with positive cooperativity involved in the occupation of the Na+ binding sites. The apparent dissociation constant of the high affinity ATP binding site determined from the ATP concentration dependence of 1/tau1 was 7.0 (+/-0.6) microM, while the apparent Kd for the low affinity site and the rate constant for the E2 to E1 conformational change evaluated in the absence of Mg2+ were 143 (+/-17) microM and </= 28 s-1. At RH421 concentrations in the micromolar range, a decrease in the value of 1/tau1 is observed. On the basis of rapid quenched-flow measurements, this inhibition can be attributed to a reaction step subsequent to phosphorylation. This accounts for previously observed kinetic discrepancies between RH421 and BIPM.

DSC studies are carried out to characterize Na + ,K + -ATPase isolated from pig kidney in its natural membrane environment as well as in its purified state upon detergent treatment. The transition temperatures of the investigated thermal protein unfolding process, observed between 43 and 64.5° C, depend on the local membrane environment as well as on pH . In addition, the transition temperature is significantly increased upon binding of different cations and ligands which are known to interact with the enzyme. Evidence for a lipid phase transition around 23 °C in the original biological membrane is reported. The application of a calorimeter equipped with removable cells appears to be more suitable for the investigation of this type of membrane sample than an instrument with fixed capillary cells. Filling the sample capillary cell with an usual syringe, consisting of a long and thin needle, can influence the experimental results. Na + , K + -ATPase acts as the receptor for cardiac glycoside binding. The thermodynamic parameters of this binding process are determined by titration calorimetry. The binding of ouabain, as a typical example, is unusually slow and is enthalpy driven. The enthalpy change upon binding enthalpy is −75 kJ mol −1 at 25 °C. The surprisingly low stoichiometric coefficient, resulting from an evaluation based on a simple one step binding model, is interpreted in terms of a dimeric receptor unit.