E. Lewitzki

Kinetic evidence for a ligand-binding-induced conformational transition in the T cell receptor.

Thermodynamics and kinetics of the interaction between T cell receptor specific for cytomegalovirus peptide (TCRCMV) 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 (3·105 to 106 M−1·s−1), whereas the second steps 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.

PHASE TRANSITIONS IN NON-IONIC DETERGENT MICELLES

Differential scanning calorimetry (DSC) studies of micellar, 60 mM solutions of the octaethyleneglycol alkylethers C14E8 and C16E8 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.

Reassignment of cation-induced population of main conformational states of FITC-Na+/K+-ATPase as detected by fluorescence spectroscopy and characterized by equilibrium binding studies

To link proposed features of the Na+/K+-ATPase reaction cycle to the molecular structure, spectroscopic studies mainly concerning the elucidation of partial reactions are carried out. This can be done by introducing fluorescence labels such as the F1TC-group (1) which enables the study of alkali ion binding as a key feature of this ion pump. According to earlier studies, a correlation between the fluorescence emission intensity and the position of the E1/E2 equilibrium is suggested: low fluorescence intensity in the presence of K+ has been attributed to E2, high intensity to E1(1–5). Evidence will be presented that this correlation concerning E1 is not fulfilled. Membrane-bound Na+/K+-ATPase has been prepared from pig kidney according to (6), the FITC-enzyme according to (1); details are given in (7).

Nucleotide/protein interaction: Energetic and structural features of Na, K-ATPase

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.

Characterizing Protein Conformational Transitions of Na,K-ATPase with Antibodies by Fluorescence Spectroscopy

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

DEDO: A specific, fluorescent inhibitor for spectroscopic investigations of Na,K-ATPase

The interaction between the fluorescent ouabain derivative DEDO and purified renal Na,K-ATPase (isolated from different animal species) is investigated. Equilibrium binding studies provide a pK value of about 7.5 and a stoichoimetric coefficient of 1. Nonmodified ouabain exhibits the same affinity to the rabbit enzyme; the enzyme originating from the other sources binds DEDO 10 times less strongly than ouabain. Kinetic studies indicate that this is the consequence of a 10 times higher dissociation rate constant of the complexes formed with DEDO. The fluorescence emission intensity of DEDO is enhanced, being dependent on the enzyme source. The single decay time of DEDO is 3 ns in the absence and 21 ns in the presence of the rabbit enzyme and 14 ns in the presence of the pig renal enzyme. This result suggests that the fluorophore of DEDO is bound to a very hydrophobic environment of the enzyme. Further characterization of the static fluorescence spectra provides evidence for energy transfer between Trp residues of the enzyme and DEDO. Distance estimations suggest that one or two Trp residues are likely to be located in the proximity of the fluorophore.

Structural Changes in the Catalytic Cycle of the Na+,K+-ATPase Studied by Infrared Spectroscopy

Pig kidney Na(+),K(+)-ATPase was studied by means of reaction-induced infrared difference spectroscopy. The reaction from E1Na(3)(+) to an E2P state was initiated by photolysis of P(3)-1-(2-nitrophenyl)ethyl ATP (NPE caged ATP) in samples that contained 3 mM free Mg(2+) and 130 mM NaCl at pH 7.5. Release of ATP from caged ATP produced highly detailed infrared difference spectra indicating structural changes of the Na(+),K(+)-ATPase. The observed transient state of the enzyme accumulated within seconds after ATP release and decayed on a timescale of minutes at 15 degrees C. Several controls ensured that the observed difference signals were due to structural changes of the Na(+),K(+)-ATPase. Samples that additionally contained 20 mM KCl showed similar spectra but less intense difference bands. The absorbance changes observed in the amide I region, reflecting conformational changes of the protein backbone, corresponded to only 0.3% of the maximum absorbance. Thus the net change of secondary structure was concluded to be very small, which is in line with movement of rigid protein segments during the catalytic cycle. Despite their small amplitude, the amide I signals unambiguously reveal the involvement of several secondary structure elements in the conformational change. Similarities and dissimilarities to corresponding spectra of the Ca(2+)-ATPase and H(+),K(+)-ATPase are discussed, and suggest characteristic bands for the E1 and E2 conformations at 1641 and 1661 cm(-1), respectively, for alphabeta heterodimeric ATPases. The spectra further indicate the participation of protonated carboxyl groups or lipid carbonyl groups in the reaction from E1Na(3)(+) to an E2P state. A negative band at 1730 cm(-1) is in line with the presence of a protonated Asp or Glu residue that coordinates Na(+) in E1Na(3)(+). Infrared signals were also detected in the absorption regions of ionized carboxyl groups.

Lipid-similar Thermal Transition of Polyethylene Glycol Alkyl Ether Detergents

A narrow, reversible endothermic main transition is found in the aqueous micellar phase of octaethylene glycol tetradecyl ether (C14E8) by DSC, characterized by a transition temperature of 41°C and a ΔH 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.

Time-resolved response of fluorescent alkali ion indicators and detection of short-lived intermediates upon binding to molecular cavities

Stopped-flow kinetic studies have been performed to determine the kinetic parameters of K+ binding to the fluorescent cryptand F222 and of Na+binding to F221 at pH 8.O. The results clearly indicate that a comparatively stable intermediate is formed before the rate-limiting binding step occurs with a rate constant around 30 s−1 under the chosen experimental conditions. The conversion of the intermediate to the final cation complex is assigned to the final penetration of the already bound, but still partially solvated cation into the ligands cavity. The main fluorescence intensity change found upon cation binding is attributed to the second reaction step, and not to the fast, initial binding reaction. The comparatively slow overall binding reaction is interpreted on the bases of a special solvate substitution mechanism which, in principle, can also account for the 1500 times slower binding of Ca 2+ to F221. With regard to time-resolved analytical Na+ and K+ determinations, the response times under the chosen conditions are around 20 ms. Differentiation between Na+ and Ca2+, for example, is possible with F221 on the basis of completely different response times.

Na+/K+-ATPase: Probing for a High Affinity Binding Site for Na+ by Spectrofluorometry

The transfer of Na+ and K+ across membranes by the Na+/K+-pump is coupled to the transition between two conformational states of the enzyme denoted E1 and E2 (1,12). In the E1 state the enzyme binds preferentially Na+, while in the E2 state it binds preferentially K+. Fluorescent probes like fluorescein-isothiocyanate (FITC) covalently bound to the protein (8) or potential-sensitive styryl dyes such as RH 421 incorporated into the membrane (2,9,13) change their fluorescent properties in the presence of Na+ or K+, and thus offer the possibility of studying the binding of these ions and conformational transitions of the enzyme spectroscopically. FITC-labeled Na+/K+-ATPase is strongly fluorescent in the presence of high Na+ concentrations, but exhibits a much lower fluorescence if K+ or one of its congeners is bound. Accordingly the two fluorescence emission intensity states have been assigned to the conformational states E1 and E2 (6,8,11). Na+ binding alone can also be studied by means of the fluorescence changes of RH 421, which has been used to study phosphorylation of the native enzyme by ATP in the presence of Na+ and Mg++ under conditions where no K+ is present (2,9,13). Titrations of the enzyme with Na+ in the presence of these dyes showed that the two different fluorescent labels report binding of Na+ to two different sites (4). The implications of these findings to the assignment of fluorescent states to conformations of the enzyme will be discussed here in more detail.