Andreas Barth

Time-resolved Infrared Spectroscopy of the Ca2+-ATPase THE ENZYME AT WORK

Changes in the vibrational spectrum of the sarcoplasmic reticulum Ca2+-ATPase in the course of its catalytic cycle were followed in real time using rapid scan Fourier transform infrared spectroscopy. In the presence of Ca2+, the cycle was induced by the photochemical release of ATP from a biologically inactive precursor (caged ATP, P3-1-(2-nitro)phenylethyladenosine 5′-triphosphate). Absorbance changes arising from ATP binding to the ATPase were observed within the first 65 ms after initiation of ATP release. After ATP binding, up to two subsequent partial reactions of the ATPase reaction cycle were observed depending on the buffer composition (10 mM CaCl2 + 330 mM KCl or 1 mM CaCl2 + 20% Me2SO): (i) formation of the ADP-sensitive phosphoenzyme (kapp = 0.79 s−1 ± 15% at 1°C, pH 7.0, 10 mM CaCl2, 330 mM KCl) and (ii) phosphoenzyme conversion to the ADP-insensitive phosphoenzyme concomitant with Ca2+ release (kapp = 0.092 s−1 ± 7% at 1°C, pH 7.0, 1 mM CaCl2, 20% Me2SO). Each reaction step could well be described by a single time constant for all associated changes in the vibrational spectrum, and no intermediates other than those mentioned above were found. In particular, there is no evidence for a delay between the transition from ADP-sensitive to ADP-insensitive phosphoenzyme and Ca2+ release. In 2H2O a kinetic isotope effect was observed: both the phosphorylation reaction and phosphoenzyme conversion were slowed down by factors of 1.5 and 3.0, respectively. The small amplitudes of the observed changes in the infrared spectrum indicate that the net change of secondary structure is very small and of the same order of magnitude for ATP binding, phosphorylation, and phosphoenzyme conversion. Therefore, our results do not support a distinction between minor and major secondary structure changes in the catalytic cycle of the ATPase, which might be expected according to the classical E1-E2 model.

Molecular changes in the sarcoplasmic reticulum calcium ATPase during catalytic activity: A Fourier transform infrared (FTIR) study using photolysis of caged ATP to trigger the reaction cycle

Fourier transform infrared spectroscopy was used to study ligand binding and conformational changes in the Ca2+‐ATPase of sarcoplasmic reticulum. Novel in infrared difference spectroscopy, the catalytic cycle in the IR sample was started by photolytic release of ATP from an inactive, photolabile ATP‐derivative (caged ATP). Small, but characteristic infrared absorbance changes were observed upon ATP release. On the basis of model spectra, the absorbance changes corresponding to the trigger and substrate reactions, i.e. to photolysis of caged ATP and hydrolysis of ATP, were separated from the absorbance changes due to the active ATPase reflecting formation of the phosphorylated Ca2E1P enzyme form. A major rearrangement of ATPase conformation as the result of catalysis can be excluded.

Changes of protein structure, nucleotide microenvironment, and Ca2+-binding states in the catalytic cycle of sarcoplasmic reticulum Ca2+-ATPase: investigation of nucleotide binding, phosphorylation and phosphoenzyme conversion by FTIR difference spectroscopy

Changes of infrared absorbance of sarcoplasmic reticulum Ca(2+)-ATPase (EC 3.6.1.38) associated with partial reactions of its catalytic cycle were investigated in the region from 1800 to 950 cm-1 in H2O and 2H2O. Starting from Ca2E1, 3 reaction steps were induced in the infrared cuvette via photolytic release of ATP and ADP: (a) nucleotide binding, (b) formation of the ADP-sensitive phosphoenzyme (Ca2E1P) and (c) formation of the ADP-insensitive phosphoenzyme (E2P). All reaction steps caused distinct changes of the infrared spectrum which were characteristic for each reaction step but comparable for all steps in the number and magnitude of the changes. Most pronounced were absorbance changes in the amide I spectral region sensitive to protein secondary structure. However, they were small--less than 1% of the total protein absorbance--indicating that the reaction steps are associated with small and local conformational changes of the polypeptide backbone instead of a large conformational rearrangement. Especially, there is no outstanding conformational change associated with the phosphoenzyme conversion Ca2E1P-->E2P. ADP-binding induces conformational changes in the ATPase polypeptide backbone with alpha-helical structures and presumably beta-sheet or beta-turn structures involved. Phosphorylation is accompanied by the appearance of a keto group vibration that can tentatively be assigned to the phosphorylated residue Asp351. Phosphoenzyme conversion and Ca(2+)-release produce difference signals which can be explained by the release of Ca2+ from carboxylate groups and a change of hydrogen bonding or protonation state of carboxyl groups.

Fourier transform infrared (FTIR) spectroscopic investigation of the nicotinic acetylcholine receptor (nAChR) Investigation of agonist binding and receptor conformational changes by flash‐induced release of ‘caged’ carbamoylcholine

The binding and interaction of carbamoylcholine with the nicotinic acetylcholine receptor was investigated using photolytically release carbamoylcholine (‘caged’ carbamoylcholine). Upon UV flash activation of this photolabile substrate analog, characteristic changes in the IR absorbance spectrum were detected. Apart from difference bands arising from the changes of molecular structure upon photolytical release, spectral features can be attributed to the agonist upon binding to the receptor as well as to conformational changes of the receptor itself. The two photo‐labile agonist analogs N‐[1‐(2‐nitrophenyl)ethyl] carbamoylcholine iodide (cage I) and N‐(α‐carboxy‐2‐nitrobenzyl) carbamoylcholine trifluoroacetate (cage II), with different structures for comparison of the 1680‐1540 cm−1 region sensitive for protein conformation, yielded consistent results. A preliminary interpretation in terms of substrate binding and local conformational changes of the receptor upon carbamoylcholine binding is provided, in analogy to the binding of acetylcholine, activation, and subsequent deactivation taking place during signal transduction.

Ca2+ Release from the Phosphorylated and the Unphosphorylated Sarcoplasmic Reticulum Ca2+ ATPase Results in Parallel Structural Changes AN INFRARED SPECTROSCOPIC STUDY

Structural changes of the sarcoplasmic reticulum Ca2+-ATPase occurring in the reaction step involving phosphoenzyme conversion and Ca2+ release (Ca2 E 1-P →E 2-P) were followed using time-resolved infrared spectroscopy in H2O and2H2O. The difference spectra measured between 1800 and 1500 cm−1 were almost identical to those of Ca2+ release from the unphosphorylated ATPase (Ca2 E 1 → E), implying that parallel structural changes occur in both steps. This suggests that characteristic structural features of the high affinity Ca2+ binding sites of Ca2 E 1 are still present in the ADP-sensitive phosphoenzyme Ca2 E 1-P. In both Ca2+release steps at least two carboxyl groups become protonated, each of them experiencing the same strength of hydrogen bonding irrespective of whether or not the Ca2+ free ATPase is phosphorylated. This suggests that the same amino acid residues are involved and that they are most likely those that participate in high affinity Ca2+ binding and H+ countertransport. We propose that during Ca2+ release from the phosphoenzyme protons from the lumenal side have access to these residues. Our results are consistent with only one pair of Ca2+ binding sites on the ATPase that serves both Ca2+ translocation and H+ countertransport.

Three Partial Reactions of the Ca2+-Pumping Cycle of the Ca2+-Atpase Studied by Time-Resolved FTIR Spectroscopy

Infrared absorbance changes of the sarcoplasmic reticulum (SR) Ca2+-ATPase arising from 3 partial reactions of its Ca2+-pumping cycle were triggered by the photochemical release of ATP from caged ATP (P 3-1-(2-nitro)phenylethyladenosine 5′-triphosphate) and were followed in real time using rapid scan FTIR spectroscopy. Each reaction was investigated in H2O and D2O and the effects of isotopic substitution of the γ-phosphate oxygens were analysed. Absorbance changes of the phosphate group and presumably Asp351 and ATP due to the phosphate transfer from ATP to Asp351 could be identified.

Changes of molecular structure and interaction in the catalytic cycle of sarcoplasmic reticulum Ca2+- ATPase

Infrared difference spectroscopy was used to get more insight into the transport mechanism of sarcoplasmic reticulum Ca2+-ATPase. Several partial reactions were induced directly in the infrared cuvette via photolytic release of ligands or substrates from photolabile precursors (1,2). From the spectra before and after the release a difference spectrum can be calculated that reflects only those changes of infrared absorption which are associated with the catalytic reactions and the triggering photolysis reaction. The accuracy of this method is extremely high, since it avoids possible inaccuracies when spectra of two samples prepared in different enzyme states are compared.

Infrared Spectroscopic Investigation of Enzyme Reactions Using Photolabile Effector Molecules

Reaction-induced infrared difference spectroscopy has long been used to study the molecular basis of light-induced reactions in pigment-protein complexes. The techniques used are either light-minus-dark Fourier transform infrared (FTIR) spectroscopy for steady states or flash-induced time-resolved IR and FTIR techniques for transient reactions, with the major focus on bacteriorhodopsin, rhodopsin, and the photosynthetic reaction center (for reviews, see [1–3]). Upon development of protein electrochemical techniques and ultra-thin-layer IR spectroelectrochemical cells, the basic concepts of IR difference spectroscopy could be extended to the large class of redox proteins and enzymes [4–6]. Yet, enzyme-substrate reactions in general, ATPases, and many other protein reactions started by an effector molecule could only be studied for very few cases by IR difference techniques, since the mixing of the effector to the enzyme presents too large a perturbation for the sensitivity needed in these experiments.

Titration of Protonable Residues in Proteins by Flash Induced H+-Release from “Caged Proton” — UV/VIS and IR Studies

Infrared spectroscopy has proven to be a valuable tool for the study of enzyme structure and function. Using differential techniques, molecular changes in the course of photoreactions and redox reactions have been studied (for a review see [1]). Recently, the use of photolabile substrate analogues, which can be released by a UV flash, has been reported for the study of ATPases [2,3]. This technique of photo-chemo-triggering a reaction can be used for many different enzymes (see Barth et al., these proceedings), provided that a suitable photolabile compound is available.

Infrared absorbance changes of sarcoplasmic reticulum (SR) Ca2+-ATPase in its catalytic cycle

Ca2+ transport from the cytoplasm of muscle cells into SR, necessary for muscle relaxation, is performed by the Ca2+-ATPase, an intrinsic membrane protein of about 110 kDa molecular mass. The energy required for this active transport is provided by hydrolysis of ATP. Although the catalytic cycle of the ATPase has been subject of detailed investigation, the molecular nature of the transport mechanism is still unknown. The reaction cycle involves successive Ca2+ binding (E→ Ca2E1), formation of an ADP-sensitive phosphoenzyme with occluded Ca2+ (Ca2E1-P), formation of an ADP-insensitive phosphoenzyme (E2-P) and concomitant Ca2+ release to the SR lumen and deposphorylation of the ATPase [1]