Acid-induced unfolding of myoglobin triggered by a laser pH jump method.

Using 1-(2-nitrophenyl)ethyl sulfate (caged sulfate) as a photoactivatable caged proton, we could induce complete acid unfolding of myoglobin with a single nanosecond laser pulse. This was possible because of the high ( approximately mM) concentration of protons released by the photolabile compound. The ability of the compound to produce a large pH jump arises because the other photoproducts (2-nitrosoacetophenone and sulfate ion) do not buffer the released protons. The complete time course of the unfolding kinetics, spanning a range from milliseconds to several seconds, could be accurately reproduced by monitoring absorbance changes in the visible spectrum at 633 nm.     

P(3)-[2-(4-hydroxyphenyl)-2-oxo]ethyl ATP for the rapid activation of the Na(+),K(+)-ATPase

P(3)-[2-(4-hydroxyphenyl)-2-oxo]ethyl ATP (pHP-caged ATP) has been investigated for its application as a phototrigger for the rapid activation of electrogenic ion pumps. The yield of ATP after irradiation with a XeCl excimer laser (lambda = 308 nm) was determined at pH 6.0-7.5. For comparison, the photolytic yields of P(3)-[1-(2-nitrophenyl)]ethyl ATP (NPE-caged ATP) and P(3)-[1, 2-diphenyl-2-oxo]ethyl ATP (desyl-caged ATP) were also measured. It was shown that at lambda = 308 nm pHP-caged ATP is superior to the other caged ATP derivatives investigated in terms of yield of ATP after irradiation. Using time-resolved single-wavelength IR spectroscopy, we determined a lower limit of 10(6) s(-1) for the rate constant of release of ATP from pHP-caged ATP at pH 7.0. Like NPE-caged ATP, pHP-caged ATP and desyl-caged ATP bind to the Na(+), K(+)-ATPase and act as competitive inhibitors of ATPase function. Using pHP-caged ATP, we investigated the charge translocation kinetics of the Na(+),K(+)-ATPase at pH 6.2-7.4. The kinetic parameters obtained from the electrical measurements are compared to those obtained with a technique that does not require caged ATP, namely parallel stopped-flow experiments using the voltage-sensitive dye RH421. It is shown that the two techniques yield identical results, provided the inhibitory properties of the caged compound are taken into account. Our results demonstrate that under physiological (pH 7.0) and slightly basic (pH 7.5) or acidic (pH 6. 0) conditions, pHP-caged ATP is a rapid, effective, and biocompatible phototrigger for ATP-driven biological systems.     

Flash photolysis of caged compounds in Limulus ventral photoreceptors

Rapid concentration jumps of Ins(1,4,5)P3 or ATP were made inside Limulus ventral photoreceptors by flash photolysis of the parent caged compounds. In intact ventral photoreceptors, the photolysis flash evokes a maximum amplitude light-activated current; therefore, a procedure was developed for uncoupling phototransduction by blocking two of the initial reactions in the cascade, rhodopsin excitation and G protein activation. Rhodopsin was inactivated by exposure to hydroxylamine and bright light. This procedure abolished the early receptor potential and reduced the quantum efficiency by 325 +/- 90-fold (mean +/- SD). G protein activation was blocked by injection of guanosine-5'-O-(2-thiodiphosphate) (GDP beta S). GDP beta S injection reduced the quantum efficiency by 1,881 +/- 1,153-fold (mean +/- SD). Together hydroxylamine exposure and GDP beta S injection reduced the quantum efficiency by 870,000 +/- 650,000-fold (mean +/- SD). After the combined treatment, photoreceptors produced quantum bumps to light that was approximately 10(6) times brighter than the intensity that produced quantum bumps before treatment. Experiments were performed with caged compounds injected into photoreceptors in which phototransduction was largely uncoupled. Photolysis of one compound, myo-inositol 1,4,5-triphosphate P4(5)-1-(2-nitrophenyl)ethyl ester (caged IP3), increased the voltage clamp current in response to the flashlamp by more than twofold without changing the latency of the response. The effect was not seen with photolysis of either adenosine-5'-triphosphate P3-1-(2-nitrophenyl)ethyl ester (caged ATP) or caged IP3 in cells preloaded with either heparin or (1,2-bis-(o-amino-phenoxy)ethane-N-N-N'-N' tetraacetic acid tetrapotassium salt (BAPTA). The results suggest that photoreleased IP3 releases calcium ions from intracellular stores and the resulting increase in [Ca2+]i enhances the amplification of the phototransduction cascade.     

Time-resolved infrared spectroscopy of pH-induced aggregation of the Alzheimer Abeta(1-28) peptide

Aggregation of the Alzheimer's disease-related Aβ_1-28 peptide was induced by a rapid, sub-millisecond pH jump and monitored by time-resolved infrared spectroscopy on the millisecond to second time-scale. The release of protons was induced by the photolysis of a caged compound, 1-(2-nitrophenyl)ethyl sulfate (NPE-sulfate). The pH jump generated in our experimental setup is used to model the Aβ peptide structural conversions that may occur in the acidic endosomal/lysosomal cell compartment system. The aggregation of the Aβ_1-28 peptide induced by the pH jump from 8.5 to < 6 yields an antiparallel β-sheet structure. The kinetics of the structural transition is biphasic, showing an initial rapid phase with a transition from random coil to an oligomeric β-sheet form with a time constant of 3.6 s. This phase is followed by a second slower transition, which yields larger aggregates during 48.0 s.     

Photolabile precursors of inositol phosphates. Preparation and properties of 1-(2-nitrophenyl)ethyl esters of myo-inositol 1,4,5-trisphosphate

1-(2-Nitrophenyl)ethyl esters of D-myo-inositol 1,4,5-trisphosphate (InsP3) have been synthesized and shown to have suitable properties for use as photolabile precursors of InsP3. Synthesis was accomplished by treatment of InsP3 with 1-(2-nitrophenyl)diazoethane in a CHCl3/water mixture. This resulted in esterification of each of the three phosphate residues in InsP3, the 1-phosphate being more reactive than the 4- or 5-phosphate. Singly esterified P-1, P-4, and P-5 esters, termed P-1, P-4, and P-5 caged InsP3, were isolated from the reaction mixture by anion-exchange HPLC and characterized by 500-MHz 1H NMR spectroscopy. Each of these caged InsP3 esters exists as a pair of diastereoisomers and was identified by examining the effects of pH and nitrophenyl ring current shielding on the chemical shifts of nonexchangeable inositol protons. 1H NMR spectra of InsP3 were analyzed for comparison. On photolysis the compounds released InsP3 with rate constants of 175 (P-1), 225 (P-4), and 280 s-1 (P-5) as determined by monitoring the aci-nitro decay reaction at pH 7.1, 0.2 M ionic strength, 21 degrees C. Quantum yields determined by steady-state near-UV photolysis were 0.65 +/- 0.08 for each compound. P-4 and P-5 caged InsP3 were the most promising biologically inactive InsP3 precursors since at concentrations up to 50 microM they did not release Ca2+ from smooth muscle sarcoplasmic reticulum (SR) and were not metabolized by vascular smooth muscle InsP3 5-phosphatase or bovine brain InsP3 3-kinase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fast transient currents in Na,K-ATPase induced by ATP concentration jumps from the P3-[1-(3',5'-dimethoxyphenyl)-2-phenyl-2-oxo]ethyl ester of ATP.

Electrogenic ion transport by Na,K-ATPase was investigated by analysis of transient currents in a model system of protein-containing membrane fragments adsorbed to planar lipid bilayers. Sodium transport was triggered by ATP concentration jumps in which ATP was released from an inactive precursor by an intense near-UV light flash. The method has been used previously with the P3-1-(2-nitrophenyl)ethyl ester of ATP (NPE-caged ATP), from which the relatively slow rate of ATP release limits analysis of processes in the pump mechanism controlled by rate constants greater than 100 s(-1) at physiological pH. Here Na,K-ATPase was reinvestigated using the P3-[1-(3,5-dimethoxyphenyl)-2-phenyl-2-oxo]ethyl ester of ATP (DMB-caged ATP), which has an ATP release rate of >10(5) s(-1). Under otherwise identical conditions, photorelease of ATP from DMB-caged ATP showed faster kinetics of the transient current compared to that from NPE-caged ATP. With DMB-caged ATP, transient currents had rate profiles that were relatively insensitive to pH and the concentration of caged compound. Rate constants of ATP binding and of the E1 to E2 conformational change were compatible with earlier studies. Rate constants of enzyme phosphorylation and ADP-dependent dephosphorylation were 600 s(-1) and 1.5 x 10(6) M(-1) s(-1), respectively, at pH 7.2 and 22 degrees C.     

Time-resolved electron microscopic analysis of the behavior of myosin heads on actin filaments after photolysis of caged ATP

The interaction between myosin subfragment 1 (S1) and actin filaments after the photolysis of P3-1-(2-nitrophenyl)ethyl ester of ATP (caged ATP) was analyzed with a newly developed freezing system using liquid helium. Actin and S1 (100 microM each) formed a ropelike double-helix characteristic of rigor in the presence of 5 mM caged ATP at room temperature. At 15 ms after photolysis, the ropelike double helix was partially disintegrated. The number of S1 attached to actin filaments gradually decreased up to 35 ms after photolysis, and no more changes were detected from 35 to 200 ms. After depletion of ATP, the ropelike double helix was reformed. Taking recent analyses of actomyosin kinetics into consideration, we concluded that most S1 observed on actin filaments at 35-200 ms are so called "weakly bound S1" (S1.ATP or S1.ADP.Pi) and that the weakly bound S1 under a rapid association- dissociation equilibrium with actin filaments can be captured by electron microscopy by means of our newly developed freezing system. This enabled us to directly compare the conformation of weakly and strongly bound S1. Within the resolution of deep-etch replica technique, there were no significant conformational differences between weakly and strongly bound S1, and neither types of S1 showed any positive cooperativity in their binding to actin filaments. Close comparison revealed that the weakly and strongly bound S1 have different angles of attachment to actin filaments. As compared to strongly bound S1, weakly bound S1 showed a significantly broader distribution of attachment angles. These results are discussed with special reference to the molecular mechanism of acto-myosin interaction in the presence of ATP.     

The efficiency of two-photon photolysis of a "caged" fluorophore, o-1-(2-nitrophenyl)ethylpyranine, in relation to photodamage of synaptic terminals

Localized photolysis of caged neurotransmitters with the two-photon effect for investigations at synaptic preparations was evaluated by determining the toxicity to synaptic transmission of pulsed near-IR laser light focused into the terminals of the snake neuromuscular junction, and measuring the extent of photolysis of a conventional caging group with similar irradiation in microcuvette experiments. Photodamage was seen in synaptic terminals as a large, irreversible increase of spontaneous synaptic activity with laser flashes of 5 ms at 1 Hz at average powers > 5 mW and was due to multiphoton absorption. Localized photolysis due to two-photon absorption was investigated for a representative caged fluorophore, the 1-(2-nitrophenyl)ethyl ether of pyranine (NPE-HPTS). Irradiation of NPE-HPTS at 5 mW with the same optical arrangement produced very low rates of photolysis. NPE-HPTS photolysis mechanisms were investigated at high laser powers by measuring (1) the kinetics of two-photon fluorescence generated by two-photon photolysis in the focal volume and (2) the rates of HPTS accumulation inside closed 2-10 microm radius vesicles, measured with one-photon excitation during two-photon photolysis by repetitive 10 micros laser exposures. The two-photon crosssection of NPE-HPTS photolysis calculated from the rates is 0.02-0.04 GM (10(-50) cm4 x s/photon) and limits the efficiency of photolysis at 5 mW. With free diffusional exchange, 50% steady-state cage depletion in the focal volume was estimated to occur only at high laser powers of ca. 72 mW, masked in experiments by multiphoton bleaching. Based on these results, the two-photon photolysis cross-section needed for 50% steady-state photolysis of a caged neurotransmitter at 5 mW is calculated as 31 GM, much higher than in existing caged compounds.     

3'-Arylazido-8-azido ATP--a cross-linking photoaffinity label for ATP binding proteins

The synthesis of 3′-O-{3-[N-(4-azido-2-nitrophenyl) amino] propionyl} 8-azido-adenosine 5′-triphosphate—a 3′-arylazido-8-azido ATP—is described. The ATP derivative is characterized by thin layer chromatography, infrared spectroscopy, and optical spectroscopy. Its photolysis upon irradiation with uv light and its stability in dependence on pH are tested. Its two photolabile azido groups allow the use of this ATP analog as a photoaffinity label for cross-linking the subunits of ATP binding proteins.     

Kinetics of proton binding to the sarcoplasmic reticulum Ca-ATPase in the E1 state

A new caged proton, 2-methoxy-5-nitrophenyl sulfate, was synthesized and used in time-resolved pH jump experiments to study proton binding in the sarcoplasmic reticulum Ca-ATPase. The major advantage of this compound is that it does not produce significant artifacts in experiments in which the fluorescent styryl dye 2BITC is used to monitor ion movements in the Ca pump. Two rate-limiting processes were resolved and their dependence on pH, Ca(2+) concentration, and temperature investigated. The faster process showed a relaxation time between 4 and 8 ms independent on pH and Ca(2+) concentration, and the time constant of the slower process varied between 31 ms (0 Ca(2+)) and 100 ms (100 microM Ca(2+)). A consistent mechanism to explain the results was derived in agreement with previous studies and the generally accepted Post-Albers scheme of the pump cycle. This mechanism requires that under physiological conditions the ion-binding sites are always occupied and two protons and a Ca(2+) ion replace each other. In the absence of ATP at low pH a nonphysiological state can be induced in which up to four protons bind to the Ca pump in the E(1) conformation. So far it could not be verified whether these additional protons bind to amino acid side chains or are coordinated as hydronium ions.     

Interactions of caged-ATP and photoreleased ATP with alkaline phosphatase

Photolytic release of ATP from inactive P(3)-[1-(2-nitrophenyl)]ethyl ester of ATP (NPE-caged ATP) provides a means to reveal molecular interactions between nucleotide and enzyme by using infrared spectroscopy. Reaction-induced infrared difference spectra of bovine intestinal alkaline phosphatase (BIAP) and of NPE-caged ATP revealed small structural alterations on the peptide backbone affecting one or two amino-acid residues. After photorelease of ATP, the substrate could be hydrolyzed sequentially by the enzyme producing three Pi, adenosine, and the photoproduct nitrosoacetophenone. It was concluded that NPE-caged ATP could bind to BIAP prior to the photolytic cleavage of ATP and that Pi could interact with BIAP after photolysis of NPE-caged ATP and hydrolysis, yielding infrared spectra with distinct structure changes of BIAP. This suggests that the molecular mechanism of ATP hydrolysis by BIAP involved small structural adjustments of the peptide backbone in the vicinity of the active site during ATP hydrolysis which continued during Pi binding.     

Excitatory signaling in bacterial probed by caged chemoeffectors.

Chemotactic excitation responses to caged ligand photorelease of rapidly swimming bacteria that reverse (Vibrio alginolyticus) or tumble (Escherichia coli and Salmonella typhimurium) have been measured by computer. Mutants were used to assess the effects of abnormal motility behavior upon signal processing times and test feasibility of kinetic analyses of the signaling pathway in intact bacteria. N-1-(2-Nitrophenyl)ethoxycarbonyl-L-serine and 2-hydroxyphenyl 1-(2-nitrophenyl) ethyl phosphate were synthesized. These compounds are a 'caged' serine and a 'caged' proton and on flash photolysis release serine and protons and attractant and repellent ligands, respectively, for Tsr, the serine receptor. The product quantum yield for serine was 0.65 (+/- 0.05) and the rate of serine release was proportional to [H+] near-neutrality with a rate constant of 17 s-1 at pH 7.0 and 21 degrees C. The product quantum yield for protons was calculated to be 0.095 on 308-nm irradiation but 0.29 (+/- 0.02) on 300-350-nm irradiation, with proton release occurring at > 10(5) s-1. The pH jumps produced were estimated using pH indicators, the pH-dependent decay of the chromophoric aci-nitro intermediate and bioassays. Receptor deletion mutants did not respond to photorelease of the caged ligands. Population responses occurred without measurable latency. Response times increased with decreased stimulus strength. Physiological or genetic perturbation of motor rotation bias leading to increased tumbling reduced response sensitivity but did not affect response times. Exceptions were found. A CheR-CheB mutant strain had normal motility, but reduced response. A CheZ mutant had tumbly motility, reduced sensitivity, and increased response time to attractant, but a normal repellent response. These observations are consistent with current ideas that motor interactions with a single parameter, namely phosphorylated CheY protein, dictate motor response to both attractant and repellent stimuli. Inverse motility motor mutants with extreme rotation bias exhibited the greatest reduction in response sensitivity but, nevertheless, had normal attractant response times. This implies that control of CheY phosphate concentration rather than motor reactions limits responses to attractants.     

TNP-AMP binding to the sarcoplasmic reticulum Ca(2+)-ATPase studied by infrared spectroscopy

Infrared spectroscopy was used to monitor the conformational change of 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-monophosphate (TNP-AMP) binding to the sarcoplasmic reticulum Ca(2+)-ATPase. TNP-AMP binding was observed in a competition experiment: TNP-AMP is initially bound to the ATPase but is then replaced by beta,gamma-iminoadenosine 5'-triphosphate (AMPPNP) after AMPPNP release from P(3)-1-(2-nitrophenyl)ethyl AMPPNP (caged AMPPNP). The resulting infrared difference spectra are compared to those of AMPPNP binding to the free ATPase, to obtain a difference spectrum that reflects solely TNP-AMP binding to the Ca(2+)-ATPase. TNP-AMP used as an ATP analog in the crystal structure of the sarcoplasmic reticulum Ca(2+)-ATPase was found to induce a conformational change upon binding to the ATPase. It binds with a binding mode that is different from that of AMPPNP, ATP, and other tri- and diphosphate nucleotides: TNP-AMP binding causes partially opposite and smaller conformational changes compared to ATP or AMPPNP. The conformation of the TNP-AMP ATPase complex is more similar to that of the E1Ca(2) state than to that of the E1ATPCa(2) state. Regarding the use of infrared spectroscopy as a technique for ligand binding studies, our results show that infrared spectroscopy is able to distinguish different binding modes.     

Kinetics of contraction initiated by flash photolysis of caged adenosine triphosphate in tonic and phasic smooth muscles

Laser flash photolysis of caged adenosine triphosphate (ATP), in the presence of Ca2+, was used to examine the time course of isometric force development from rigor states in glycerinated tonic (rabbit trachealis) and phasic (guinea-pig ileum and portal vein) smooth muscles. Photolytic liberation of ATP from caged ATP initiated force development, at 20 degrees C, with half-time (t1/2) of 5.4 s in trachealis and 1.2-2.2 s in the phasic muscles. Prior to photolysis, some muscles were phosphorylated with ATP plus okadaic acid (an inhibitor of myosin light-chain phosphatase) or thiophosphorylated with ATP gamma S to fully activate the regulatory system, before turning on the contractile apparatus. In these prephosphorylated muscles, force development, after caged ATP photolysis, was more rapid than in the unphosphorylated muscles, but the t1/2 values for trachealis (0.8-1.1 s) were still longer than for ileum and portal-vein muscles (0.20-0.25 s). The results suggest that both the contractile machinery and the regulatory system are slower in the tonic than in the phasic smooth muscles. The time course of force development for each muscle type was sigmoidal, with an initial delay (td) of approximately 10% of the t1/2 value. Some possible chemical and mechanical origins of the delay are discussed.     

Fluorescence polarization of skeletal muscle fibers labeled with rhodamine isomers on the myosin heavy chain.

Fluorescence polarization was used to examine orientational changes of Rhodamine probes in single, skinned muscle fibers from rabbit psoas muscle following either photolysis of caged nucleotides or rapid length changes. Fibers were extensively and predominantly labeled at SH1 (Cys-707) of the myosin heavy chain with either the 5- or the 6-isomer of iodoacetamidotetramethylrhodamine. Results from spectroscopic experiments utilizing the two Rhodamine isomers were quite similar. Following photolysis of either caged ATP or caged ADP, probes promptly reoriented toward the muscle fiber axis. Changes in the fluorescence polarization signals with transients elicited by the photolysis of caged ATP in the presence of saturating Ca2+ greatly preceded active force generation. Photolysis of caged ADP caused only a small, rapid decrease in force but elicited changes in the fluorescence polarization signals with time course and amplitude similar to those following photolysis of caged ATP. Fluorescence polarization signals were virtually unchanged by rapid length steps in both rigor and active muscle fibers. These results indicate that structural changes monitored by Rhodamine probes at SH1 are not associated directly with the force-generating event of muscle contraction. However, the fluorescence polarization transients were slightly faster than the estimated rate of cross-bridge detachment following photolysis of caged ATP, suggesting that the observed structural changes at SH1 may be involved in the communication pathway between the nucleotide- and actin-binding sites of myosin.     

Photochemical reaction mechanisms of 2-nitrobenzyl compounds: 2-nitrobenzyl alcohols form 2-nitroso hydrates by dual proton transfer.

Irradiation of 2-nitrobenzyl alcohol (1, R = H) and 1-(2-nitrophenyl)ethanol (1, R = Me) in various solvents yields 2-nitroso benzaldehyde (4, R = H) and 2-nitroso acetophenone (4 R = Me), respectively, with quantum yields of about 60%. The mechanism of this reaction, known since 1918, was investigated using laser flash photolysis, time-resolved infrared spectroscopy (TRIR), and 18O-labeling experiments. The primary aci-nitro photoproducts 2 react by two competing paths. The balance between the two depends on the reaction medium. Reaction via hydrated nitroso compounds 3 formed by proton transfer prevails in aprotic solvents and in aqueous acid and base. In water, pH 3-8, the classical mechanism of cyclization to benzisoxazolidine intermediates 5, followed by ring opening to carbonyl hydrates 6, predominates. The transient intermediates 3 and 6 were identified by TRIR. Potential energy surfaces for these reactions were mapped by density functional calculations.     

Infrared spectroscopic signals arising from ligand binding and conformational changes in the catalytic cycle of sarcoplasmic reticulum calcium ATPase.

Fourier transform infrared spectroscopy was used to investigate ligand binding and conformational changes in the Ca2(+)-ATPase of sarcoplasmic reticulum during the catalytic cycle. The ATPase reaction was started in the infrared sample by release of ATP from the inactive, photolabile ATP derivative P3-1-(2-nitro)phenylethyladenosine 5'-triphosphate (caged ATP). Absorption spectroscopy in the visible spectral region using the Ca2(+)-sensitive dye Antipyrylazo III ensured that the infrared samples were able to transport Ca2+ in spite of their low water content, which is required for mid-infrared measurements (1800-950 cm-1). Small, but characteristic and highly reproducible infrared absorbance changes were observed upon ATP release. These infrared absorbance changes exhibit different kinetic properties. Comparison with model compound infrared spectra indicates that they are related to photolysis of caged ATP, hydrolysis of ATP in consequence of ATPase activity and to molecular changes in the active ATPase. The absorbance changes due to alterations in the ATPase were observed mainly in the region of Amide I and Amide II protein absorbance and presumably reflect the molecular processes upon phosphoenzyme formation. Since the absorbance changes were small compared to the overall ATPase absorbance, no major rearrangement of ATPase conformation as the result of catalysis could be detected.     

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.