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1.
J Am Chem Soc ; 143(3): 1513-1520, 2021 01 27.
Article in English | MEDLINE | ID: mdl-33449695

ABSTRACT

Photopharmacology addresses the challenge of drug selectivity and side effects through creation of photoresponsive molecules activated with light with high spatiotemporal precision. This is achieved through incorporation of molecular photoswitches and photocages into the pharmacophore. However, the structural basis for the light-induced modulation of inhibitory potency in general is still missing, which poses a major design challenge for this emerging field of research. Here we solved crystal structures of the glutamate transporter homologue GltTk in complex with photoresponsive transport inhibitors-azobenzene derivative of TBOA (both in trans and cis configuration) and with the photocaged compound ONB-hydroxyaspartate. The essential role of glutamate transporters in the functioning of the central nervous system renders them potential therapeutic targets in the treatment of neurodegenerative diseases. The obtained structures provide a clear structural insight into the origins of photocontrol in photopharmacology and lay the foundation for application of photocontrolled ligands to study the transporter dynamics by using time-resolved X-ray crystallography.


Subject(s)
Amino Acid Transport System X-AG/antagonists & inhibitors , Amino Acid Transport System X-AG/metabolism , Aspartic Acid/analogs & derivatives , Aspartic Acid/metabolism , Azo Compounds/metabolism , Amino Acid Transport System X-AG/chemistry , Aspartic Acid/radiation effects , Azo Compounds/chemistry , Azo Compounds/radiation effects , Crystallography, X-Ray , Protein Binding , Stereoisomerism , Thermococcus/chemistry , Ultraviolet Rays
2.
Amino Acids ; 39(2): 599-604, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20130939

ABSTRACT

The synthesis of N-protected glycosyl amino acids from amines has been investigated and it was found that, under microwave conditions, glycosylamines could be hydrolyzed leading to new products containing a glycosyl ester linkage. The efficiency of the microwave-induced glycosylation of aspartic acid was studied comparing the microwave activity between amide and ester bond formation. Different sugar moieties have been employed to demonstrate the simple and reproducible coupling methodology. New glycosyl ester compounds were further characterized by NMR spectroscopy.


Subject(s)
Amines/chemistry , Aspartic Acid/radiation effects , Microwaves , Amines/radiation effects , Glucosamine/analogs & derivatives , Glucosamine/chemistry , Glucosamine/radiation effects , Glycosylation , Nuclear Magnetic Resonance, Biomolecular
3.
Biophys Chem ; 125(1): 92-103, 2007 Jan.
Article in English | MEDLINE | ID: mdl-16887255

ABSTRACT

Aspartic acid (Asp) and asparagine (Asn) are vulnerable amino acids. One-electron addition or withdrawal reactions initiate many deleterious processes involving these amino acids. To study these redox processes we have irradiated by gamma-rays asparagine or aspartic acid in the solid state. The nature of the resulting free radicals was determined by electron paramagnetic resonance (EPR) and by calculations using DFT methods in various environments. Reactions initiated by electron transfer are different for both amino acids: Asn anion loses hydrogen atom whereas the cation undergoes decarboxylation. Conversely, Asp cation loses hydrogen atom from amine group, which triggers decarboxylation.


Subject(s)
Asparagine/radiation effects , Aspartic Acid/radiation effects , Asparagine/chemistry , Aspartic Acid/chemistry , Decarboxylation , Electron Spin Resonance Spectroscopy , Free Radicals/chemistry , Gamma Rays , Molecular Conformation/radiation effects
4.
Eur Radiol ; 13(5): 994-1000, 2003 May.
Article in English | MEDLINE | ID: mdl-12695820

ABSTRACT

Proton MR spectroscopy was used to observe long-term post-irradiation metabolic changes in epileptogenic tissue and in the contralateral parts of the brain which are not available with conventional imaging methods. We studied these changes in the temporal lobe in six patients, following radiosurgery on the amygdala and hippocampus. (1)H MR spectroscopy at 1.5 T with short and long echo times (TE=10 and 135 ms) were used together with standard MR imaging sequences (T1-, T2-weighted). The treatment was performed by Leksell Gamma Knife with a dose of 50 Gy to the center and a 50% isodose to the margin of the target, represented by the mean volume of approximately 7.5 ml. Magnetic resonance imaging and MR spectroscopy examinations were performed at least once per year for 3 years. The most significant changes in spectra were observed approximately 1 year after the irradiation when edema in irradiated area was observed and strong signal of lipids was identified. Later, edema and lipid signals disappeared and follow-up was characterized by a decrease of NAA, Cr, and Cho concentrations in the ipsilateral region of the brain to the irradiation (LCModel calculation from voxel of interest 3.8-4.5 ml positioned into the centrum of target volume). The concentration of NAA, Cr, and Cho after radiosurgery was significantly different from control values ( p<0.05) and also from concentrations in the contralateral part of the brain ( p<0.05). In the contralateral part, the concentration of NAA was significantly increased ( p<0.05) (NAA: before treatment 8.81, after treatment 11.33 mM). No radiotoxic changes were observed in the contralateral part of the brain or behind the area of target volume. The MR spectroscopy findings precluded MRI observation and MRS results completed data about the development of radiotoxic changes in the target volume.


Subject(s)
Aspartic Acid/analogs & derivatives , Epilepsy, Temporal Lobe/diagnosis , Epilepsy, Temporal Lobe/surgery , Radiosurgery , Adult , Aspartic Acid/metabolism , Aspartic Acid/radiation effects , Brain/metabolism , Brain/radiation effects , Brain Neoplasms/diagnosis , Brain Neoplasms/metabolism , Brain Neoplasms/surgery , Choline/metabolism , Choline/radiation effects , Creatine/metabolism , Creatine/radiation effects , Epilepsy, Temporal Lobe/metabolism , Female , Follow-Up Studies , Glutamic Acid/metabolism , Glutamic Acid/radiation effects , Glutamine/metabolism , Glutamine/radiation effects , Humans , Lipid Metabolism , Lipids/radiation effects , Magnetic Resonance Imaging , Magnetic Resonance Spectroscopy , Male , Postoperative Complications/diagnosis , Postoperative Complications/etiology , Postoperative Complications/metabolism , Radiation Injuries/diagnosis , Radiation Injuries/etiology , Radiation Injuries/metabolism , Radiation Tolerance/physiology , Radiosurgery/adverse effects , Seizures/diagnosis , Seizures/etiology , Seizures/metabolism , Time Factors , Treatment Failure
5.
Orig Life Evol Biosph ; 32(2): 91-8, 2002 Apr.
Article in English | MEDLINE | ID: mdl-12185677

ABSTRACT

The photochemistry of a mixture of urea and maleic acid, which are thought to have been widely present on the primitive Earth, was studied in order to examine a possibility of the formation of amino acids. When an aqueous solution of urea and maleic acid was irradiated with an ultraviolet light of wavelength 172 nm, urea was revealed to be rather resistant to photochemical decomposition. In contrast, maleic acid was completely decomposed within 4 h, reflecting the reactivity of a C-C double bond in the molecule. In the reaction mixture, 2-isoureidosuccinic acid was detected. The acid was considered to be formed by addition of an isoureido radical which had been produced from urea by the action of a hydroxyl radical, to a C-C double bond of maleic acid. The isoureido group of the product was revealed to undergo thermal rearrangement to afford 2-ureidosuccinic acid (N-carbamoylaspartic acid). The result suggested a novel pathway leading to the formation of aspartic acid from non-amino acid precursors, possibly effected by UV-light on the primitive Earth. The formation of ureidocarboxylic acids is of another significance, since they are capable of undergoing thermal polymerization, resulting in formation of polyamino acids.


Subject(s)
Aspartic Acid/radiation effects , Maleates/chemistry , Ultraviolet Rays , Urea/chemistry , Aspartic Acid/chemistry , Maleates/radiation effects , Models, Molecular , Molecular Structure , Urea/radiation effects
6.
Photochem Photobiol ; 74(3): 477-82, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11594064

ABSTRACT

Alpha-crystallin possesses a molecular chaperone-like activity that prevents proteins from aggregating; however, the mechanism of this activity is not well known. Here we have taken gamma-irradiated alpha-crystallin and studied the relationship between the decrease in chaperone-like activity and the modifications such as oxidation, isomerization and racemization of amino acids in this molecule. We found that the chaperone-like activity of alpha-crystallin decreased with increasing gamma irradiation. After 4000 Gy gamma irradiation the activity of alpha-crystallin was reduced to 40% of the level of nonirradiated, native alpha-crystallin. The circular dichroism spectrum showed that the secondary structure of the irradiated alpha-crystallin had not changed. However, its tertiary structure appeared to change following more than 1000 Gy irradiation. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis also indicated that cross-linking of alpha-crystallin increased with increasing radiation doses. Irradiated and nonirradiated alpha-crystallin was subjected to trypsin digestion and peptide analysis by reverse-phase high-performance liquid chromatography and mass and sequence analysis. Depending on the radiation dose, Met-1 of alpha A-crystallin was oxidized to methionine sulfoxide. In addition, Asp-151 of alpha A-crystallin was isomerized to the beta-Asp form after irradiation, and racemization of Asp-151 decreased. Thus, the loss of the chaperone-like activity of alpha-crystallin is related to changes in its isomerization, oxidation and racemization.


Subject(s)
Crystallins/chemistry , Crystallins/radiation effects , Molecular Chaperones/chemistry , Molecular Chaperones/radiation effects , Animals , Aspartic Acid/chemistry , Aspartic Acid/radiation effects , Cattle , Gamma Rays/adverse effects , In Vitro Techniques , Isomerism , Oxidation-Reduction , Photochemistry , Protein Structure, Secondary/radiation effects , Protein Structure, Tertiary/radiation effects , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Stereoisomerism
8.
Biophys J ; 76(3): 1706-19, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10049350

ABSTRACT

Computer-assisted motion analysis coupled to flash photolysis of caged chemoeffectors provides a means for time-resolved analysis of bacterial chemotaxis. Escherichia coli taxis toward the amino acid attractant L-aspartate is mediated by the Tar receptor. The physiology of this response, as well as Tar structure and biochemistry, has been studied extensively. The beta-2, 6-dinitrobenzyl ester of L-aspartic acid and the 1-(2-nitrophenyl)ethyl ether of 8-hydroxypyrene-1,3,6-tris-sulfonic acid were synthesized. These compounds liberated L-aspartate and the fluorophore 8-hydroxypyrene 1,3,6-tris-sulfonic acid (pyranine) upon irradiation with near-UV light. Photorelease of the fluorophore was used to define the amplitude and temporal stability of the aspartate jumps employed in chemotaxis experiments. The dependence of chemotactic adaptation times on aspartate concentration, determined in mixing experiments, was best fit by two Tar aspartate-binding sites. Signal processing (excitation) times, amplitudes, and adaptive recovery of responses elicited by aspartate jumps producing less than 20% change in receptor occupancy were characterized in photorelease assays. Aspartate concentration jumps in the nanomolar range elicited measurable responses. The response threshold and sensitivity of swimming bacteria matched those of bacteria tethered to glass by a single flagellum. Stimuli of similar magnitude, delivered either by rapid mixing or photorelease, evoked responses of similar strength, as assessed by recovery time measurements. These times remained proportional to change in receptor occupancy close to threshold, irrespective of prior occupancy. Motor excitation responses decayed exponentially with time. Rates of excitation responses near threshold ranged from 2 to 7 s-1. These values are consistent with control of excitation signaling by decay of phosphorylated pools of the response regulator protein, CheY. Excitation response rates increased slightly with stimulus size up to values limited by the instrumentation; the most rapid was measured to be 16 +/- 3 (SE) s-1. This increase may reflect simultaneous activation of CheY dephosphorylation, together with inhibition of its phosphorylation.


Subject(s)
Aspartic Acid/pharmacology , Chemotaxis/drug effects , Escherichia coli Proteins , Escherichia coli/drug effects , Escherichia coli/physiology , Receptors, Cell Surface , Adaptation, Physiological , Aspartic Acid/radiation effects , Bacterial Proteins/drug effects , Bacterial Proteins/physiology , Biophysical Phenomena , Biophysics , Chemoreceptor Cells , Chemotaxis/radiation effects , Escherichia coli/radiation effects , Fluorescent Dyes , Kinetics , Membrane Proteins/drug effects , Membrane Proteins/physiology , Photochemistry , Photolysis , Spectrometry, Fluorescence
9.
Biophys J ; 75(3): 1446-54, 1998 Sep.
Article in English | MEDLINE | ID: mdl-9726946

ABSTRACT

Structural intermediates occurring in the photocycle of wild-type bacteriorhodopsin are trapped by illuminating hydrated, glucose-embedded purple membrane at 170 K, 220 K, 230 K, and 240 K. We characterize light-induced changes in protein conformation by electron diffraction difference Fourier maps, and relate these to previous work on photocycle intermediates by infrared (FTIR) spectroscopy. Samples illuminated at 170 K are confirmed by FTIR spectroscopy to be in the L state; a difference Fourier projection map shows no structural change within the 0.35-nm resolution limit of our data. Difference maps obtained with samples illuminated at 220 K, 230 K, and 240 K, respectively, reveal a progressively larger structural response in helix F when the protein is still in the M state, as judged by the FTIR spectra. Consistent with previous structural studies, an adjustment in the position or in the degree of ordering of helix G accompanies this motion. The model of the photocycle emerging from this and previous studies is that bacteriorhodopsin experiences minimal change in protein structure until a proton is transferred from the Schiff base to Asp85. The M intermediate then undergoes a conformational evolution that opens a hydrated "half-channel," allowing the subsequent reprotonation of the Schiff base by Asp96.


Subject(s)
Bacteriorhodopsins/chemistry , Bacteriorhodopsins/radiation effects , Aspartic Acid/chemistry , Aspartic Acid/radiation effects , Biophysical Phenomena , Biophysics , Halobacterium salinarum/chemistry , Halobacterium salinarum/radiation effects , Light , Photochemistry , Protein Conformation/radiation effects , Protein Structure, Secondary , Protons , Schiff Bases/chemistry , Schiff Bases/radiation effects , Spectroscopy, Fourier Transform Infrared
10.
Biophys J ; 75(3): 1455-65, 1998 Sep.
Article in English | MEDLINE | ID: mdl-9726947

ABSTRACT

In the recently proposed local-access model for proton transfers in the bacteriorhodopsin transport cycle (Brown et al. 1998. Biochemistry. 37:3982-3993), connection between the retinal Schiff base and Asp85 (in the extracellular direction) and Asp96 (in the cytoplasmic direction)is maintained as long as the retinal is in its photoisomerized state. The directionality of the proton translocation is determined by influences in the protein that make Asp85 a proton acceptor and, subsequently, Asp96 a proton donor. The idea of concurrent local access of the Schiff base in the two directions is now put to a test in the photocycle of the D115N/D96N mutant. The kinetics had suggested that there is a single sequence of intermediates, L<-->M1<-->M2<-->N, and the M2-->M1 reaction depends on whether a proton is released to the extracellular surface. This is now confirmed. We find that at pH 5, where proton release does not occur, but not at higher pH, the photostationary state created by illumination with yellow light contains not only the M1 and M2 states, but also the L and the N intermediates. Because the L and M1 states decay rapidly, they can be present only if they are in equilibrium with later intermediates of the photocycle. Perturbation of this mixture with a blue flash caused depletion of the M intermediate, followed by its partial recovery at the expense of the L state. The change in the amplitude of the C=O stretch band at 1759 cm-1 demonstrated protonation of Asp85 in this process. Thus, during the reequilibration the Schiff base lost its proton to Asp85. Because the N state, also present in the mixture, arises by protonation of the Schiff base from the cytoplasmic surface, these results fulfill the expectation that under the conditions tested the extracellular access of the Schiff base would not be lost at the time when there is access in the cytoplasmic direction. Instead, the connectivity of the Schiff base flickers rapidly (with the time constant of the M1<-->M2 equilibration) between the two directions during the entire L-to-N segment of the photocycle.


Subject(s)
Bacteriorhodopsins/chemistry , Bacteriorhodopsins/radiation effects , Aspartic Acid/chemistry , Aspartic Acid/radiation effects , Bacteriorhodopsins/genetics , Biophysical Phenomena , Biophysics , Halobacterium salinarum/chemistry , Halobacterium salinarum/genetics , Halobacterium salinarum/radiation effects , Hydrogen-Ion Concentration , Kinetics , Light , Models, Chemical , Mutagenesis, Site-Directed , Photochemistry , Protein Conformation/radiation effects , Protons , Retinaldehyde/chemistry , Retinaldehyde/radiation effects , Schiff Bases/chemistry , Schiff Bases/radiation effects , Spectroscopy, Fourier Transform Infrared
12.
Exp Eye Res ; 65(1): 99-104, 1997 Jul.
Article in English | MEDLINE | ID: mdl-9237870

ABSTRACT

We have reported that the aspartyl (Asp)-151 residue in alphaA-crystallin in human eye lens was inverted to the D-isomer and isomerized to beta-Asp residue with age. We report here that ultraviolet (UV)-B irradiation induces the racemization and isomerization of the Asp-151 residue of alphaA-crystallin from lenses of 6-week-old rats to form D-isomer and beta-Asp residue. Simultaneous racemization and isomerization of the specific Asp residue indicate that the reaction proceeds via formation of a succinimide intermediate. This modification was not observed in UV-A irradiated and normal lenses. UV-B irradiation induced the racemization of only the Asp-151 residue and did not affect the other Asp residues in alphaA-crystallin. On the other hand, the high molecular weight fraction of the lens protein increased upon UV-B irradiation. Modification of the Asp residue would affect the three-dimensional packing array of the lens protein.


Subject(s)
Aspartic Acid/radiation effects , Crystallins/radiation effects , Ultraviolet Rays , Amino Acid Sequence , Animals , Asparagine/chemistry , Asparagine/radiation effects , Aspartic Acid/chemistry , Crystallins/chemistry , Isomerism , Molecular Sequence Data , Rats , Rats, Wistar
13.
Int J Radiat Oncol Biol Phys ; 33(3): 719-24, 1995 Oct 15.
Article in English | MEDLINE | ID: mdl-7558964

ABSTRACT

PURPOSE: External radiation therapy for brain tumors exposes healthy areas of brain to considerable doses of radiation. This may cause cognitive and psychological impairment, which indicate neuronal dysfunction. 1H-magnetic resonance spectroscopy (MRS) was used to study brain metabolites in the adjacent regions 0.5-13 years after exposure to therapeutic irradiation. METHODS AND MATERIALS: Eight patients with irradiated brain tumors were examined by means of in vivo 1H-MRS using a point-resolved spectroscopy (PRESS) sequence with echo times of 60 or 270 ms. The metabolites were quantified by using brain water concentration as internal reference. The volume of interest (VOI) was positioned in irradiated brain areas excluding, however, scar and recurrent tumor. The respective radiation doses were measured based on radiation therapy plans, simulator films, and localization MR images. RESULTS: The concentration of the neuron-specific metabolite N-acetyl-L-aspartate (NAA) was 13.2 +/- 1.4 mmol/l in controls, whereas it was reduced in the brains of treated patients to 8.6 +/- 0.9 mmol/l (total radiation dose 59-62 Gy). Concentrations of creatine and choline-containing compounds were unchanged. The T2 of water was longer in irradiated than in unexposed brain areas. CONCLUSION: Therapeutic brain irradiation causes neuronal damage, which is reflected by reduction of N-acetyl-L-aspartate (NAA) concentrations. 1H-MRS could serve clinically as a means of evaluating adverse effects in the central nervous system, enabling intervention and rehabilitation.


Subject(s)
Aspartic Acid/analogs & derivatives , Brain Chemistry/radiation effects , Brain/radiation effects , Magnetic Resonance Spectroscopy , Adult , Aged , Aspartic Acid/analysis , Aspartic Acid/radiation effects , Brain Neoplasms/radiotherapy , Female , Humans , Magnetic Resonance Imaging , Male , Middle Aged , Radiotherapy Dosage
14.
Radiat Res ; 131(3): 266-71, 1992 Sep.
Article in English | MEDLINE | ID: mdl-1332107

ABSTRACT

Paramagnetic molecular centers produced by gamma irradiation at 77 K and at room temperature in the novel compound Al5(OH)15(Asp)3.3H2O were studied by ESR spectroscopy. The g value of 2.0034 and the lack of such lines in pure aluminum hydroxide suggested that all the paramagnetic centers observed are related to the aspartic acid molecule. However, none of the paramagnetic centers gave an ESR spectrum characteristic for gamma-irradiated pure aspartic acid powder. The influence of the oxygen on the formation of the paramagnetic centers was noticed. The extreme stability of the paramagnetic molecular centers formed in Al5(OH)15(Asp)3.3H2O suggests that aspartic acid complexed in aluminum hydroxide is a good trap for gamma-radiation energy.


Subject(s)
Aluminum Hydroxide/radiation effects , Aspartic Acid/radiation effects , Electron Spin Resonance Spectroscopy , Gamma Rays , Temperature
15.
Orig Life Evol Biosph ; 21(4): 219-23, 1991.
Article in English | MEDLINE | ID: mdl-1688138

ABSTRACT

Sunlight photolysis of the amino acids glutamate and aspartate were carried out on 0.1 M aqueous solutions at pH = 7.0. The non-volatile products were identified by GC-MS analysis of derived methyl esters. The major product from glutamic acid was succinic acid, and, analogously, aspartic acid photolyzed to malonic acid. The photochemical oxidative decarboxylation of glutamate parallels its metabolism in modern cells and may provide an evolutionary link between simple amino acids and reactions of the citric acid cycle.


Subject(s)
Aspartic Acid/radiation effects , Citric Acid Cycle , Glutamates/radiation effects , Sunlight , Gas Chromatography-Mass Spectrometry , Glutamic Acid , Hydrogen-Ion Concentration , Photolysis
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