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Structural determinants of substrate specificity and Cl-role in two invertebrate cotransporters.
No full textThe two highly homologous neutral amino acid transporters KAAT1 and CAATCH1, cloned from the midgut epithelium of the larva of the lepidoptera Manduca sexta have been classified as members of the Na+/Cldependent transporter family (SLC6A gene family) (9; 11). These two cotransporters represent an interesting tool for investigating structurefunction relationships concerning ion and substrate selectivity in the SLC6 transporter family. At variance with the strict Na+ dependence of known vertebrate transporters, KAAT1 and CAATCH1 are able to exploit in addition a K+ electrochemical gradient to power up the uptake of neutral amino acids from the intestinal lumen.
In fact, KAAT1 and CAATCH1 give rise to specific kinds of current depending on the transported amino acid, the cotransported ion, pH and the membrane voltage. Transportassociated currents with their own characteristics are induced by different organic substrates, which are notably distinct between the two proteins. Differences in amplitude, kinetics and voltagedependence of the transportassociated
currents have been observed, as well as different substrate selectivity patterns, measured by radioactive amino acid uptake assays (9, 4, 23 ) In this work the specificity of KAAT1 was compared to that of CAATCH1, especially with respect to leucine, which in physiological conditions is transported by KAAT1, but not by CAATCH1, where it acts as a blocker. In order to identify the determinants involved in these phenomena, we have mutated the single different residue between the two transporters, the serine 308, that corresponds to that S290 participating in the leucine binding site of LeuTAa, a bacterial member of the SLC6A family of ioncoupled cotransporters (19). This amino acid is conserved only in the members of the family able to transport leucine ( 3), and its substitution in KAAT1 with threonine, blocks the current induced by leucine in sodium, and causes a reduction of about 70% of the current in potassium. The behaviour of CAATCH1 is mimicked by the S308T mutant form of KAAT1, that indicates the participation of this residue in the leucine binding site. The reverse mutation T308S in CAATCH1 conferred to this transporter the ability to transport leucine in presence of K+. The substrate specificity of KAAT1 has been investigated using electrophysiological and radiotracer methods. Competition experiments between different substrates indicate that both transporters bind leucine more strongly than threonine and proline, the difference between KAAT1 and CAATCH1 residing in the incapacity of the latter to complete the transport cycle in presence of leucine. These results may be interpreted by a kinetic scheme in which in presence of Na+ the leucinebound state of the transporter is relatively stable, while in presence of K+ and at negative potentials the progression of the leucinebound form along the cycle is favoured. In this context serine 308 appears to be important in allowing the change to the inwardfacing conformation of the transporter, rather than in determining the specificity of leucine binding. Recent homology modeling work, based on the atomic structure of LeuTAa (19), has pinpointed the possible site of interaction of these proteins with chloride ions (25; 26 ). However, not all transporters in this family are Cldependent, and this difference appears to be due to the presence (or absence) of a critical negative charge in this region. This charge which might be provided either by the intrinsic glutamate side chain in the chlorideindependent transporters, or by a chloride ions in those transporters that have a nonconservative substitution in this position. The different chloride sensitivity observed in KAAT1 and CAATCH1, in spite of an identical sequence in the putative chloride binding site prompted us to re-examine this point. We have then performed electrophysiological experiments in which chloride was replaced either by iodide or by gluconate. Significant transportassociated current reductions were seen with gluconate replacement, while strong potentiation was induced by iodide. These observations indicate that for KAAT1 and CAATCH1 complete chloride dependency can be ruled out
Theoretical aspects of Quantum Accelerator Modes.
No full textThis thesis is devoted to the study of some theoretical aspects of the problem of Quantum Accelerator Modes (QAMs), which has received considerable interest in recent years both by experimentalists and theorists in the fields of Quantum Chaos, Quantum Optics and cold-ultracold atomic physics.
QAMs, recently observed in cold atom optics, are formed by exposing cold alkali atoms to periodic kicks in the direction of the gravitational field. The kicks are generated by a pulsed standing wave of light. A QAM is characterized by a momentum transfer, which increases linearly with the number of pulses, to a substantial fraction of the atoms. QAMs arise for values of the pulse period close to a integer multiple of half of a characteristic time TB (the Talbot time), typical of the kind of atoms used.
A detailed theoretical explanation of this quantum effect is presented. The system can be modelled by a variant of the well-known quantum Kicked Rotator, in which the effects of a static force, produced by the earth gravitational field, are taken into account. A pseudo-classical theory has been formulated, in which the role of Planck constant is played by the detuning of the kicking period from the resonant value: in the asymptotic limit for a vanishing detuning, QAMs are described by the stable periodic orbit of a “formally” classical map on the 2-torus. An analysis of parametric dependence of the map is performed perturbatively; the parameter regions where the motion is stable and periodic exhibit Arnol’d tongues-like structures. The ordering of Arnol’d tongues and hence the classification of experimentally observed accelerator modes is provided by a number-theoretic construction known as the Farey Tree.
Quantum decay of the modes from classical stable islands immersed in a chaotic sea is analysed and its relations to the famous Wannier-Stark problem are investigated. Theoretical estimates for the lifetimes of the metastable states as a function of the effective Planck constant are obtained.
QAM dynamics in a Bose-Einstein condensate is then considered. Modifications on transport phenomena, imposed by nonlinearities of Gross-Pitaevskii type, which describe the atomic interactions in a mean-field approach, are examined by computer-assisted analysis.
Finally, the problem of QAMs in the vicinity of a generic quantum resonance, namely for kicking period sufficiently close to any rational multiple of the Talbot time, is studied.
The theoretical framework is nontrivially generalized in terms of spinor dynamics: an ansatz of the Born-Oppenheimer type allows to decouple the dynamics of spinor and orbital degrees of freedom and a description of the orbital motion by means of “formally” classical equations is achieved. New rich families of experimental observable QAMs are predicted
Mappe della memoria: identita’ e rappresentazioni autobiografiche nella Germania unificata.
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Sintesi di sistemi eterociclici azotati mediante processi intramolecolari catalizzati da palladio.
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Design, synthesis and structural evaluation of peptidomimetics with a defined secondary structure.
No full textThe efficient synthesis of a g-butyrolactone based d-amino acid was realised starting from furan methyl ester.
Its enantioselecive cyclopropanation by copper triflate and a chiral bis-oxazoline led to the formation of an enantiopure cyclopropanated furan product which could be subjected to ozonolysis affording a cyclopropane aldehyde having three chiral centers. A Sakurai allylation was performed efficiently in high d.r. The lactonisation of the allylated cyclopropane alcohol could be realised with barium hydroxide. This reaction could be scaled up using triethylamine in the same conditions. The reductive amination of the resulting
lactonaldehyde could be realised in good yields in mild conditions. The Boc protection of the secondary amine and the PMB cleavage were performed affording the lactone with a protected primary amine. The
allylic double bond could be oxidised by ruthenium chloride and sodium periodate affording the d-amino acid 199 in good yields.
Modification of the lactone by a-substitution was investigated to obtain a better NMR dispersion. The a-methylation with LDA and methyl iodide led to poor diastereoselectivities (d.r. 3/1). The a-fluorination was not diastereoselective as well (d.r. 2/1) and yields were poor. The a-dimethylation of the lactone could be
performed in good yields and the resulting d-amino acid showed an excellent NMR dispersion for further foldamers studies.
The liquid-phase synthesis of the desired tetramer of dimethylated d-amino acid could not be performed, even by using efficient coupling reagents. The synthesis of mixed a-d-peptides with the non-methylated building block was realised. First, an alternated a-d-pentapeptide containing L-Phe and the lactone building block was efficiently synthesised and its secondary structure analysed by various techniques. Despite
overlapping of some protons in NMR spectroscopy, some NOE contacts could be observed and allowed computational studies using NOE constraints. The calculations predicted an extended loop conformation of
the pentapeptide and other analyses such as IR in solution or CD spectroscopy are in favour of intramolecular hydrogen bonding. CD spectra refences missing for that type of alternated peptide, no comparison could be done with similar peptides. The secondary structure could not be determined with
certainty and attemps to crystallise the peptide in a stable form failed.
Secondly, a heptapeptide containing only one d-amino acid was synthesised in order to know whether this building block could induce hairpin secondary structures. IR in solution revealed the presence of
intramolecular hydrogen bonding and CD spectroscopy showed interesting features suggesting the presence of a stable secondary structure although its CD spectrum looked a little disorganised. Its 2D-NMR analysis revealed interstrand contacts and showed that this heptapeptide was forming, among other things, a 13-membered hydrogen-bonded ring. As a consequence, we came to the conclusion that it did not adopt a
hairpin conformation but a disorganised helicoidal structure.
For the second part of this project, the ability of cis-DKP in b-bend ribbon structure was investigated. The trimer of DKP could be synthesised and fully characterised and revealed a partial b-bend ribbon conformation. The tetramer, on the other hand, could not be fully characterised by 2D-NMR, but its CD spectrum showed the presence of a stable secondary structure and comparison with other b-bend ribbon CD
spectra in the literature showed a similar behaviour in terms of shapes and intensities. The CD data collected, in addition to proton-deuterium exchange analysis are in favour of a stable b-bend ribbon secondary structure.
A short cyclic peptide based on a trans-DKP scaffold was successfully synthesised and fully characterised by 2D NMR spectroscopy. The presence of a g-turn in the peptide was detected and confirmed preliminary computational studies realised on a similar peptide. It is very probable that a pentapeptide containing a RGD sequence and the trans-DKP scaffold would as well induce a g-turn, whose presence is primordial for its activity against integrins avb3.
Finally, two potential tripeptidic organocatalysts were synthesised, each of them containing a cis-DKP and being linked by an amide or an ester bond to a proline. Their investigation in organocatalysed aldol reaction and Michael addition is now in progress
