Margarita Morillo

Poly(α-alkyl γ-glutamate)s of microbial origin: I. Ester derivatization of poly(γ-glutamic acid) and thermal degradation

A series of poly(α-alkyl γ-glutamate)s (PAAG-n with n=1–10) were prepared from poly(γ-glutamic acid) (PGGA) of microbial origin by a two-steps synthesis procedure. The poly(α-ethyl γ-glutamate) was firstly obtained by esterification of PGGA and then transesterified with the selected alkanol to obtain the corresponding PAAG-n. The modification reactions were found to occur without alteration of the original enantiomeric composition but with a significant reduction in molecular weight. The thermal degradation of PAAG-n was examined by thermogravimetric analysis combined with IR and NMR spectroscopy. At low temperatures (<300°C) decomposition was found to take place by depolymerization with releasing of the corresponding alkyl pyroglutamate. Decarboxylation of PAAG-n to an unsaturated polyamide seemed to be the main process occurring at high temperatures (400–450°C).

Ultrasonic degradation of polyaspartates and polyglutamates

In both cases molecular weights of severalof unraveling the mechanism responsible for chain hundreds of thousands daltons or even above the mil-breaking, the value of the method for reducing the mo- lion are typically obtained. Such high molecular sizeslecular weight of polymers without increasing poli- represent a serious inconvenience when these polymersdispersity is of prime practical importance. are handled for processing. In such a case moderateThis work is concerned with the ultrasonic degrada- molecular weights samples are highly desired. Becausetion of a certain group of polyamides that have as dis- amide bonds in the main chain are more reluctant tohydrolysis than ester side groups, cleavage of thesetinctive features to be stereoregularly substituted atpolyamides is unfeasible bychemical means. Ultrasonicthe backbone carbon adjacent to the NH and to containirradiation appears therefore to be an attractivea high density of amide groups in the main chain. Themethod to reduce the molecular weight of these polyam-selected polymers are: (a) the polypeptide poly(g-ben-ides without altering their chemical constitution.zyl-a-

Synthesis and structure of random and block copoly(β,l-aspartate)s containing short and long alkyl side chains

Copoly(α-alkyl-β,l-aspartate)s containing n-octadecyl and n-butyl side groups at different ratios were prepared by anionic ring-opening polymerization of the corresponding optically pure (S)-4-alkoxycarbonyl-2-azetidinones. Random copolymers were obtained by polymerization of comonomer mixtures. Diblock copolymers were achieved by sequential copolymerization using the living poly(α-n-octadecyl-β,l-aspartate) block to initiate the polymerization of (s)-4-n-butoxycarbonyl-2-azetidinone as a second block. Composition and sequence distribution were characterized by NMR with the help of a model copoly(β,l-aspartate) made of α-n-dodecyl and α-benzyl β,l-aspartate units. All the copolymers were found to adopt the layered structure made of 13/4 helices described previously for comb-like poly(α-n-alkyl-β,l-aspartate) homopolymers. Copolymers containing at least 50% of n-octadecyl side groups have these groups crystallized in an interlayer microphase and aligned normal to the main helical chain. Melting of the paraffinic crystallites happened within the 40–60°C temperature range with formation of a liquid-crystal phase in which side chains are molten but retain the alignment of the low temperature phase. Different from what is known to happen to poly(α-n-octadecyl-β,l-aspartate), no indications on the occurrence of a second structural transition were observed at higher temperatures.

Structural coupling of 11-cis-7-methyl-retinal and amino acids at the ligand binding pocket of rhodopsin.

It was previously shown that opsin can be regenerated with the newly synthesized 11‐cis‐7‐methyl‐retinal forming an artificial visual pigment. We now extend this study to include mutants at positions close to the retinal to further dissect the interactions of native and artificial chromophores with opsin. Several mutants at M207, W265 and Y268 have been obtained and regenerated with 11‐cis‐retinal and the 7‐methyl analog. M207 is the site of the point mutation M207R associated with the retinal degenerative disease retinitis pigmentosa. All the studied mutants regenerated with 11‐cis‐retinal except for M207C which proved to be completely misfolded. The naturally occurring M207R mutant formed a pigment with an unprotonated Schiff base linkage, altered photobleaching and low MetarhodopsinII stability. Mutants regenerated with the 7‐methyl analog showed altered photobleaching reflecting a structural perturbation in the vicinity of M207. The newly obtained mutants at M207 also showed reduced levels of transducin activation with M207R showing essentially no transducin activation. Our results highlight the tight coupling of the vicinity of C7 of retinal and M207 and support the involvement of this amino acid residue in the conformational changes associated with rhodopsin photoactivation.

Mercury-induced dark-state instability and photobleaching alterations of the visual G-protein coupled receptor rhodopsin

Mercuric compounds were previously shown to affect the visual phototransduction cascade, and this could result in vision impairment. We have analyzed the effect of mercuric chloride on the structure and stability of the dim light vision photoreceptor rhodopsin. For this purpose, we have used both native rhodopsin immunopurified from bovine retinas and a recombinant mutant rhodopsin carrying several Cys to Ser substitutions in order to investigate the potential binding site of mercury on the receptor. Our results show that mercuric chloride dramatically reduces the stability of dark-state rhodopsin and alters the molecular features of the photoactived conformation obtained upon illumination by eliciting the formation of an altered photointermediate. The thermal bleaching kinetics of native and mutant rhodopsin is markedly accelerated by mercury in a concentration-dependent manner, and its chromophore regeneration ability is severely reduced without significantly affecting its G-protein activation capacity. Furthermore, fluorescence spectroscopic measurements on the retinal release process, ensuing illumination, suggest that mercury impairs complete retinal release from the receptor binding pocket. Our results provide further support for the capacity of mercury as a hazardous metal ion with reported deleterious effect on vision and provide a molecular explanation for such an effect at the rhodopsin photoreceptor level. We suggest that mercury could alter vision by acting in a specific manner on the molecular components of the retinoid cycle, particularly by modifying the ability of the visual photoreceptor protein rhodopsin to be regenerated and to be normally photoactivated by light.

A methyl group at C7 of 11-cis-retinal allows chromophore formation but affects rhodopsin activation

The newly synthesized 11-cis-7-methylretinal can form an artificial visual pigment with kinetic and spectroscopic properties similar to the native pigment in the dark-state. However, its photobleaching behavior is altered, showing a Meta I-like photoproduct. This behavior reflects a steric constraint imposed by the 7-methyl group that affects the conformational change in the binding pocket as a result of retinal photoisomerization. Transducin activation is reduced, when compared to the native pigment with 11-cis-retinal. Molecular dynamics simulations suggest coupling of the C7 methyl group and the beta-ionone ring with Met207 in transmembrane helix 5 in agreement with recent experimental results.

Human Blue Cone Opsin Regeneration Involves Secondary Retinal Binding with Analog Specificity

Human color vision is mediated by the red, green, and blue cone visual pigments. Cone opsins are G-protein-coupled receptors consisting of an opsin apoprotein covalently linked to the 11-cis-retinal chromophore. All visual pigments share a common evolutionary origin, and red and green cone opsins exhibit a higher homology, whereas blue cone opsin shows more resemblance to the dim light receptor rhodopsin. Here we show that chromophore regeneration in photoactivated blue cone opsin exhibits intermediate transient conformations and a secondary retinoid binding event with slower binding kinetics. We also detected a fine-tuning of the conformational change in the photoactivated blue cone opsin binding site that alters the retinal isomer binding specificity. Furthermore, the molecular models of active and inactive blue cone opsins show specific molecular interactions in the retinal binding site that are not present in other opsins. These findings highlight the differential conformational versatility of human cone opsin pigments in the chromophore regeneration process, particularly compared to rhodopsin, and point to relevant functional, unexpected roles other than spectral tuning for the cone visual pigments.

Synthesis of Poly(α‐alkyl β,L‐aspartate)s by Transesterification

A new procedure for the preparation of poly(α-alkyl β,L-aspartate)s based on the transesterification of poly(α-benzyl β,L-aspartate) with alcohols in the presence of titanium tetrabutoxide is described. The reaction proceeded to almost total conversion without substantial racemization or imidation. Thermal properties of the resulting polymers were comparable to those of their homologues obtained by anionic ring-opening polymerization of β-lactams and their thermal stability is even higher.