Una aproximación a la conformación proteica a través de la fotomarcación de superficies acoplada a resonancia magnética nuclear
The solvent accessible surface area (SASA) in proteins is a geometric parameter of\ncentral importance in the thermodynamics of folding and interaction. The use of\ndiazirine (DZN) as a mimetic probe of the aqueous solvent allowed us to estimate the\nmagnitude and nature of SASA, by generating stabl...
Guardado en:
| Autor principal: | |
|---|---|
| Otros Autores: | |
| Formato: | Tesis doctoral acceptedVersion |
| Lenguaje: | Español |
| Publicado: |
Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica
2022
|
| Materias: | |
| Acceso en línea: | http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=posgraafa&cl=CL1&d=HWA_6733 https://repositoriouba.sisbi.uba.ar/gsdl/collect/posgraafa/index/assoc/HWA_6733.dir/6733.PDF |
| Aporte de: |
| Sumario: | The solvent accessible surface area (SASA) in proteins is a geometric parameter of\ncentral importance in the thermodynamics of folding and interaction. The use of\ndiazirine (DZN) as a mimetic probe of the aqueous solvent allowed us to estimate the\nmagnitude and nature of SASA, by generating stable methylated products. The novel\nuse of NMR as an analytical technique coupled with DZN labeling yields site-specific\ninformation without the need to fragment the sample, due to the possibility of\nunambiguously assigning and identifying the labeled sites. Using E. coli thioredoxin\n(EcTRX) as a model protein for its labeling with DZN, we observed that the dominant\nmodification phenomenon involves the sparse methylation of amino acid side chains,\na fact supported by the enrichment of the aliphatic region observed in the 1H NMR\nspectra. In the unfolded state (8M urea), EcTRX shows a generalized higher\nmethylation profile, consistent with the extensive solvent exposure of the polypeptide\nchain.\n1H, 15N HSQC spectra reveal the differential impact of the methylation reaction on\nbackbone amide bond environments. From the quantitative analysis of the variation\nin the intensity associated with each cross peak (N-H) of the labeled samples with\nrespect to the control sample, it was possible to obtain a direct measure of the\ndegree of modification of each amino acid. In parallel, the 1H, 13C HSQC spectra of\nEcTRX labeled with DZN show new spots caused by the appearance of methyl\ngroups exposed to water. The relative intensity of the cross peaks corresponding to\nCH? allowed to determine the degree of methylation at the level of individual amino\nacids. In addition, the analysis of the signals of CH?, CH? and CH? show details of\nthe methylation of the side chain, observing the following trend:\nCH?>CH?>CH?>CH?. Thus, a fully consistent pattern emerges from both the HN\nand HC regions.\nThe analysis of results obtained in the context of the three-dimensional structure of\nEcTRX made it possible to define that the amino acids that show to the greatest\nextent the impact of methylation are part of the ?2 helix and secondary structure\nmotifs spatially close to that helix: amino acids that are components of the ?2, ?4\nstrands and the loop connecting the ?5 strand with the ?4 helix. The significance of\nthis finding lies in the fact that, precisely, this area embodies a main triad of cavities\nin EcTRX, capable of lodging the DZN reagent (host-guest interaction). By their\nnature, these internal surface elements are preferential targets for the methylation\nreaction.\nAs an overall conclusion, we show here that methylene carbene modification\ncoupled to NMR detection offers a rich and useful new source of information\nfor the mapping of a folded polypeptide, the study of conformational changes,\nand the identification of protein interaction surfaces. |
|---|