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, un système permettant d'accéder à des informations sur le(s) état(s) intermédiaire(s) de dépliement de la protéine substrat GFPssrA. La stratégie envisagée était de bloquer le processus de dépliement par PAN en greffant un module streptavidine à l'extrémité N-terminale ou C-terminale de la GFPssrA, La première partie du chapitre décrit les expériences biochimiques effectuées afin de caractériser l'activité du protéasome PAN-20S avec les constructions GFPssrA_Nter-streptavidine et GFPssrA_Cter-streptavidine dans les conditions d'études nécessaires aux mesures TR-SANS. Les mesures TR-SANS réalisées sur ces mêmes systèmes sont décrites dans la seconde partie de ce chapitre
, Contexte de l'étude 1.1. Stratégie employée dans la littérature
, L'hypothèse de cette étude était que si le processus de dépliement d'un substrat était une conséquence de son transfert au sein de PAN, alors la fixation d'une molécule volumineuse, l'avidine, à la GFPssrA devrait bloquer son transfert et empêcher son dépliement. Pour tester cette hypothèse, les auteurs ont utilisé une stratégie consistant à lier une molécule de biotine, Les travaux présentés dans ce chapitre sont basés sur un article publié en 2001 par Navon & Goldberg 317
,
, Une molécule d'avidine peut fixer quatre molécules de biotine, et donc quatre GFPssrA. B & C. Représentation artistique Tous les échantillons souhaités ont néanmoins pu être mesurés au cours de ces deux sessions et les résultats sont présentés dans la partie suivante, Figure, vol.106
, Des mesures TR-SANS ont été réalisées sur la d-GFPssrA_Nter-biotine isolée (figure 116.A), en présence de h-PAN (figure 116.C) et en présence de h-PAN
, Tous les échantillons ont été préparés avec une concentration initiale de 100 mM ATP & 200 mM MgCl2. Les réactions ont été activées lors de leur disposition dans le porte-échantillon à 55°C et suivies pendant 30 minutes avec un point de mesure SANS toutes les minutes et un point de mesure de la fluorescence
, et de la fluorescence à 509 nm (vert) au cours du temps. Dans le tampon à 42% D2O, les signaux provenant de la biotine, streptavidine monovalente, protéine PAN et particule 20S hydrogénées sont masqués. Le R g et I(0) correspondent alors exclusivement au signal de la GFPssrA deutérée et à ses formes dérivées. Les informations à déduire des valeurs R g et de I(0) sont de la même nature que celles décrites dans la partie 2, La figure 116 représente, pour chacun des échantillons, l'évolution de l'intensité à angle nul I(0) (noir), du rayon de giration R g (bleu)
,
, On sait que la réaction de dépliement et de dégradation a lieu lors des premières minutes de réaction. De plus, les valeurs initiales du I(0) dans les échantillons de d-GFPssrA_Nter-biotine isolée et en présence de h-PAN (voir tableau 22) indiquent qu'il y a eu une perte importante de la valeur du I(0) au cours des trois premières minutes de réaction, qui a ensuite continué de diminuer progressivement jusqu'à une perte d'environ 75%. La fluorescence diminue quant à elle d'environ 77%. La d-GFPssrA est, Cependant, il est important de noter que les données des trois premières minutes de réaction n'ont pas pu être enregistrées
Tout comme la réaction contrôle avec la d-GFPssrA_Nter-biotine, la valeur de I(0) diminue au cours du temps, avec une perte totale de d'environ 55% (voir tableau 22). La fluorescence diminue en parallèle d'environ 79%. On peut donc en conclure qu'environ 69% des d-GFPssrA dénaturées par h-PAN, vol.116 ,
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