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Functional and structural analysis o...

Chaudhry, Charu.

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Functional and structural analysis of the ATP-driven GroEL-GroES reaction: Role of the gamma-phosphate of ATP as a crucial trigger of the work of protein folding.

紀錄類型:	書目-電子資源 : Monograph/item
正題名/作者:	Functional and structural analysis of the ATP-driven GroEL-GroES reaction: Role of the gamma-phosphate of ATP as a crucial trigger of the work of protein folding./
作者:	Chaudhry, Charu.
面頁冊數:	186 p.
附註:	Source: Dissertation Abstracts International, Volume: 64-10, Section: B, page: 4766.
Contained By:	Dissertation Abstracts International64-10B.
標題:	Biology, Molecular. -
電子資源:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3109380
ISBN:	0496569223

Functional and structural analysis of the ATP-driven GroEL-GroES reaction: Role of the gamma-phosphate of ATP as a crucial trigger of the work of protein folding.
Chaudhry, Charu.

Functional and structural analysis of the ATP-driven GroEL-GroES reaction: Role of the gamma-phosphate of ATP as a crucial trigger of the work of protein folding. - 186 p.

Source: Dissertation Abstracts International, Volume: 64-10, Section: B, page: 4766.

Thesis (Ph.D.)--Yale University, 2003.

The work of cellular protein folding is carried out by the chaperonin GroEL, an ATP-driven double toroid molecular machine. Productive folding by GroEL is triggered by the binding of 7 ATP molecules along with the cochaperonin GroES to the same ring as the non-native polypeptide, ejecting polypeptide into an encapsulated hydrophilic (cis) chamber. The specific contribution of the gamma-phosphate to the activation process was established by using an ADP adduct containing aluminum fluoride (ADP · AlF 3), an analog of ATP. This analog, when added with GroES to a GroEL-polypeptide complex, triggers productive cis folding. Remarkably, the productivity of the nucleotide-metallofluoride complex could be duplicated by adding aluminum fluoride (AlF3) alone to an already-formed, folding-inactive GroEL-GroES-ADP-polypeptide complex. Thus, underscoring the remarkable plasticity of the cis ADP chamber, the chaperonin complex could undergo the transition from a folding-inactive to active state without GroES release, by introducing the gamma-phosphate of ATP in an independent step. This allowed an estimation of the energetic contribution of the gamma-phosphate to productive folding, amounting to &sim;46 kcal/mole rings, determined from aluminum fluoride ligand binding measurements. Surprisingly, a crystallographic study of GroEL-GroES-ADP · AlF3 at 2.8 A revealed that aluminum fluoride binding, while producing seven new interactions locally at the gamma-phosphate site, did not appear to be associated with a major structural change in the asymmetric GroEL-GroES complex. This was validated by independent structure determinations from a 30 A cryo-EM reconstruction and a 7.5 A sparsely contacted crystal lattice. Aluminum fluoride binding was, however, associated with release of polypeptide into the central cavity, detected by fluorescence anisotropy, and a strong stabilization of the GroEL-GroES complex, features observed previously when GroEL-GroES complexes were formed with ATP. These data in total show that binding of the gamma-phosphate moiety of ATP is the crucial trigger of the work carried out and lead to a model where the sizable free energy of gamma-phosphate binding ejects polypeptide from the cavity wall, and results in the assumption of a very stable state of the GroEL-GroES complex observed crystallographically, that supports ongoing folding in the central cavity.

ISBN: 0496569223Subjects--Topical Terms:

1017719
Biology, Molecular.

Functional and structural analysis of the ATP-driven GroEL-GroES reaction: Role of the gamma-phosphate of ATP as a crucial trigger of the work of protein folding.
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500 $a Source: Dissertation Abstracts International, Volume: 64-10, Section: B, page: 4766.
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520 $a The work of cellular protein folding is carried out by the chaperonin GroEL, an ATP-driven double toroid molecular machine. Productive folding by GroEL is triggered by the binding of 7 ATP molecules along with the cochaperonin GroES to the same ring as the non-native polypeptide, ejecting polypeptide into an encapsulated hydrophilic (cis) chamber. The specific contribution of the gamma-phosphate to the activation process was established by using an ADP adduct containing aluminum fluoride (ADP · AlF 3), an analog of ATP. This analog, when added with GroES to a GroEL-polypeptide complex, triggers productive cis folding. Remarkably, the productivity of the nucleotide-metallofluoride complex could be duplicated by adding aluminum fluoride (AlF3) alone to an already-formed, folding-inactive GroEL-GroES-ADP-polypeptide complex. Thus, underscoring the remarkable plasticity of the cis ADP chamber, the chaperonin complex could undergo the transition from a folding-inactive to active state without GroES release, by introducing the gamma-phosphate of ATP in an independent step. This allowed an estimation of the energetic contribution of the gamma-phosphate to productive folding, amounting to &sim;46 kcal/mole rings, determined from aluminum fluoride ligand binding measurements. Surprisingly, a crystallographic study of GroEL-GroES-ADP · AlF3 at 2.8 A revealed that aluminum fluoride binding, while producing seven new interactions locally at the gamma-phosphate site, did not appear to be associated with a major structural change in the asymmetric GroEL-GroES complex. This was validated by independent structure determinations from a 30 A cryo-EM reconstruction and a 7.5 A sparsely contacted crystal lattice. Aluminum fluoride binding was, however, associated with release of polypeptide into the central cavity, detected by fluorescence anisotropy, and a strong stabilization of the GroEL-GroES complex, features observed previously when GroEL-GroES complexes were formed with ATP. These data in total show that binding of the gamma-phosphate moiety of ATP is the crucial trigger of the work carried out and lead to a model where the sizable free energy of gamma-phosphate binding ejects polypeptide from the cavity wall, and results in the assumption of a very stable state of the GroEL-GroES complex observed crystallographically, that supports ongoing folding in the central cavity.
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