SANS simulation of aggregated protein in aqueous solution

Sugiyama, Masaaki; Hamada, Kensaku; Kato, Koichi; Kurimoto, Eiji; Okamoto, Kenta; Morimoto, Yukio; Ikeda, Susumu; Naito, Sachio; Furusaka, Michihiko; Itoh, Keiji; Mori, Kazuhiro; Fukunaga, Toshiharu

doi:10.1016/j.nima.2008.11.121

Cited by 9 publications

(17 citation statements)

References 10 publications

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“…Among human proteasome α subunits, α7 exhibits a unique feature of in vitro self-assembly into a homo-tetradecamer with a double ring structure 9 10 . This raises the question whether the α7 homo-tetradecamer is an off-pathway, dead-end product during proteasome formation.…”

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confidence: 99%

Disassembly of the self-assembled, double-ring structure of proteasome α7 homo-tetradecamer by α6

Ishii

Noda

Yagi

et al. 2015

Sci Rep

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The 20S core particle of the eukaryotic proteasome is composed of two α- and two β-rings, each of which is a hetero-heptamer composed of seven homologous but distinct subunits. Although formation of the eukaryotic proteasome is a highly ordered process assisted by assembly chaperones, α7, an α-ring component, has the unique property of self-assembling into a homo-tetradecamer. We used biophysical methods to characterize the oligomeric states of this proteasome subunit and its interaction with α6, which makes direct contacts with α7 in the proteasome α-ring. We determined a crystal structure of the α7 tetradecamer, which has a double-ring structure. Sedimentation velocity analytical ultracentrifugation and mass spectrometric analysis under non-denaturing conditions revealed that α7 exclusively exists as homo-tetradecamer in solution and that its double-ring structure is disassembled upon the addition of α6, resulting in a 1:7 hetero-octameric α6–α7 complex. Our findings suggest that proteasome formation involves the disassembly of non-native oligomers, which are assembly intermediates.

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confidence: 99%

Disassembly of the self-assembled, double-ring structure of proteasome α7 homo-tetradecamer by α6

Ishii

Noda

Yagi

et al. 2015

Sci Rep

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“…In conclusion, the study demonstrated that only one subunit is exchangeable among the seven a7 subunits constituting one heptameric ring. This means that the a7 heptameric ring is probably not sevenfold symmetric, although the previous structural studies could not predict such an asymmetric property because of low spatial resolution (22,23). The kinetic asymmetry of subunit exchange of the a7 tetradecamer not only provides important clues to the underlying mechanisms of proteasome subunit assembly but also offers general insights into the formation and dynamics of quaternary structures of homooligomeric proteins.…”

Section: Kinetics Of Subunit Exchangementioning

confidence: 96%

“…2 A), confirming that 1), the h-a7 and the d-a7 tetradecamers have the same absolute value of scattering contrast in 81% D 2 O solution and 2), they are FIGURE 1 Three-dimensional (3D) structural model and neutron scattering length densities of the tetradecamer of proteasomal a7 subunit computed from the 3D structural model. (A) Top and side views of the double-ring structure of the a7 tetradecamer derived from the SANS data in conjunction with the crystal structure of the 20S core particle (19,23). Each a7 heptameric ring with sevenfold symmetry is drawn in green or cyan.…”

Section: Detection Of Subunit Exchangementioning

confidence: 99%

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Kinetic Asymmetry of Subunit Exchange of Homooligomeric Protein as Revealed by Deuteration-Assisted Small-Angle Neutron Scattering

Sugiyama

Kurimoto

Yagi

et al. 2011

Biophysical Journal

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We developed a novel, to our knowledge, technique for real-time monitoring of subunit exchange in homooligomeric proteins, using deuteration-assisted small-angle neutron scattering (SANS), and applied it to the tetradecamer of the proteasome α7 subunit. Isotopically normal and deuterated tetradecamers exhibited identical SANS profiles in 81% D(2)O solution. After mixing these solutions, the isotope sensitive SANS intensity in the low-q region gradually decreased, indicating subunit exchange, whereas the small-angle x-ray scattering profile remained unchanged confirming the structural integrity of the tetradecamer particles during the exchange. Kinetic analysis of zero-angle scattering intensity indicated that 1), only two of the 14 subunits were exchanged in each tetradecamer and 2), the exchange process involves at least two steps. This study underscores the usefulness of deuteration-assisted SANS, which can provide quantitative information not only on the molecular sizes and shapes of homooligomeric proteins, but also on their kinetic properties.

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“…Unique self-assembling properties of eukaryotic proteasome subunits Experiments using negative-stain EM and small-angle neutron scattering (SANS) have shown that Trypanosoma brucei α5 and human proteasome α7 subunits have unique properties of self-assembling into four and two stacked heptameric rings, respectively (Gerards et al 1997;Sugiyama et al 2009Sugiyama et al , 2011Yao et al 1999). Our recent X-ray crystallographic data revealed that the α7 subunits make a double-ring tetradecameric structure in which the single-ring architecture of α7 is similar to those of the archaeal homoheptameric and eukaryotic heteroheptameric α rings (Fig.…”

Section: Eukaryotic Proteasome Assemblymentioning

confidence: 99%

Structural insights on the dynamics of proteasome formation

Kato

Satoh

2017

Biophys Rev

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Molecular organization in biological systems comprises elaborately programmed processes involving metastable complex formation of biomolecules. This is exemplified by the formation of the proteasome, which is one of the largest and most complicated biological supramolecular complexes. This biomolecular machinery comprises approximately 70 subunits, including structurally homologous, but functionally distinct, ones, thereby exerting versatile proteolytic functions. In eukaryotes, proteasome formation is non-autonomous and is assisted by assembly chaperones, which transiently associate with assembly intermediates, operating as molecular matchmakers and checkpoints for the correct assembly of proteasome subunits. Accumulated data also suggest that eukaryotic proteasome formation involves scrap-and-build mechanisms. However, unlike the eukaryotic proteasome subunits, the archaeal subunits show little structural divergence and spontaneously assemble into functional machinery. Nevertheless, the archaeal genomes encode homologs of eukaryotic proteasome assembly chaperones. Recent structural and functional studies of these proteins have advanced our understanding of the evolution of molecular mechanisms involved in proteasome biogenesis. This knowledge, in turn, provides a guiding principle in designing molecular machineries using protein engineering approaches and de novo synthesis of artificial molecular systems.

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SANS simulation of aggregated protein in aqueous solution

Cited by 9 publications

References 10 publications

Disassembly of the self-assembled, double-ring structure of proteasome α7 homo-tetradecamer by α6

Disassembly of the self-assembled, double-ring structure of proteasome α7 homo-tetradecamer by α6

Kinetic Asymmetry of Subunit Exchange of Homooligomeric Protein as Revealed by Deuteration-Assisted Small-Angle Neutron Scattering

Structural insights on the dynamics of proteasome formation

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