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Small-angle neutron scattering reveals the assembly mode and oligomeric architecture of TET, a large, dodecameric aminopeptidase.

Appolaire A, Girard E, Colombo M, Durá MA, Moulin M, Härtlein M, Franzetti B, Gabel F - Acta Crystallogr. D Biol. Crystallogr. (2014)

Bottom Line: Furthermore, in the case of homo-oligomeric complexes, or complexes with very similar building blocks, the respective positions of subunits and their assembly pathways are difficult to determine using many structural biology techniques.Here, an elegant and powerful approach based on small-angle neutron scattering is applied, in combination with deuterium labelling and contrast variation, to elucidate the oligomeric organization of the quaternary structure and the assembly pathways of 468 kDa, hetero-oligomeric and symmetric Pyrococcus horikoshii TET2-TET3 aminopeptidase complexes.The results reveal that the topology of the PhTET2 and PhTET3 dimeric building blocks within the complexes is not casual but rather suggests that their quaternary arrangement optimizes the catalytic efficiency towards peptide substrates.

View Article: PubMed Central - HTML - PubMed

Affiliation: Université Grenoble Alpes, IBS, 38044 Grenoble, France.

ABSTRACT
The specific self-association of proteins into oligomeric complexes is a common phenomenon in biological systems to optimize and regulate their function. However, de novo structure determination of these important complexes is often very challenging for atomic-resolution techniques. Furthermore, in the case of homo-oligomeric complexes, or complexes with very similar building blocks, the respective positions of subunits and their assembly pathways are difficult to determine using many structural biology techniques. Here, an elegant and powerful approach based on small-angle neutron scattering is applied, in combination with deuterium labelling and contrast variation, to elucidate the oligomeric organization of the quaternary structure and the assembly pathways of 468 kDa, hetero-oligomeric and symmetric Pyrococcus horikoshii TET2-TET3 aminopeptidase complexes. The results reveal that the topology of the PhTET2 and PhTET3 dimeric building blocks within the complexes is not casual but rather suggests that their quaternary arrangement optimizes the catalytic efficiency towards peptide substrates. This approach bears important potential for the determination of quaternary structures and assembly pathways of large oligomeric and symmetric complexes in biological systems.

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SANS curves and p(r) functions of peak 1 and peak 2. (a) SANS curves at four different contrast conditions at 20°C. All data sets are drawn without applying scaling. The total protein concentrations of all samples were identical (4.5 mg ml−1). Guinier fits to the 0, 42 (49) and 100% D2O data are shown as an inset (the 70% D2O data were not fitted using the Guinier approach owing to their elevated noise level). (b) Pair-distance distribution functions p(r) of the SANS data in arbitrary units generated with GNOM (Svergun, 1992 ▶). The two 0% D2O data sets are very similar (cf. Supplementary Fig. S6). The 0, 42, 49 and 100% D2O data are normalized to the second peak and the 70% D2O data to the first peak (using a different scaling factor for clarity).
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fig4: SANS curves and p(r) functions of peak 1 and peak 2. (a) SANS curves at four different contrast conditions at 20°C. All data sets are drawn without applying scaling. The total protein concentrations of all samples were identical (4.5 mg ml−1). Guinier fits to the 0, 42 (49) and 100% D2O data are shown as an inset (the 70% D2O data were not fitted using the Guinier approach owing to their elevated noise level). (b) Pair-distance distribution functions p(r) of the SANS data in arbitrary units generated with GNOM (Svergun, 1992 ▶). The two 0% D2O data sets are very similar (cf. Supplementary Fig. S6). The 0, 42, 49 and 100% D2O data are normalized to the second peak and the 70% D2O data to the first peak (using a different scaling factor for clarity).

Mentions: The samples from both peaks yielded very specific and distinct SANS curves (Fig. 4 ▶a) and model-free parameters I(0) and Rg (Table 1 ▶) at the four contrast conditions. At 0% D2O both resemble the homododecameric reference curves (Supplementary Fig. S6) most since both dPhTET2 and hPhTET3 moieties have positive contrast with respect to the solvent (Supplementary Fig. S4). The 70% D2O data have the lowest relative intensities since the hydrogenated and deuterated building blocks are of opposite contrast and result in a decrease in the overall scattered intensities. At 42 (49) and 100% D2O the scattered signals are almost exclusively owing to the deuterated and hydrogenated building blocks of the particles, respectively. Since small but significant variations of the curves at 0, 42 (49), 70 and 100% D2O are observed for both peaks, it can be concluded that the geometric arrangement of dPhTET2 and hPhTET3 building blocks are different within the particles constituting peak 1 and peak 2.


Small-angle neutron scattering reveals the assembly mode and oligomeric architecture of TET, a large, dodecameric aminopeptidase.

Appolaire A, Girard E, Colombo M, Durá MA, Moulin M, Härtlein M, Franzetti B, Gabel F - Acta Crystallogr. D Biol. Crystallogr. (2014)

SANS curves and p(r) functions of peak 1 and peak 2. (a) SANS curves at four different contrast conditions at 20°C. All data sets are drawn without applying scaling. The total protein concentrations of all samples were identical (4.5 mg ml−1). Guinier fits to the 0, 42 (49) and 100% D2O data are shown as an inset (the 70% D2O data were not fitted using the Guinier approach owing to their elevated noise level). (b) Pair-distance distribution functions p(r) of the SANS data in arbitrary units generated with GNOM (Svergun, 1992 ▶). The two 0% D2O data sets are very similar (cf. Supplementary Fig. S6). The 0, 42, 49 and 100% D2O data are normalized to the second peak and the 70% D2O data to the first peak (using a different scaling factor for clarity).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4220976&req=5

fig4: SANS curves and p(r) functions of peak 1 and peak 2. (a) SANS curves at four different contrast conditions at 20°C. All data sets are drawn without applying scaling. The total protein concentrations of all samples were identical (4.5 mg ml−1). Guinier fits to the 0, 42 (49) and 100% D2O data are shown as an inset (the 70% D2O data were not fitted using the Guinier approach owing to their elevated noise level). (b) Pair-distance distribution functions p(r) of the SANS data in arbitrary units generated with GNOM (Svergun, 1992 ▶). The two 0% D2O data sets are very similar (cf. Supplementary Fig. S6). The 0, 42, 49 and 100% D2O data are normalized to the second peak and the 70% D2O data to the first peak (using a different scaling factor for clarity).
Mentions: The samples from both peaks yielded very specific and distinct SANS curves (Fig. 4 ▶a) and model-free parameters I(0) and Rg (Table 1 ▶) at the four contrast conditions. At 0% D2O both resemble the homododecameric reference curves (Supplementary Fig. S6) most since both dPhTET2 and hPhTET3 moieties have positive contrast with respect to the solvent (Supplementary Fig. S4). The 70% D2O data have the lowest relative intensities since the hydrogenated and deuterated building blocks are of opposite contrast and result in a decrease in the overall scattered intensities. At 42 (49) and 100% D2O the scattered signals are almost exclusively owing to the deuterated and hydrogenated building blocks of the particles, respectively. Since small but significant variations of the curves at 0, 42 (49), 70 and 100% D2O are observed for both peaks, it can be concluded that the geometric arrangement of dPhTET2 and hPhTET3 building blocks are different within the particles constituting peak 1 and peak 2.

Bottom Line: Furthermore, in the case of homo-oligomeric complexes, or complexes with very similar building blocks, the respective positions of subunits and their assembly pathways are difficult to determine using many structural biology techniques.Here, an elegant and powerful approach based on small-angle neutron scattering is applied, in combination with deuterium labelling and contrast variation, to elucidate the oligomeric organization of the quaternary structure and the assembly pathways of 468 kDa, hetero-oligomeric and symmetric Pyrococcus horikoshii TET2-TET3 aminopeptidase complexes.The results reveal that the topology of the PhTET2 and PhTET3 dimeric building blocks within the complexes is not casual but rather suggests that their quaternary arrangement optimizes the catalytic efficiency towards peptide substrates.

View Article: PubMed Central - HTML - PubMed

Affiliation: Université Grenoble Alpes, IBS, 38044 Grenoble, France.

ABSTRACT
The specific self-association of proteins into oligomeric complexes is a common phenomenon in biological systems to optimize and regulate their function. However, de novo structure determination of these important complexes is often very challenging for atomic-resolution techniques. Furthermore, in the case of homo-oligomeric complexes, or complexes with very similar building blocks, the respective positions of subunits and their assembly pathways are difficult to determine using many structural biology techniques. Here, an elegant and powerful approach based on small-angle neutron scattering is applied, in combination with deuterium labelling and contrast variation, to elucidate the oligomeric organization of the quaternary structure and the assembly pathways of 468 kDa, hetero-oligomeric and symmetric Pyrococcus horikoshii TET2-TET3 aminopeptidase complexes. The results reveal that the topology of the PhTET2 and PhTET3 dimeric building blocks within the complexes is not casual but rather suggests that their quaternary arrangement optimizes the catalytic efficiency towards peptide substrates. This approach bears important potential for the determination of quaternary structures and assembly pathways of large oligomeric and symmetric complexes in biological systems.

Show MeSH
Related in: MedlinePlus