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Folding and assembly of large macromolecular complexes monitored by hydrogen-deuterium exchange and mass spectrometry.

Suchanova B, Tuma R - Microb. Cell Fact. (2008)

Bottom Line: HDX-MS became a valuable tool to follow protein folding, assembly and aggregation.The methodology has a wide range of applications in biotechnology ranging from quality control for over-expressed proteins and their complexes to screening of potential ligands and inhibitors.This review provides an introduction to protein folding and assembly followed by the principles of HDX and MS detection, and concludes with selected examples of applications that might be of interest to the biotechnology community.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Biotechnology, University of Helsinki, Finland. r.tuma@leeds.ac.uk.

ABSTRACT
Recent advances in protein mass spectrometry (MS) have enabled determinations of hydrogen deuterium exchange (HDX) in large macromolecular complexes. HDX-MS became a valuable tool to follow protein folding, assembly and aggregation. The methodology has a wide range of applications in biotechnology ranging from quality control for over-expressed proteins and their complexes to screening of potential ligands and inhibitors. This review provides an introduction to protein folding and assembly followed by the principles of HDX and MS detection, and concludes with selected examples of applications that might be of interest to the biotechnology community.

No MeSH data available.


Mapping subunit interfaces and dynamics in large viral assembly- P4 hexamer within ϕ12 procapsid [94]. (A) Schematics of the viral procapsid (PC) and packaging of ssRNA precursors by the P4 hexamers associated with the capsid vertices (left). The right panel illustrates the problem of determining the orientation of the hexamer with respect to the procapsid. (B) Bar representation of the isotopic envelopes during progressive deuteration, free hexamer in red and PC-bound in blue. (C) The HDX kinetics derived from centroid positions of the isotopic envelopes in panel B (and from additional data not shown). (D) Mapping of average exchange rates onto P4 structure in surface color representation (color scale on the bottom right). (E) Fitting of the P4 hexamer into the EM-derived electron density using the correct orientation from HDX experiment (adopted from [113]). (F) Average exchange rates mapped onto the ribbon diagram of P4 subunit for free (left) and PC-bound (middle) hexamer together with the scaled crystallographic temperature factors (B-factors, right panel). Color scale as in panel D. (G) Schematics of ssRNA loading into the hexameric packaging motor via ring opening mechanism. Top: A specific viral RNA structure (packaging signal) is recognized by the major capsid protein P1 which brings the polynucleotide strand to the vicinity of P4. Middle: P4 ring opens and lets the RNA slip in between subunit interfaces into the central channel. Bottom: The ring topologically encloses the bound RNA and translocates RNA in 5' to 3' direction into the capsid at the expense of ATP hydrolysis (packaging).
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Figure 7: Mapping subunit interfaces and dynamics in large viral assembly- P4 hexamer within ϕ12 procapsid [94]. (A) Schematics of the viral procapsid (PC) and packaging of ssRNA precursors by the P4 hexamers associated with the capsid vertices (left). The right panel illustrates the problem of determining the orientation of the hexamer with respect to the procapsid. (B) Bar representation of the isotopic envelopes during progressive deuteration, free hexamer in red and PC-bound in blue. (C) The HDX kinetics derived from centroid positions of the isotopic envelopes in panel B (and from additional data not shown). (D) Mapping of average exchange rates onto P4 structure in surface color representation (color scale on the bottom right). (E) Fitting of the P4 hexamer into the EM-derived electron density using the correct orientation from HDX experiment (adopted from [113]). (F) Average exchange rates mapped onto the ribbon diagram of P4 subunit for free (left) and PC-bound (middle) hexamer together with the scaled crystallographic temperature factors (B-factors, right panel). Color scale as in panel D. (G) Schematics of ssRNA loading into the hexameric packaging motor via ring opening mechanism. Top: A specific viral RNA structure (packaging signal) is recognized by the major capsid protein P1 which brings the polynucleotide strand to the vicinity of P4. Middle: P4 ring opens and lets the RNA slip in between subunit interfaces into the central channel. Bottom: The ring topologically encloses the bound RNA and translocates RNA in 5' to 3' direction into the capsid at the expense of ATP hydrolysis (packaging).

Mentions: The first step towards functional macromolecular complex is assembly of subunits guided by subunit-subunit interactions. In many instances only the high-resolution structures of subunits are available while a medium resolution electron density of the whole assembly is readily obtained by cryo-EM. The fitting of the subunit structures into the EM density is greatly facilitated by knowing the subunit interfaces within the complex. This strategy was adopted for bacteriophage ϕ12 which is a dsRNA virus belonging to the Cystoviridae family and is structurally related to members of the Reoviridae family [110]. Although the high-resolution structure of the virus-associated packaging ATPase, a hexameric molecular motor P4, had been known [111] and the hexameric structure had been resolved by cryo-EM [112,113] it was not possible to determine which way the hexamer interacted with the viral procapsid (Fig. 7A). HDX-MS was used to map the subunit interfaces [94]. Fig. 7B shows comparison of the isotopic patterns for the C-terminal helix in the free hexamer (red) and the PC-bound motor (blue). A substantial increase in protection (lower final plateau in Fig. 7C) and slower kinetic (smaller initial slope) is clearly apparent for the assembled state. Mapping of the average exchange rates for all resolved fragments onto the surface representation of the hexamer confirmed that P4 associates with the procapsid using its C-terminal facet (Fig. 7D). This information was subsequently used to fit the atomic model of the hexamer into a refined asymmetric cryo-EM reconstruction of a related virus ϕ6 [113] (Fig. 7E).


Folding and assembly of large macromolecular complexes monitored by hydrogen-deuterium exchange and mass spectrometry.

Suchanova B, Tuma R - Microb. Cell Fact. (2008)

Mapping subunit interfaces and dynamics in large viral assembly- P4 hexamer within ϕ12 procapsid [94]. (A) Schematics of the viral procapsid (PC) and packaging of ssRNA precursors by the P4 hexamers associated with the capsid vertices (left). The right panel illustrates the problem of determining the orientation of the hexamer with respect to the procapsid. (B) Bar representation of the isotopic envelopes during progressive deuteration, free hexamer in red and PC-bound in blue. (C) The HDX kinetics derived from centroid positions of the isotopic envelopes in panel B (and from additional data not shown). (D) Mapping of average exchange rates onto P4 structure in surface color representation (color scale on the bottom right). (E) Fitting of the P4 hexamer into the EM-derived electron density using the correct orientation from HDX experiment (adopted from [113]). (F) Average exchange rates mapped onto the ribbon diagram of P4 subunit for free (left) and PC-bound (middle) hexamer together with the scaled crystallographic temperature factors (B-factors, right panel). Color scale as in panel D. (G) Schematics of ssRNA loading into the hexameric packaging motor via ring opening mechanism. Top: A specific viral RNA structure (packaging signal) is recognized by the major capsid protein P1 which brings the polynucleotide strand to the vicinity of P4. Middle: P4 ring opens and lets the RNA slip in between subunit interfaces into the central channel. Bottom: The ring topologically encloses the bound RNA and translocates RNA in 5' to 3' direction into the capsid at the expense of ATP hydrolysis (packaging).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 7: Mapping subunit interfaces and dynamics in large viral assembly- P4 hexamer within ϕ12 procapsid [94]. (A) Schematics of the viral procapsid (PC) and packaging of ssRNA precursors by the P4 hexamers associated with the capsid vertices (left). The right panel illustrates the problem of determining the orientation of the hexamer with respect to the procapsid. (B) Bar representation of the isotopic envelopes during progressive deuteration, free hexamer in red and PC-bound in blue. (C) The HDX kinetics derived from centroid positions of the isotopic envelopes in panel B (and from additional data not shown). (D) Mapping of average exchange rates onto P4 structure in surface color representation (color scale on the bottom right). (E) Fitting of the P4 hexamer into the EM-derived electron density using the correct orientation from HDX experiment (adopted from [113]). (F) Average exchange rates mapped onto the ribbon diagram of P4 subunit for free (left) and PC-bound (middle) hexamer together with the scaled crystallographic temperature factors (B-factors, right panel). Color scale as in panel D. (G) Schematics of ssRNA loading into the hexameric packaging motor via ring opening mechanism. Top: A specific viral RNA structure (packaging signal) is recognized by the major capsid protein P1 which brings the polynucleotide strand to the vicinity of P4. Middle: P4 ring opens and lets the RNA slip in between subunit interfaces into the central channel. Bottom: The ring topologically encloses the bound RNA and translocates RNA in 5' to 3' direction into the capsid at the expense of ATP hydrolysis (packaging).
Mentions: The first step towards functional macromolecular complex is assembly of subunits guided by subunit-subunit interactions. In many instances only the high-resolution structures of subunits are available while a medium resolution electron density of the whole assembly is readily obtained by cryo-EM. The fitting of the subunit structures into the EM density is greatly facilitated by knowing the subunit interfaces within the complex. This strategy was adopted for bacteriophage ϕ12 which is a dsRNA virus belonging to the Cystoviridae family and is structurally related to members of the Reoviridae family [110]. Although the high-resolution structure of the virus-associated packaging ATPase, a hexameric molecular motor P4, had been known [111] and the hexameric structure had been resolved by cryo-EM [112,113] it was not possible to determine which way the hexamer interacted with the viral procapsid (Fig. 7A). HDX-MS was used to map the subunit interfaces [94]. Fig. 7B shows comparison of the isotopic patterns for the C-terminal helix in the free hexamer (red) and the PC-bound motor (blue). A substantial increase in protection (lower final plateau in Fig. 7C) and slower kinetic (smaller initial slope) is clearly apparent for the assembled state. Mapping of the average exchange rates for all resolved fragments onto the surface representation of the hexamer confirmed that P4 associates with the procapsid using its C-terminal facet (Fig. 7D). This information was subsequently used to fit the atomic model of the hexamer into a refined asymmetric cryo-EM reconstruction of a related virus ϕ6 [113] (Fig. 7E).

Bottom Line: HDX-MS became a valuable tool to follow protein folding, assembly and aggregation.The methodology has a wide range of applications in biotechnology ranging from quality control for over-expressed proteins and their complexes to screening of potential ligands and inhibitors.This review provides an introduction to protein folding and assembly followed by the principles of HDX and MS detection, and concludes with selected examples of applications that might be of interest to the biotechnology community.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Biotechnology, University of Helsinki, Finland. r.tuma@leeds.ac.uk.

ABSTRACT
Recent advances in protein mass spectrometry (MS) have enabled determinations of hydrogen deuterium exchange (HDX) in large macromolecular complexes. HDX-MS became a valuable tool to follow protein folding, assembly and aggregation. The methodology has a wide range of applications in biotechnology ranging from quality control for over-expressed proteins and their complexes to screening of potential ligands and inhibitors. This review provides an introduction to protein folding and assembly followed by the principles of HDX and MS detection, and concludes with selected examples of applications that might be of interest to the biotechnology community.

No MeSH data available.