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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 functional dynamics in the hexamer of P4 from bacteriophage ϕ8 [114]. Mass/charge (m/z) spectra corresponding to the peptic fragment encompassing residues 139–158 (m = 2210.14 Da, z = 3) during H/D exchange (only interval 0 to 60 min shown). (B) Increase of deuterium content in the peptide (symbols) and the corresponding maximum entropy fit (MEM) for P4 alone (black circles, solid line), P4+1 mM poly(C) (blue triangles, dotted), P4 + 1 mM poly(C) + 1 mM ATP (red squares, dashed line), P4 + 1 mM poly(C) + 1 mM AMP-PNP (green diamonds, dash-dot-dot) and P4 + 1 mM ADP (cyan inverted triangles, dash-dot). Standard deviations (error bars) were estimated from three independent experiments. (C) Rate distributions obtained by MEM fitting of data in panel B. The color bar under the panel indicates the three integration regions which were used to obtain the number of sites within each rate class (blue = slow/protected, green = intermediate, red = fast/unprotected). (D) Number of amide sites in the three classes and under different conditions (nucleotide di/triphosphates, RNA binding) obtained from data in panel C, bar colors as in panel C. (E) RGB representation of the HDX kinetics for subunit interfaces. The two facets (left and right) represent the facing interfaces from the neighboring subunits in a surface representation. Bound ATP molecule is shown in yellow ball-and-stick representation. Several regions of interest are delineated: NT-nucleotide binding pocket; L2H-loop 2 and α-helix 6 which constitute the moving lever of the motor; IH-interfacial helix which becomes transiently exposed during ring opening and RNA loading. (F) Three-color, RGB scale for number of amides exchanging in the three classes.
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Figure 9: Mapping functional dynamics in the hexamer of P4 from bacteriophage ϕ8 [114]. Mass/charge (m/z) spectra corresponding to the peptic fragment encompassing residues 139–158 (m = 2210.14 Da, z = 3) during H/D exchange (only interval 0 to 60 min shown). (B) Increase of deuterium content in the peptide (symbols) and the corresponding maximum entropy fit (MEM) for P4 alone (black circles, solid line), P4+1 mM poly(C) (blue triangles, dotted), P4 + 1 mM poly(C) + 1 mM ATP (red squares, dashed line), P4 + 1 mM poly(C) + 1 mM AMP-PNP (green diamonds, dash-dot-dot) and P4 + 1 mM ADP (cyan inverted triangles, dash-dot). Standard deviations (error bars) were estimated from three independent experiments. (C) Rate distributions obtained by MEM fitting of data in panel B. The color bar under the panel indicates the three integration regions which were used to obtain the number of sites within each rate class (blue = slow/protected, green = intermediate, red = fast/unprotected). (D) Number of amide sites in the three classes and under different conditions (nucleotide di/triphosphates, RNA binding) obtained from data in panel C, bar colors as in panel C. (E) RGB representation of the HDX kinetics for subunit interfaces. The two facets (left and right) represent the facing interfaces from the neighboring subunits in a surface representation. Bound ATP molecule is shown in yellow ball-and-stick representation. Several regions of interest are delineated: NT-nucleotide binding pocket; L2H-loop 2 and α-helix 6 which constitute the moving lever of the motor; IH-interfacial helix which becomes transiently exposed during ring opening and RNA loading. (F) Three-color, RGB scale for number of amides exchanging in the three classes.

Mentions: HDX-MS was used for mapping the interactions of proteins with ligands [137,138] and enabled to unravel subtle structural changes in troponin C upon Ca2+ binding. These changes are important for regulation but escaped detection by other techniques [139]. The concept is illustrated in Fig. 9A–D for binding of nucleotide di- and tri-phosphates and RNA to the hexameric viral packaging motor, protein P4 (see above). The exchange of a peptide fragment originating from the vicinity of the ATP binding site exhibited slower rate in the presence of ATP or ADP but is was largely unaffected by RNA binding (Fig. 9B–D) [114]. Further insight was obtained by computing the distribution of exchange rates by a maximum entropy method [140] (Fig. 9C) and by classification of the amide sites into three groups: fast (exchanged before the first time point, i.e. 30 sec), intermediate (exchanging on the time scale of the experiment), and protected (not exchanged during the whole duration of the experiment, i.e. 8 h) (Fig. 9D) [114]. The three-class analysis revealed that reversible ligand binding affected mostly the fast exchanging amides and provided only partial protection. This is understandable given that ATP and ADP binding and dissociation both happen on millisecond-to-second time scale.


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

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

Mapping functional dynamics in the hexamer of P4 from bacteriophage ϕ8 [114]. Mass/charge (m/z) spectra corresponding to the peptic fragment encompassing residues 139–158 (m = 2210.14 Da, z = 3) during H/D exchange (only interval 0 to 60 min shown). (B) Increase of deuterium content in the peptide (symbols) and the corresponding maximum entropy fit (MEM) for P4 alone (black circles, solid line), P4+1 mM poly(C) (blue triangles, dotted), P4 + 1 mM poly(C) + 1 mM ATP (red squares, dashed line), P4 + 1 mM poly(C) + 1 mM AMP-PNP (green diamonds, dash-dot-dot) and P4 + 1 mM ADP (cyan inverted triangles, dash-dot). Standard deviations (error bars) were estimated from three independent experiments. (C) Rate distributions obtained by MEM fitting of data in panel B. The color bar under the panel indicates the three integration regions which were used to obtain the number of sites within each rate class (blue = slow/protected, green = intermediate, red = fast/unprotected). (D) Number of amide sites in the three classes and under different conditions (nucleotide di/triphosphates, RNA binding) obtained from data in panel C, bar colors as in panel C. (E) RGB representation of the HDX kinetics for subunit interfaces. The two facets (left and right) represent the facing interfaces from the neighboring subunits in a surface representation. Bound ATP molecule is shown in yellow ball-and-stick representation. Several regions of interest are delineated: NT-nucleotide binding pocket; L2H-loop 2 and α-helix 6 which constitute the moving lever of the motor; IH-interfacial helix which becomes transiently exposed during ring opening and RNA loading. (F) Three-color, RGB scale for number of amides exchanging in the three classes.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 9: Mapping functional dynamics in the hexamer of P4 from bacteriophage ϕ8 [114]. Mass/charge (m/z) spectra corresponding to the peptic fragment encompassing residues 139–158 (m = 2210.14 Da, z = 3) during H/D exchange (only interval 0 to 60 min shown). (B) Increase of deuterium content in the peptide (symbols) and the corresponding maximum entropy fit (MEM) for P4 alone (black circles, solid line), P4+1 mM poly(C) (blue triangles, dotted), P4 + 1 mM poly(C) + 1 mM ATP (red squares, dashed line), P4 + 1 mM poly(C) + 1 mM AMP-PNP (green diamonds, dash-dot-dot) and P4 + 1 mM ADP (cyan inverted triangles, dash-dot). Standard deviations (error bars) were estimated from three independent experiments. (C) Rate distributions obtained by MEM fitting of data in panel B. The color bar under the panel indicates the three integration regions which were used to obtain the number of sites within each rate class (blue = slow/protected, green = intermediate, red = fast/unprotected). (D) Number of amide sites in the three classes and under different conditions (nucleotide di/triphosphates, RNA binding) obtained from data in panel C, bar colors as in panel C. (E) RGB representation of the HDX kinetics for subunit interfaces. The two facets (left and right) represent the facing interfaces from the neighboring subunits in a surface representation. Bound ATP molecule is shown in yellow ball-and-stick representation. Several regions of interest are delineated: NT-nucleotide binding pocket; L2H-loop 2 and α-helix 6 which constitute the moving lever of the motor; IH-interfacial helix which becomes transiently exposed during ring opening and RNA loading. (F) Three-color, RGB scale for number of amides exchanging in the three classes.
Mentions: HDX-MS was used for mapping the interactions of proteins with ligands [137,138] and enabled to unravel subtle structural changes in troponin C upon Ca2+ binding. These changes are important for regulation but escaped detection by other techniques [139]. The concept is illustrated in Fig. 9A–D for binding of nucleotide di- and tri-phosphates and RNA to the hexameric viral packaging motor, protein P4 (see above). The exchange of a peptide fragment originating from the vicinity of the ATP binding site exhibited slower rate in the presence of ATP or ADP but is was largely unaffected by RNA binding (Fig. 9B–D) [114]. Further insight was obtained by computing the distribution of exchange rates by a maximum entropy method [140] (Fig. 9C) and by classification of the amide sites into three groups: fast (exchanged before the first time point, i.e. 30 sec), intermediate (exchanging on the time scale of the experiment), and protected (not exchanged during the whole duration of the experiment, i.e. 8 h) (Fig. 9D) [114]. The three-class analysis revealed that reversible ligand binding affected mostly the fast exchanging amides and provided only partial protection. This is understandable given that ATP and ADP binding and dissociation both happen on millisecond-to-second time scale.

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.