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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.


Related in: MedlinePlus

Mechanism of HDX. (A) Mechanism of the chemical step. Inset on the right illustrates the pH and temperature (solid line 25°C, dashed line 0°C) dependence of the intrinsic exchange rate (B) Influence of quaternary, tertiary and secondary structure on HDX. (C) Overall reaction scheme and the two limiting cases.
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Figure 2: Mechanism of HDX. (A) Mechanism of the chemical step. Inset on the right illustrates the pH and temperature (solid line 25°C, dashed line 0°C) dependence of the intrinsic exchange rate (B) Influence of quaternary, tertiary and secondary structure on HDX. (C) Overall reaction scheme and the two limiting cases.

Mentions: HDX probes the exchange kinetics of the main-chain amide (NH) hydrogens for deuterium in samples exposed to D2O (Fig. 2A). The advantage is that, in principle, it provides site-specific probes along the whole polypeptide chain (except prolines and the N-terminal amino group). Under extreme pH conditions (pH<1 or pH>14) a free amide group can be either protonated or ionized [45]. Amide hydrogens can exchange via those states albeit slowly under physiologically relevant conditions. The reaction is greatly accelerated by an acid or a base. In aqueous solutions the hydronium ion (H3O+) or the hydroxide anion (OH-) plays the role of the acid or the base, respectively [45]. The acid- or base-catalyzed substitution of the unprotected (free) amide hydrogen, i.e. the chemical step of the exchange reaction, is strongly pH- and temperature-dependent (Fig. 2A right panel). This means that an exposed amide group would exchange on millisecond time scale at room temperature and neutral pH. Conversely, it would take about an hour to exchange the same exposed NH at low pH = 2.5 and 0°C [46]. The strong pH dependence of the chemical step allows preserving the pattern of labeling that was attained under native, neutral pH conditions by shifting the sample to pH 2.5 and low temperature. This, in turn provides a window of opportunity for the analysis of isotope incorporation by mass spectrometry [46].


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

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

Mechanism of HDX. (A) Mechanism of the chemical step. Inset on the right illustrates the pH and temperature (solid line 25°C, dashed line 0°C) dependence of the intrinsic exchange rate (B) Influence of quaternary, tertiary and secondary structure on HDX. (C) Overall reaction scheme and the two limiting cases.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2365927&req=5

Figure 2: Mechanism of HDX. (A) Mechanism of the chemical step. Inset on the right illustrates the pH and temperature (solid line 25°C, dashed line 0°C) dependence of the intrinsic exchange rate (B) Influence of quaternary, tertiary and secondary structure on HDX. (C) Overall reaction scheme and the two limiting cases.
Mentions: HDX probes the exchange kinetics of the main-chain amide (NH) hydrogens for deuterium in samples exposed to D2O (Fig. 2A). The advantage is that, in principle, it provides site-specific probes along the whole polypeptide chain (except prolines and the N-terminal amino group). Under extreme pH conditions (pH<1 or pH>14) a free amide group can be either protonated or ionized [45]. Amide hydrogens can exchange via those states albeit slowly under physiologically relevant conditions. The reaction is greatly accelerated by an acid or a base. In aqueous solutions the hydronium ion (H3O+) or the hydroxide anion (OH-) plays the role of the acid or the base, respectively [45]. The acid- or base-catalyzed substitution of the unprotected (free) amide hydrogen, i.e. the chemical step of the exchange reaction, is strongly pH- and temperature-dependent (Fig. 2A right panel). This means that an exposed amide group would exchange on millisecond time scale at room temperature and neutral pH. Conversely, it would take about an hour to exchange the same exposed NH at low pH = 2.5 and 0°C [46]. The strong pH dependence of the chemical step allows preserving the pattern of labeling that was attained under native, neutral pH conditions by shifting the sample to pH 2.5 and low temperature. This, in turn provides a window of opportunity for the analysis of isotope incorporation by mass spectrometry [46].

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.


Related in: MedlinePlus