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Electrostatic stabilization of a native protein structure in the gas phase.

Breuker K, Brüschweiler S, Tollinger M - Angew. Chem. Int. Ed. Engl. (2010)

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Affiliation: Institut für Organische Chemie and Center for Molecular Biosciences Innsbruck, Universität Innsbruck, Innrain 52a, 6020 Innsbruck, Austria. kathrin.breuker@uibk.ac.at

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Recently, a general picture has been proposed of how long, and to what extent, native protein structure can be retained in the gas phase... In particular, molecular dynamics simulations suggest that salt bridges and ionic hydrogen bonds on the protein surface can transiently stabilize the global fold shortly after desolvation... We demonstrate that in the gas phase, the most stable regions are those stabilized by salt bridges and ionic hydrogen bonds... The data in Figure 2 provide substantial evidence for a correlation between the solution- and gas-phase structures of KIX... This supposition is corroborated by ECD of 12+ ions generated by nano-ESI from a solution (in H2O at pH 4.5) that better resembles the native protein environment, which gave decreased - and -ion yields in the α2 and α3 regions (see Figure S4 in the Supporting Information), along with a smaller total fragment ion yield (37 %) relative to that resulting from ECD of 12+ ions from ESI of solutions in H2O/CH3OH (80:20) at pH 4 (total fragment-ion yield: 49 %; see Figure S3 in the Supporting Information)... This behavior is also reflected in the site-specific transition charge values from analysis of site-specific - and -ion yields (see Figure S6 in the Supporting Information), which generally increase from the N to the C terminus (Figure 4b)... Transition charge values for cleavage sites between helix regions (31–41, 62–64) are similar to values for adjacent helix ends, indicating that helix separation does not precede helix unraveling... This observation strongly suggests that interactions involving charged residues, that is, ionic hydrogen bonds and salt bridges, largely determine helix stability in the gas phase... The density of salt bridges correlates (r=0.9999) with transition charge values (Figure 6b) even better than the density of charged residues, suggesting that salt bridges are major determinants for protein structural stabilization in the gas phase... However, this conclusion does not exclude additional stabilization by ionic hydrogen bonds as well as charge–dipole interactions... We show here that electrostatic interactions can compensate for the loss of hydrophobic bonding and stabilize the native three-helix bundle structure of KIX in the gas phase on a timescale of at least 4 s... Among these interactions, salt bridges were found to play a dominant role... However, a high number of surface-exposed charged residues alone does not guarantee protein stability in the gas phase: equine Cytochrome c has 24 basic and 12 acidic residues, with the number of salt bridges on the protein surface increasing from 6 in solution to an average value of 17.3 in the gas phase within 10 ps after desolvation, yet its native fold disintegrates on a timescale of milliseconds., The outstanding stability of gaseous KIX ions observed in this study must be attributed to the combination of favorable electrostatic interactions, including salt bridges, neutral and ionic hydrogen bonds, as well as charge–dipole interactions.

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Yields of c (black bars) and z• (open bars) fragment ions from ECD of (M + n H)n+ ions of KIX versus backbone cleavage site; helix regions are shaded gray. Ions with n=7–12 and n=13–16 were electrosprayed from quasinative (80:20 H2O/CH3OH, pH 4) and denaturing (50:50 H2O/CH3OH, pH 2.5) protein solutions (1–2 μm), respectively.
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fig02: Yields of c (black bars) and z• (open bars) fragment ions from ECD of (M + n H)n+ ions of KIX versus backbone cleavage site; helix regions are shaded gray. Ions with n=7–12 and n=13–16 were electrosprayed from quasinative (80:20 H2O/CH3OH, pH 4) and denaturing (50:50 H2O/CH3OH, pH 2.5) protein solutions (1–2 μm), respectively.

Mentions: Figure 2 shows site-specific yields of c and z• fragment ions6 from ECD of (M + n H)n+ ions of KIX (see Figure S1 in the Supporting Information) formed by electrospray ionization (ESI).7 For the 7+ ions, separated c and z• products were observed only from backbone cleavage near the termini (residues 1–13 and 89–91), but not from the three-helix bundle region, which forms a globular fold around a hydrophobic core (residues 16–88).5 This observation is consistent with intramolecular interactions in the three-helix bundle region preventing separation of c and z• backbone-cleavage products3a–c in the gaseous 7+ ions. Collisional activation of the 7+ ions (laboratory-frame energy: 28 eV) prior to ECD effected only marginal unfolding near the N terminus (see Figure S2 in the Supporting Information), revealing a notable stability of the three-helix bundle in the absence of solvent.


Electrostatic stabilization of a native protein structure in the gas phase.

Breuker K, Brüschweiler S, Tollinger M - Angew. Chem. Int. Ed. Engl. (2010)

Yields of c (black bars) and z• (open bars) fragment ions from ECD of (M + n H)n+ ions of KIX versus backbone cleavage site; helix regions are shaded gray. Ions with n=7–12 and n=13–16 were electrosprayed from quasinative (80:20 H2O/CH3OH, pH 4) and denaturing (50:50 H2O/CH3OH, pH 2.5) protein solutions (1–2 μm), respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3045662&req=5

fig02: Yields of c (black bars) and z• (open bars) fragment ions from ECD of (M + n H)n+ ions of KIX versus backbone cleavage site; helix regions are shaded gray. Ions with n=7–12 and n=13–16 were electrosprayed from quasinative (80:20 H2O/CH3OH, pH 4) and denaturing (50:50 H2O/CH3OH, pH 2.5) protein solutions (1–2 μm), respectively.
Mentions: Figure 2 shows site-specific yields of c and z• fragment ions6 from ECD of (M + n H)n+ ions of KIX (see Figure S1 in the Supporting Information) formed by electrospray ionization (ESI).7 For the 7+ ions, separated c and z• products were observed only from backbone cleavage near the termini (residues 1–13 and 89–91), but not from the three-helix bundle region, which forms a globular fold around a hydrophobic core (residues 16–88).5 This observation is consistent with intramolecular interactions in the three-helix bundle region preventing separation of c and z• backbone-cleavage products3a–c in the gaseous 7+ ions. Collisional activation of the 7+ ions (laboratory-frame energy: 28 eV) prior to ECD effected only marginal unfolding near the N terminus (see Figure S2 in the Supporting Information), revealing a notable stability of the three-helix bundle in the absence of solvent.

View Article: PubMed Central - PubMed

Affiliation: Institut für Organische Chemie and Center for Molecular Biosciences Innsbruck, Universität Innsbruck, Innrain 52a, 6020 Innsbruck, Austria. kathrin.breuker@uibk.ac.at

AUTOMATICALLY GENERATED EXCERPT
Please rate it.

Recently, a general picture has been proposed of how long, and to what extent, native protein structure can be retained in the gas phase... In particular, molecular dynamics simulations suggest that salt bridges and ionic hydrogen bonds on the protein surface can transiently stabilize the global fold shortly after desolvation... We demonstrate that in the gas phase, the most stable regions are those stabilized by salt bridges and ionic hydrogen bonds... The data in Figure 2 provide substantial evidence for a correlation between the solution- and gas-phase structures of KIX... This supposition is corroborated by ECD of 12+ ions generated by nano-ESI from a solution (in H2O at pH 4.5) that better resembles the native protein environment, which gave decreased - and -ion yields in the α2 and α3 regions (see Figure S4 in the Supporting Information), along with a smaller total fragment ion yield (37 %) relative to that resulting from ECD of 12+ ions from ESI of solutions in H2O/CH3OH (80:20) at pH 4 (total fragment-ion yield: 49 %; see Figure S3 in the Supporting Information)... This behavior is also reflected in the site-specific transition charge values from analysis of site-specific - and -ion yields (see Figure S6 in the Supporting Information), which generally increase from the N to the C terminus (Figure 4b)... Transition charge values for cleavage sites between helix regions (31–41, 62–64) are similar to values for adjacent helix ends, indicating that helix separation does not precede helix unraveling... This observation strongly suggests that interactions involving charged residues, that is, ionic hydrogen bonds and salt bridges, largely determine helix stability in the gas phase... The density of salt bridges correlates (r=0.9999) with transition charge values (Figure 6b) even better than the density of charged residues, suggesting that salt bridges are major determinants for protein structural stabilization in the gas phase... However, this conclusion does not exclude additional stabilization by ionic hydrogen bonds as well as charge–dipole interactions... We show here that electrostatic interactions can compensate for the loss of hydrophobic bonding and stabilize the native three-helix bundle structure of KIX in the gas phase on a timescale of at least 4 s... Among these interactions, salt bridges were found to play a dominant role... However, a high number of surface-exposed charged residues alone does not guarantee protein stability in the gas phase: equine Cytochrome c has 24 basic and 12 acidic residues, with the number of salt bridges on the protein surface increasing from 6 in solution to an average value of 17.3 in the gas phase within 10 ps after desolvation, yet its native fold disintegrates on a timescale of milliseconds., The outstanding stability of gaseous KIX ions observed in this study must be attributed to the combination of favorable electrostatic interactions, including salt bridges, neutral and ionic hydrogen bonds, as well as charge–dipole interactions.

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