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, 13 (11), 2878-87

Application of Electrospray Ionization Mass Spectrometry to Study the Hydrophobic Interaction Between the Epsilon and Theta Subunits of DNA Polymerase III

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Application of Electrospray Ionization Mass Spectrometry to Study the Hydrophobic Interaction Between the Epsilon and Theta Subunits of DNA Polymerase III

Rajesh Gupta et al. Protein Sci.

Abstract

The interactions between the N-terminal domain of the epsilon (epsilon186) and theta subunits of DNA polymerase III of Escherichia coli were investigated using electrospray ionization mass spectrometry. The epsilon186-theta complex was stable in 9 M ammonium actetate (pH 8), suggesting that hydrophobic interactions have a predominant contribution to the stability of the complex. Addition of primary alkanols to epsilon186-theta in 0.1 M ammonium acetate (pH 8), led to dissociation of the complex, as observed in the mass spectrometer. The concentrations of methanol, ethanol, and 1-propanol required to dissociate 50% of the complex were 8.9 M, 4.8 M, and 1.7 M, respectively. Closer scrutiny of the effect of alkanols on epsilon186, theta, and epsilon186-theta showed that epsilon186 formed soluble aggregates prior to precipitation, and that the association of epsilon186 with theta stabilized epsilon186. In-source collision-induced dissociation experiments and other results suggested that the epsilon186-theta complex dissociated in the mass spectrometer, and that the stability (with respect to dissociation) of the complex in vacuo was dependent on the solution from which it was sampled.

Figures

Figure 1.
Figure 1.
Positive ion ESI mass spectra of ɛ186–θ acquired under different conditions. (A) ɛ186–θ in 0.1 M NH4OAc at pH 8; cone 30 V. (B) ɛ186–θ in 0.1 M NH4OAc at pH 8; cone 60 V. (C) ɛ186– θ in 9 M NH4OAc at pH 8; cone 30 V. Ions from: θ (•); ɛ186 (▪); ɛ186–θ (♦).
Figure 2.
Figure 2.
Stability of ɛ186–θ in various solvents as a function of cone voltage. The intensities of ions from ɛ186–θ were summed and expressed as a percentage of the total intensities of all ions in ESI mass spectra. 2.0 M NH4OAc at pH 8 (▴); 0.1 M NH4OAc at pH 8 (▪); 0.01 M NH4OAc at pH 8 (•).
Figure 3.
Figure 3.
Positive ion ESI mass spectra of ɛ186–θ acquired using concentrations of 1-propanol. (A) ɛ186–θ in 0.1 M NH4OAc at pH 8, 1.0 M in 1-propanol. (B) ɛ186–θ in 0.1 M NH4OAc at pH 8, 1.4 M in 1-propanol. (C) ɛ186–θ in 0.1 M NH4OAc at pH 8, 1.8 M 1-propanol. (D) ɛ186–θ in 0.1 M NH4OAc at pH 8, 2.0 M 1-propanol. Ions from: θ (•); ɛ186 (▪); ɛ186–θ (♦).
Figure 4.
Figure 4.
Stability of ɛ186–θ in various alkanols as a function of alkanol concentration. The intensities of ions from ɛ186–θ were summed and expressed as a percentage of the total intensities of all ions in ESI mass spectra.
Figure 5.
Figure 5.
A360 of various solutions containing ɛ186–θ, ɛ186, or θ (2 μM). (A) ɛ186 in 2.0 M 1-propanol in 0.1 M NH4OAc at pH 8; (B) ɛ186 in 1.6 M 1-propanol in 0.1 M NH4OAc at pH 8; (C) θ in 2.0 M 1-propanol in 0.1 M NH4OAc at pH 8; (D) ɛ186–θ in 2.0 M 1-propanol in 0.1 M NH4OAc at pH 8; (E) ɛ186 in 0.1 M NH4OAc at pH 8.
Figure 6.
Figure 6.
Stabilization of ɛ186 by θ as judged by ESI-MS. (A) ɛ186–θ in 0.1 M NH4OAc at pH 8. (B) ɛ186 treated with 1.6 M 1-propanol (in 0.1 M NH4OAc at pH 8) with θ added after 1 min. (C) θ treated with 1.6 M 1-propanol (in 0.1 M NH4OAc at pH 8) with ɛ186 added after 1 min. (D) ɛ186 treated with 1.8 M 1-propanol (in 0.1 M NH4OAc at pH 8) with θ added after 1 min. (E) θ treated with 1.8 M 1-propanol (in 0.1 M NH4OAc at pH 8) with ɛ186 added after 1 min. Ions from: θ (•); ɛ186 (▪); ɛ186–θ (♦).
Figure 7.
Figure 7.
Stabilization of ɛ186 by θ as judged by ESI-MS monitored after 9 h. (A) ɛ186–θ in 0.1 M NH4OAc at pH 8. (B) ɛ186 treated with 1.8 M 1-propanol (in 0.1 M NH4OAc at pH 8) with θ added after 1 min. (C) θ treated with 1.8 M 1-propanol (in 0.1 M NH4OAc at pH 8) with ɛ186 added after 1 min. Ions from: θ (•); ɛ186 (▪); ɛ186–θ (♦).

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