Monitoring the zinc affinity of the metallo-β-lactamase CphA by automated nanoESI-MS

Abstract

Metallo-␤-lactamases are zinc containing enzymes that are able to hydrolyze and inactivate ␤-lactam antibiotics. The subclass B2 enzyme CphA of Aeromonas hydrophila is a unique metallo-␤-lactamase because it degrades only carbapenems efficiently and is only active when it has one zinc ion bound. A zinc titration experiment was used to study the zinc affinity of the wild-type and of several mutant CphA enzymes. It shows that a second Zn 2ϩ is also bound at high ion concentrations. All samples were analyzed using mass spectrometry in combination with an automated nanoESI source. The metal-free enzyme has a bimodal charge distribution indicative of two conformational states. A completely folded enzyme is detected when the apo-enzyme has bound the first zinc. Intensity ratios of the different enzyme forms were used to deduce the zinc affinities. CphA enzymes mutated in metal ligands show decreased zinc affinity compared to wild-type, especially D120 mutants. (J Am Soc Mass Spectrom 2006, 17, 180 -188)

Estrogen receptor–ligand complexes measured by chip‐based nanoelectrospray mass spectrometry: An approach for the screening of endocrine disruptors

“…Several reports have shown the potential of ESI-MS to characterize the binding of NR ligands to their receptor in terms of stochiometry, specificity, and stability (Greschik et al 2002;Lengqvist et al 2002Lengqvist et al , 2004Lengqvist et al , 2005Bitsch et al 2003;Potier et al 2003;Stehlin-Gaon et al 2003;Sanglier et al 2004). ESI-MS has been successfully used to assess noncovalent complex stability in the vacuum of the mass spectrometer by analyzing their resistance to dissociation Wu et al 1997;van der Kerk-van Hoof and Heck 1999) and for measuring solution binding constants (Jorgensen et al 1998;Sannes-Lowery et al 2000;Daniel et al 2003;Gabelica et al 2003;Wendt et al 2003;Tjernberg et al 2004;Wortmann et al 2005;De Vriendt et al 2006). By using correction factors or assumptions to derive solution concentrations from the ion intensities of the different species, a number of these studies have demonstrated that binding affinities determined by ESI are in good agreement with solution-phase data.…”

The pH Dependence of Product Ion Spectra Obtained from Precursor Ions with the Same Charge Number in ESI of Carbonic Anhydrase 2

“…On the other hand, the folding state of the lower charged protein appears to exist in relatively compact folding states, which is consistent with previous studies. [1][2][3] In addition, multiply charged apo-CA2 ions with a higher charge state distribution with a maximum at 29+ (m/z 1001.8) were observed when a mixture of 0.1% formic acid (pH 2.6) and acetonitrile was employed as a solvent (Fig. 1c).…”

“…This charge state distribution is known to be dependent on the nature of the sample solution, and is affected by conditions, such as pH, temperature, and the type of solvent used. [1][2][3][4][5] The charge state distribution in the ESI MS of a protein is considered to reflect the folding states in the gas-phase, the solvent system used is known to be an influencing factor. The charge state distribution resulting from a denaturing acidic solvent results in the production of ion peaks corresponding to a higher charge state (lower m/z) and vice versa.…”

“…The charge state distribution resulting from a denaturing acidic solvent results in the production of ion peaks corresponding to a higher charge state (lower m/z) and vice versa. [1][2][3]6,7) However, characterizing the folding state of a proteins in solution and the gas-phase remains a controversial issue, and several studies related to this have appeared in the literature. [8][9][10] Specifically, Robinson reported a correlation between the degree of folding and charge state.…”

Mass Spectrometric Determination of Zn²⁺ Binding/Dissociation Constant for Zinc Finger Peptides

“…Mass spectrometry (MS) has been used widely to study metal ions interactions with oligonucleotides, peptides, and proteins. [8][9][10][11][12][13][14][15][16][17] For zinc fingers, a variety of MS experiments have been used to study, for example, the stoichiometry for zinc ion transfer from metallothionein to zinc finger peptides, Zn 2+ chelation by consensus zinc-finger arrays, the Zn 2+ binding sites, and relative affinity for other transition metals.…”