Metal binding properties of the zinc finger metallome – insights into variations in stability

Abstract

Zinc is one of the most widespread metal ions found in biological systems. Of the expected 3000 zinc proteins in the human proteome, most contain zinc in structural sites. Among these structures, the most important are zinc fingers, which are well suited to facilitate interactions with DNA, RNA, proteins and lipid molecules. Knowledge regarding their stability is a critical issue in understanding the function of zinc fingers and their reactivity under fluxing cellular Zn(II) availability and different redox states. Zinc stability constants that have been determined using a variety of methods demonstrate wide diversity. Recent studies on the stability of consensus zinc fingers have demonstrated that the known metal-ion affinities for zinc fingers may have been underestimated by as much as three or more orders of magnitude. Here, using four natural ββα zinc fingers, we compare in detail several different methods that have been used for the determination of zinc finger stability constants, such as common reverse-titration, potentiometry, competition with metal chelators, and a new approach based on a three-step spectrophotometric titration. We discuss why the stabilities of zinc fingers that are determined spectrophotometrically are frequently underestimated due to the lack of effective equilibrium competition, which leads to large errors during the processing of the titration data. The literature stability constants of many natural zinc fingers have been underestimated, and they are significantly lower when compared with the consensus peptides. Our data show that in the cell, some naturally occurring zinc fingers may potentially be unoccupied and are instead loaded transiently with Zn(II). Large variations in stability within the same class of zinc fingers have demonstrated that the thermodynamic effects hidden in the sequence and structure are the key elements responsible for the differentiation of the stability of the zinc finger metallome.

Crosstalk of the structural and zinc buffering properties of mammalian metallothionein-2

“…Calculations of absolute surface area (ASA) of each residue in apo-MT2 obtained by MD after 4 ns showed cysteine residues from Cys6 up to Cys20 and from Cys34 to Cys42 to be less accessible for solvent (Table S4, ESI †). These results are in agreement with those obtained from tryptic digestion and match fragments [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] and [32][33][34][35][36][37][38][39][40][41][42][43] in which alkylation was not completed, possibly due to the lower solvent accessibility.…”

“…S2b and Table S5, ESI †). Additionally, MS/MS spectra show that the [32][33][34][35][36][37][38][39][40][41][42][43] region is more resistant to alkylation since it showed a tryptic fragment with fewer modifications (2 M) compared to the metal-free protein sample (3 M) ( Table 1). MS/MS spectra of selected tryptic fragments revealed Cys36, Cys48 and Cys60 residues as highly protected, so more likely involved in Zn(II) ion coordination ( Fig.…”

“…The MALDI spectrum recorded for unmodified MT2 demonstrates one major signal of 6043.7 m/z (Table S1 and [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] and [32][33][34][35][36][37][38][39][40][41][42][43], but incomplete modification was also observed in other peptides (Table 1, Tables S2 and S3, ESI †). The pK a values of cysteine thiols predicted by PROPKA method revealed an acidity shift of Cys residues, which ranges from 6.9 to 11.9.…”

“…32,33 The resinattached peptide was cleaved from the resin by 2 h incubation with a mixture of TFA/thioanisole/EDT/anisole (90/5/3/2, v/v/v/v), followed by precipitation in cold (À20 1C) Et 2 O. The crude peptide was purified by HPLC (Dionex Ultimate 3000 system) on a Phenomenex C18 column (Gemini-NX 5 mm, 110 Å) using 0.1% TFA with a 0-45% ACN gradient in 30 min and then lyophilized.…”

“…S7, ESI †). Fragment [32][33][34][35][36][37][38][39][40][41][42][43] exhibits an almost unchanged modification mode as compared to the protein with two Zn(II) equivalents, so Cys33, 34, 36 and 37 are visible as modified and unmodified (Table 1). MS/MS data for singly and doubly modified tryptic fragments [44][45][46][47][48][49][50][51] show that one or two cysteines are involved in Zn(II) binding, i.e.…”