Femtomolar Zn(ii) affinity of minimal zinc hook peptides – a promising small tag for protein engineering

Abstract

The minimal zinc hook peptide of Rad50 and its alanine mutants form highly stable Zn(II) complexes. These peptides were successfully used as a small, efficient tag for reversible Zn(II)-mediated protein homodimerization. The high stability, its biological consequences and potential applications in protein engineering are discussed.

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.…”

“…Tryptic fragments [32][33][34][35][36][37][38][39][40][41][42][43] and [44][45][46][47][48][49][50][51] show decreased heterogeneity regarding the number of cysteine modifications in the a-cluster (with a range of 0 to 2 and 0 to 1 modified cysteines, respectively) ( Table 1). Interestingly, a new pattern is observed when adding 5 Zn(II) equivalents compared to previous titrations regarding tryptic fragment [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] since 0 to 2 cysteine residues are modified (0-2 M) ( Table 1). So, a minimum of 3 and a maximum of 5 cysteine residues would be involved in Zn(II) coordination.…”

“…Fig. 6 shows the comparison of two tryptic fragments [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] with no modification and with one modification, which indicates that Cys21 might be modified. Moreover, the MS/MS spectrum of the singly modified fragment strongly confirms localization of the acetamide modification on the Cys21 residue (Fig.…”

“…7). Specifically, the fragment exists in unmodified form only, while [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] and [21][22][23][24][25][26][27][28][29][30][31] both demonstrate an accompanying singly modified form. The obtained structure based on MD simulations displayed Cys5 and Cys29 free of Zn(II) coordination (Fig.…”

“…S2e, ESI †), but one should note that the presence of particular Zn(II)-depleted species must be taken qualitatively not quantitatively. Tryptic fragment [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] is triply modified, which means two cysteine residues resistant to alkylation reside in the b-domain and probably coordinate to the same Zn(II) ion. Tryptic fragments [32][33][34][35][36][37][38][39][40][41][42][43], [44][45][46][47][48][49][50][51] and [52][53][54][55][56][57][58][59][60][61] exist i...…”