Arsenite binding to synthetic peptides: The effect of increasing length between two cysteines

Abstract

We utilized radioactive 73As-labeled arsenite and vacuum filtration methodology to determine the binding affinity of arsenite to eight synthetic peptides ranging from 13 to 24 amino acids long and containing one or two cysteines separated by 0-17 intervening amino acids. Six of the eight peptides were highly similar in amino acid sequence and were based on cysteine containing regions of the hormone-binding site of the human estrogen receptor-alpha (e.g., the sequence of peptide 28 is LEGAWCGKGVEGTEHLYSMKCKNV). The peptides with 0-14 intervening amino acids between two cysteines bound arsenite with Kd values of 2.7-20.1 uM and with Bmax values from 36 to 103 nmol/mg protein (from 0.083 to 0.19 nmol/nmol of protein). Thus, increasing the number of intervening amino acids from 0 to 14 made very little difference in the observed Kd values for arsenite, a surprising finding. Therefore, these peptides are flexible in solution and effectively contain a dithiol high affinity binding site for arsenite. Peptide 17 with two C separated by 19 amino acids bound arsenite with a Kd of 123 uM and a Bmax of 41.8 nmol/mg. The monothiol peptide 19 bound arsenite with a Kd of 124 uM and a Bmax of 26 nmol/mg protein. All experimental binding curves fit well to a one site binding model.

Arsenite Interacts Selectively with Zinc Finger Proteins Containing C3H1 or C4 Motifs

“…Although it has been suggested that vicinal cysteine residues are required for high affinity arsenite binding (33), our current findings indicate that vicinal cysteines are not necessarily sufficient for arsenite binding to peptides (Figs. 4 and 5) or disruption of zinc binding in cells (Figs.…”

“…Our studies demonstrate arsenite binding selectivity in both peptide and protein with a link between interaction and functional consequences for protein activity. Although arsenite may be capable of binding to a C2H2 zinc finger (32,33,43), the final product may not persist for a sufficient length of time to be detected by MALDI-TOF-MS. This difference in chemical kinetics could have major impact in cells, where a more stable interaction of arsenite with a C3 or C4 zinc finger protein may be necessary to sustain biological impact.…”

“…These findings indicated functional interaction between arsenite and zinc within a specific target protein and suggested questions regarding determinants of arsenic target selectivity. Cysteine content and vicinal cysteine residues have been postulated as key factors for arsenic interaction with proteins (32,33). One study using synthetic peptides based on the estrogen receptor demonstrated that cysteine is essential for arsenite binding and that trivalent arsenite binding to peptides favors those with two or more cysteines (32 demonstrated that 10 M arsenite can strongly disrupt the oxidative folding of riboflavin-binding protein (a protein with 18 cysteines) even in the presence of 5 mM reduced glutathione (a monothiol compound), indicating poor interaction of arsenite with single thiol groups (34).…”

Dissociation of arsenite‐peptide complexes: Triphasic nature, rate constants, half‐lives, and biological importance

“…Overall, this and prior studies [13,27] have given us a more comprehensive view (both in respect to kinetics of dissociation and association of complexes and the K d and B max values) of the intermolecular and intramolecular way in which arsenite binds to cysteine-containing peptides and thus, proteins.…”

“…For triand bi-dentate arsenicals (i.e., arsenite and MMA(III)), binding can crosslink two or three different peptides or proteins forming new homo or hetero dimer or trimer forms of the original peptides and proteins. Arsenite is capable of binding with high affinity to two cysteine moieties that are a surprisingly large distance (14 intervening amino acids) apart [27].…”