Prothymosin-α is a small, multifunctional intrinsically disordered protein associated with cell survival and proliferation which binds multiple Zn ions and undergoes partial folding. The interaction between prothymosin-α and at least two of its protein targets is significantly enhanced in the presence of Zn ions, suggesting that Zn binding plays a role in the protein's function. The primary sequence of prothymosin-α is highly acidic, with almost 50% comprised of Asp and Glu, and is unusual for a Zn-binding protein as it lacks Cys and His residues. To gain a better understanding of the nature of the Zn-prothymosin-α interactions and the protein's ability to discriminate Zn over other divalent cations (e.g., Ca, Co, Mg) we synthesized a set of three model peptides and characterized the effect of metal binding using electrospray ionization mass spectrometry (ESI MS) and circular dichroism (CD) spectroscopy. ESI MS data reveal that the native peptide model of the glutamic acid rich region binds 4 Zn ions with apparent, stepwise K values that are, at highest, in the tens of micromolar range. A peptide model with the same amino acid composition as the native sequence, but with the residues arranged randomly, showed no evidence of structural change by CD upon introduction of Zn. These results suggest that the high net negative charge of the glutamic acid-rich region of prothymosin-α is not a sufficient criterion for Zn to induce a structural change; rather, Zn binding to prothymosin-α is sequence specific, providing important insight into the behavior of intrinsically disordered proteins.

The relaxation rates for the three different carbon types in EDTA (carbonyl, CH2 central, and CH2 lateral) were measured with and without Zn(2+) as a function of field strength and temperature. The use of different field strengths in combination with NOE measurements allowed for the contribution of each relaxation mechanism (chemical shift anisotropy; spin rotation; dipole-dipole) to the total relaxation rate for each carbon to be determined. Temperature studies allowed for determination of the activation energy (Ea) for the motions of each carbon type. The most surprising result was the observation that the τ(c) decreases significantly for the lateral carbon upon addition of Zn(2+) at neutral pH, going from 54 to 8.6 ps at 298 K. This appears to be a pH-dependent phenomenon as other reports indicate that τ(c) increases for the lateral carbon upon addition of Zn(2+) under strongly basic conditions.

Use of the 4-pyridylmethyl ester group for side-chain protection of glutamic acid residues in solid-phase peptide synthesis enables switching of the charge state of a peptide from negative to positive, thus making detection by positive ion mode ESI-MS possible. The pyridylmethyl ester moiety is readily removed from peptides in high yield by hydrogenation. Combining the 4-pyridylmethyl ester protecting group with benzyl ester protection reduces the number of the former needed to produce a net positive charge and allows for purification by RP HPLC. This protecting group is useful in the synthesis of highly acidic peptide sequences, which are often beset by problems with purification by standard RP HPLC and characterization by ESI-MS.