Zn-binding in the glutamate-rich region of the intrinsically disordered protein prothymosin-alpha.

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.

Calorimetric investigation of copper binding in the N-terminal region of the prion protein at low copper loading: evidence for an entropically favorable first binding event.

Although the Cu(2+)-binding sites of the prion protein have been well studied when the protein is fully saturated by Cu(2+), the Cu(2+)-loading mechanism is just beginning to come into view. Because the Cu(2+)-binding modes at low and intermediate Cu(2+) occupancy necessarily represent the highest-affinity binding modes, these are very likely populated under physiological conditions, and it is thus essential to characterize them in order to understand better the biological function of copper-prion interactions. Besides binding-affinity data, almost no other thermodynamic parameters (e.

Field- and temperature-dependent 13C NMR studies of the EDTA-Zn2+ complex: insight into structure and dynamics via relaxation measurements.

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 4-pyridylmethyl ester as a protecting group for glutamic and aspartic acids: 'flipping' peptide charge states for characterization by positive ion mode ESI-MS.

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.

Peculiarities of copper binding to alpha-synuclein.

Heavy metals have been implicated as the causative agents for the pathogenesis of the most prevalent neurodegenerative disease. Various mechanisms have been proposed to explain the toxic effects of metals ranging from metal-induced oxidation of protein to metal-induced changes in the protein conformation. Aggregation of a-synuclein is implicated in Parkinson's disease (PD), and various metals, including copper, constitute a prominent group of alpha-synuclein aggregation enhancers.

Prothymosin-alpha inhibits HIV-1 via Toll-like receptor 4-mediated type I interferon induction.

Induction of type I interferons (IFN) is a central feature of innate immune responses to microbial pathogens and is mediated via Toll-like receptor (TLR)-dependent and -independent pathways. Prothymosin-alpha (ProTalpha), a small acidic protein produced and released by CD8(+) T cells, inhibits HIV-1, although the mechanism for its antiviral activity was not known. We demonstrate that exogenous ProTalpha acts as a ligand for TLR4 and stimulates type I IFN production to potently suppress HIV-1 after entry into cells.

Zinc deposition during ESI-MS analysis of peptide-zinc complexes.

Electrospray ionization (ESI) mass spectrometry (MS) has proven to be an extremely powerful technique for studying the stoichiometry and binding strength of peptide-metal complexes. We have found a significant new problem in the ESI-MS of zinc-peptide systems involving the deposition of zinc in the ESI emitter. This deposition of zinc during the experiment removes a significant amount of zinc ions from the solution, impacting the resulting mass spectral intensities used to quantify the amount of the zinc-bound species.

The manganese transporter MntH is a critical virulence determinant for Brucella abortus 2308 in experimentally infected mice.

The gene designated BAB1_1460 in the Brucella abortus 2308 genome sequence is predicted to encode the manganese transporter MntH. Phenotypic analysis of an isogenic mntH mutant indicates that MntH is the sole high-affinity manganese transporter in this bacterium but that MntH does not play a detectable role in the transport of Fe(2+), Zn(2+), Co(2+), or Ni(2+). Consistent with the apparent selectivity of the corresponding gene product, the expression of the mntH gene in B.

Identification of the copper(II) coordinating residues in the prion protein by metal-catalyzed oxidation mass spectrometry: evidence for multiple isomers at low copper(II) loadings.

While the Cu(II) binding sites of the prion protein have been well studied under Cu-saturation conditions, the identity of the residues involved in coordinating Cu(II) at low stoichiometries and the order in which the binding sites load with Cu(II) remain unresolved. In this study, we have used two mass spectrometry based methods to gather insight into Cu(II)-prion binding under different stoichiometric loadings of Cu(II). The first method uses metal-catalyzed oxidation reactions to site specifically modify the residues bound to Cu(II) in solution, and the second method determines Cu binding sites based on the protection of His from modification by diethyl pyrocarbonate when this residue binds Cu(II) in solution.

Influence of prothymosin-alpha on HIV-1 target cells.

The important role of CD8(+) T cells in controlling HIV-1 infection through the innate as well as the adaptive immune system is well established. In addition to the major histocompatibility complex (MHC)-dependent cytotoxic activity of CD8(+) T cells, they produce soluble factors that suppress HIV-1 replication in an MHC-independent manner. Several of those factors have been identified, including beta-chemokines, Rantes, MIP-1alpha, MIP-1beta, and MDC.

Copper and zinc promote interactions between membrane-anchored peptides of the metal binding domain of the prion protein.

The prion protein (PrP) has been identified as a metalloprotein capable of binding multiple copper ions and possibly zinc. Recent studies now indicate that prion self-recognition may be an important factor in both the normal function and misfunction of this protein. We have developed fluorescently labeled models of the prion protein that allow prion-prion interactions and metal binding to be investigated on the molecular level.

Purification and characterization of the central segment of prothymosin-alpha: methodology for handling highly acidic peptides.

Prothymosin-alpha is a highly acidic protein consisting of 110 amino acids. The central segment of this protein, residues 51-89, is thought to be involved in metal binding which may be necessary for its physiological function. To carry out studies of this peptide, this central segment was synthesized in a linear fashion using Fmoc-based methods on rink amide MBHA resin.

Copper coordination in the full-length, recombinant prion protein.

The prion protein (PrP) binds divalent copper at physiologically relevant conditions and is believed to participate in copper regulation or act as a copper-dependent enzyme. Ongoing studies aim at determining the molecular features of the copper binding sites. The emerging consensus is that most copper binds in the octarepeat domain, which is composed of four or more copies of the fundamental sequence PHGGGWGQ.

Molecular features of the copper binding sites in the octarepeat domain of the prion protein.

Recent evidence suggests that the prion protein (PrP) is a copper binding protein. The N-terminal region of human PrP contains four sequential copies of the highly conserved octarepeat sequence PHGGGWGQ spanning residues 60-91. This region selectively binds Cu2+ in vivo.