Disruption of zinc (II) binding and dimeric protein structure of the XIAP-RING domain by copper (I) ions.

Modulation of metalloprotein structure and function via metal ion substitution may constitute a molecular basis for metal ion toxicity and/or metal-mediated functional control. The X-linked Inhibitor of Apoptosis Protein (XIAP) is a metalloprotein that requires zinc for proper structure and function. In addition to its role as a modulator of apoptosis, XIAP has been implicated in copper homeostasis. Given the similar coordination preferences of copper and zinc, investigation of XIAP structure and function upon interaction with copper is relevant. The Really Interesting New Gene (RING) domain of XIAP is representative of a class of zinc finger proteins that utilize a bi-nuclear zinc-binding motif to maintain proper structure and ubiquitin ligase function. Herein, we report the characterization of copper (I) binding to the Zn2-RING domain of XIAP. Electronic absorption studies that monitor copper-thiolate interactions demonstrate that the RING domain of XIAP binds 5-6 Cu(I) ions and that copper is thermodynamically preferred relative to zinc. Repetition of the experiments in the presence of the Zn(II)-specific dye Mag-Fura2 shows that Cu(I) addition results in Zn(II) ejection from the protein, even in the presence of glutathione. Loss of dimeric structure of the RING domain, which is a requirement for its ubiquitin ligase activity, upon copper substitution at the zinc-binding sites, was readily observed via size exclusion chromatography. These results provide a molecular basis for the modulation of RING function by copper and add to the growing body of literature that describe the impact of Cu(I) on zinc metalloprotein structure and function.

Emerging investigator series: characterization of silver and silver nanoparticle interactions with zinc finger peptides.

In biological systems, chemical and physical transformations of engineered silver nanomaterials (AgENMs) are mediated, in part, by proteins and other biomolecules. Metalloprotein interactions with AgENMs are also central in understanding toxicity and antimicrobial and resistance mechanisms. Despite their readily available thiolate and amine ligands, zinc finger (ZF) peptides have thus far escaped study in reaction with AgENMs and their Ag(I) oxidative dissolution product. We report spectroscopic studies that characterize AgENM and Ag(I) interactions with two ZF peptides that differ in sequence, but not in metal binding ligands: the ZF consensus peptide CP-CCHC and the C-terminal zinc finger domain of HIV-1 nucleocapsid protein p7 (NCp7_C). Both ZF peptides catalyze AgENM (10 and 40 nm, citrate coated) dissolution and agglomeration, two important AgENM transformations that impact bioreactivity. AgENMs and their oxidative dissolution product, Ag(I)(aq), mediate changes to ZF peptide structure and metalation as well. Spectroscopic titrations of Ag(I) into apo-ZF peptides show an Ag(I)-thiolate charge transfer band, indicative of Ag(I)-ZF binding. Fluorescence studies of the Zn(II)-NCp_7 complex indicate that the Ag(I) also effectively competes with the Zn(II) to drive Zn(II) displacement from the ZFs. Upon interaction with AgENMs, Zn(II) bound ZF peptides show a secondary structural change in circular dichroism spectroscopy toward an apo-like structure. The results suggest that Ag(I) and AgENMs may alter ZF protein function within the cell.

An Artificial Heme Enzyme for Cyclopropanation Reactions.

An artificial heme enzyme was created through self-assembly from hemin and the lactococcal multidrug resistance regulator (LmrR). The crystal structure shows the heme bound inside the hydrophobic pore of the protein, where it appears inaccessible for substrates. However, good catalytic activity and moderate enantioselectivity was observed in an abiological cyclopropanation reaction. We propose that the dynamic nature of the structure of the LmrR protein is key to the observed activity. This was supported by molecular dynamics simulations, which showed transient formation of opened conformations that allow the binding of substrates and the formation of pre-catalytic structures.

Cu(I) Disrupts the Structure and Function of the Nonclassical Zinc Finger Protein Tristetraprolin (TTP).

Tristetraprolin (TTP) is a nonclassical zinc finger (ZF) protein that plays a key role in regulating inflammatory response. TTP regulates cytokines at the mRNA level by binding to AU-rich sequences present at the 3'-untranslated region, forming a complex that is then degraded. TTP contains two conserved CCCH domains with the sequence CysX8CysX5CysX3His that are activated to bind RNA when zinc is coordinated. During inflammation, copper levels are elevated, which is associated with increased inflammatory response. A potential target for Cu(I) during inflammation is TTP. To determine whether Cu(I) binds to TTP and how Cu(I) can affect TTP/RNA binding, two TTP constructs were prepared. One construct contained just the first CCCH domain (TTP-1D) and serves as a peptide model for a CCCH domain; the second construct contains both CCCH domains (TTP-2D) and is functional (binds RNA) when Zn(II) is coordinated. Cu(I) binding to TTP-1D was assessed via electronic absorption spectroscopy titrations, and Cu(I) binding to TTP-2D was assessed via both absorption spectroscopy and a spin filter/inductively coupled plasma mass spectrometry (ICP-MS) assay. Cu(I) binds to TTP-1D with a 1:1 stoichiometry and to TTP-2D with a 3:1 stoichiometry. The CD spectrum of Cu(I)-TTP-2D did not exhibit any secondary structure, matching that of apo-TTP-2D, while Zn(II)-TTP-2D exhibited a secondary structure. Measurement of RNA binding via fluorescence anisotropy revealed that Cu(I)-TTP-2D does not bind to the TTP-2D RNA target sequence UUUAUUUAUUU with any measurable affinity, while Zn(II)-TTP-2D binds to this site with nanomolar affinity. Similarly, addition of Cu(I) to the Zn(II)-TTP-2D/RNA complex resulted in inhibition of RNA binding. Together, these data indicate that, while Cu(I) binds to TTP-2D, it does not result in a folded or functional protein and that Cu(I) inhibits Zn(II)-TTP-2D/RNA binding.

Copper-binding properties of the BIR2 and BIR3 domains of the X-linked inhibitor of apoptosis protein.

The X-linked inhibitor of apoptosis protein (XIAP) is a zinc metalloprotein that has recently been implicated in copper homeostasis. XIAP mediates apoptosis via the inhibition of caspase enzymes through multiple baculovirus IAP repeat (BIR) domains, wherein zinc is coordinated by three cysteine amino acids and one histidine amino acid. XIAP binds copper ions directly at one or more unspecified sites, indicating that the protein may function as a copper sensor. We report the copper-binding properties of an XIAP construct containing the BIR2 and BIR3 domains. Absorption and emission spectroscopic measurements show that XIAP exhibits only a low-to-moderate affinity for Cu(II), but a strong affinity for Cu(I). Cu(I) is observed to bind at multiple sites within the BIR2 and BIR3 domains, including the CXXC motifs of the zinc structural sites and multiple BIR2 surface sites. Mutagenesis-based experiments reveal that surface cysteine residues mediate binding in the BIR2 domain and induce protein oligomerization under elevated copper concentrations. These results constitute the first spectroscopic evidence of copper-XIAP interactions.

Spectroscopic characterization of copper(I) binding to apo and metal-reconstituted zinc finger peptides.

Cu(I) exhibits high affinity for thiolate ligands, suggesting that thiol-rich zinc or iron binding sites may be subject to disruption during copper stress conditions. Zinc fingers constitute a large class of metalloproteins that use a combination of cysteine and histidine residues that bind Zn(II) as a structural element. Despite the shared preference of both copper and zinc for thiolate and amine coordination, the susceptibility of zinc finger domains toward copper substitution is not well studied. We report spectroscopic studies that characterize the Cu(I) binding properties of the zinc finger consensus peptides CP-CCHH, CP-CCHC, and CP-CCCC and the C-terminal zinc finger domain of HIV-1 nucleocapsid protein p7 (NCp7_C). Cu(I) binds to both the apopeptides and the Co(II)-substituted peptides, and the stoichiometry of Cu(I) binding is dependent on the number of cysteine thiols at the metal binding site. Fluorescence studies of the Zn(II)-NCp7_C complex indicate that Cu(I) also effectively competes with Zn(II) at the metal binding site, despite the high affinity of Zn(II) for the CCHC binding motif. Circular dichroism studies on both CP-CCHC and NCp7_C show that the conformations of the Cu(I)-bound complexes differ substantially from those of the Zn(II) species, implying that Cu(I) substitution is likely to impact zinc finger function. These results show that for the peptides studied here, Cu(I) is the thermodynamically favored metal despite the known high Zn(II) affinity of zinc finger domains, suggesting that Cu(I)-substituted zinc finger domains might be relevant in the context of both copper toxicity mechanisms and copper-responsive transcription factors.

Characterization of a heterodimeric Smac-based peptide that features sequences specific to both the BIR2 and BIR3 domains of the X-linked inhibitor of apoptosis protein.

XIAP, an important regulator of apoptosis, has emerged as a target for the development of cancer therapeutics. The homodimeric Smac protein simultaneously binds to both the BIR2 and BIR3 domains of XIAP. Peptide-based dimeric compounds that mimic the binding mode of Smac show promise as XIAP antagonists. Herein we characterize the first example of a Smac mimetic that incorporates a peptide sequence specific for BIR2. We show that the tetrapeptide motif Ala-Glu-Ala-Val has a higher affinity for BIR2 than the BIR3-specific sequence Ala-Val-Pro-Phe, and we compare the binding characteristics of a heterodimeric peptide containing both tetrapeptide motifs to those of a homodimeric peptide featuring only AVPF. Despite the enhanced affinity of AEAV (relative to AVPF) for BIR2, the heterodimeric peptide displays only a slightly higher affinity for XIAP relative to its homodimeric counterpart. Enhanced affinity of both dimers relative to the tetrapeptide AVPF is largely maintained even when the BIR2 binding groove is modified, implying that hydrophobic contacts afforded by the second peptide motif need not necessarily be made at the BIR2 binding groove to contribute substantial binding energy. Finally, we use mutagenesis to show that the difference in sequence specificity observed between the two domains is primarily owing to steric bulk introduced at the BIR2 site by lysine 206. Replacement of K206 at BIR2 with glycine, the corresponding residue in BIR3, restores the majority of the affinity for the AVPF motif exhibited by BIR3. The implications of these finding in the development of XIAP antagonists are discussed. © 2011 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 98: 122-130, 2012.

Photophysical investigation of neutral and diprotonated free-base bis(arylethynyl)porphyrins.

The photophysical properties for a series of free-base arylethynyl porphyrins and the corresponding trans-disubstituted tetraphenylporphyrin (H(2)TPP) derivatives lacking arylethynyl functionalities have been studied via electronic absorption and emission spectroscopy in both neutral and diacid forms. Enhanced substituent effects on porphyrin absorption spectra are observed in the arylethynyl porphyrins relative to the H(2)TPP derivatives, owing to the presence of the ethynyl spacer that allows for a coplanar geometry between the porphyrin macrocycle and the appended phenyl substituents. Upon protonation, both series of porphyrins exhibit substantially red shifted absorption and emission spectra and enhanced oscillator strengths, with the magnitude of the spectral shifts being more substantial in the presence of the ethynyl functionalities. Spectral features of the arylethynyl porphyrin bearing p-dimethylamino substituents closely resemble those previously classified as "hyperporphyrin spectra" and are indicative of excited-state charge-transfer character. Protonation of both series of porphyrins results in reduced fluorescence lifetimes and enhanced nonradiative decay rates, and the impact of protonation on these parameters is attenuated in the presence of the arylethynyl functionalities. Our results coupled with previous structural data showing that arylethynyl porphyrins exhibit less structural distortion upon diacid formation relative to H(2)TPP further substantiate the proposal that significant alteration of porphyrin photophysical properties upon diacid formation can be attributed to nonplanar structural distortions induced by protonation.

In vitro assays for the determination of aminoacyl-tRNA synthetase editing activity.

Aminoacyl-tRNA synthetases are essential enzymes that help to ensure the fidelity of protein translation by accurately aminoacylating (or "charging") specific tRNA substrates with cognate amino acids. Many synthetases have an additional catalytic activity to confer amino acid editing or proofreading. This activity relieves ambiguities during translation of the genetic code that result from one synthetase activating multiple amino acid substrates. In this review, we describe methods that have been developed for assaying both pre- and post-transfer editing activities. Pre-transfer editing is defined as hydrolysis of a misactivated aminoacyl-adenylate prior to transfer to the tRNA. This reaction has been reported to occur either in the aminoacylation active site or in a separate editing domain. Post-transfer editing refers to the hydrolysis reaction that cleaves the aminoacyl-ester linkage formed between the carbonyl carbon of the amino acid and the 2' or 3' hydroxyl group of the ribose on the terminal adenosine. Post-transfer editing takes place in a hydrolytic active site that is distinct from the site of amino acid activation. Here, we focus on methods for determination of steady-state reaction rates using editing assays developed for both classes of synthetases.

Transfer RNA modulates the editing mechanism used by class II prolyl-tRNA synthetase.

Aminoacyl-tRNA synthetases catalyze the attachment of amino acids to their cognate tRNAs. To prevent errors in protein synthesis, many synthetases have evolved editing pathways by which misactivated amino acids (pre-transfer editing) and misacylated tRNAs (post-transfer editing) are hydrolyzed. Previous studies have shown that class II prolyl-tRNA synthetase (ProRS) possesses both pre- and post-transfer editing functions against noncognate alanine. To assess the relative contributions of pre- and post-transfer editing, presented herein are kinetic studies of an Escherichia coli ProRS mutant in which post-transfer editing is selectively inactivated, effectively isolating the pre-transfer editing pathway. When post-transfer editing is abolished, substantial levels of alanine mischarging are observed under saturating amino acid conditions, indicating that pre-transfer editing alone cannot prevent the formation of Ala-tRNA Pro. Steady-state kinetic parameters for aminoacylation measured under these conditions reveal that the preference for proline over alanine is 2000-fold, which is well within the regime where editing is required. Simultaneous measurement of AMP and Ala-tRNA Pro formation in the presence of tRNA Pro suggested that misactivated alanine is efficiently transferred to tRNA to form the mischarged product. In the absence of tRNA, enzyme-catalyzed Ala-AMP hydrolysis is the dominant form of editing, with "selective release" of noncognate adenylate from the active site constituting a minor pathway. Studies with human and Methanococcus jannaschii ProRS, which lack a post-transfer editing domain, suggest that enzymatic pre-transfer editing occurs within the aminoacylation active site. Taken together, the results reported herein illustrate how both pre- and post-transfer editing pathways work in concert to ensure accurate aminoacylation by ProRS.

Biochemical basis for enhanced binding of peptide dimers to X-linked inhibitor of apoptosis protein.

XIAP (X-linked inhibitor of apoptosis protein) is involved in the mediation of programmed cell death and, therefore, is a target for the development of cancer therapeutics. Peptide mimetics based upon Smac, the natural binding partner of XIAP, and specifically, dimeric peptides, have shown great promise in drug development. In the present work, the basis for enhanced dimer efficacy has been explored. Comparisons are made between the peptide binding site on the BIR3 domain of XIAP alone (residues 238-358) and a less truncated construct that includes both BIR2 and BIR3 domains (residues 151-350). This contingency differentially enhances the binding of dimeric tetrapeptides, potentially by providing additional hydrophobic binding surface. The effect of BIR2 on the BIR3 binding site is sustained, even if the BIR2 binding site is disrupted by mutagenesis, as shown by both a fluorescent competition assay and a polarity sensitive dye, badan. FRET measurements reveal an observed separation of >or=45 A between the BIR2 and BIR3 peptide binding pockets, thereby precluding a direct simultaneous interaction of the dimer molecules with both binding domains. Furthermore, variations in the linker length between dimeric tetrapeptides did not show a predictable trend in binding affinities, suggesting that local concentration effects were also an unlikely explanation for the enhanced dimeric affinities. Taken together, the results suggest that enhanced binding of dimeric peptides likely reflects the increased hydrophobic surface area on or near the BIR3 site and have significant ramifications for the design of therapeutics that target this class of proteins.

Permeable nonaggregating porphyrin thin films that display enhanced photophysical properties.

Porphyrins bearing bulky alkoxyphenyl substituents at two of the four meso-positions and phenyl phosphonates at the other two have been prepared and used as building blocks for layer-by-layer assembly of conductive-glass-supported thin films via zirconium phosphonate chemistry. Thin-film characterization shows that the addition of sterically demanding 2,6-di(n-hexoxy)phenyl substituents to the meso-positions of the porphyrin skeleton can successfully prevent molecular aggregation. Both absorption and emission studies of multilayer thin films provide strong evidence that the new compounds have the ability to form thin films in which very little molecular (chromophore) interaction is present, relative to porphyrins that are not sterically hindered. Furthermore, the films are found to be permeable to selected small redox probes but blocking toward larger ones. Taken together, the sharp absorption spectra, increased emission yields, and permeability are expected to be advantageous for various materials-based applications such as photovoltaics and sensors.

Analysis of molecular square size and purity via pulsed-field gradient NMR spectroscopy.

The size (volume) of a large tetrametallic molecular square, that has resisted characterization by mass spectrometry, has been determined by pulsed-field gradient NMR spectroscopy, a technique that reports on self-diffusion coefficients. These scale inversely with hydrodynamic radii, which in turn scale approximately as the cube of the assembly's mass. The technique has also been used to determine whether NMR spectral complexities observed for the new compound are due to contamination with chemically related assemblies, or instead reflect the intrinsic structural complexities of the compound itself.

Synthesis, characterization, and preliminary intramolecular energy transfer studies of rigid, emissive, rhenium-linked porphyrin dimers.

The reaction of pyridyl functionalized porphyrins with Re(CO)(5)Cl in THF results in the formation of porphyrin dimers which, despite incorporation of rhenium into the assemblies, remain fluorescent. The rigid compounds provide an efficient geometry and/or orbital pathway for singlet energy transfer, rendering these compounds suitable, in principle, for the study of both through-bond and through-space energy transfer. Derivatives containing both metallated and freebase porphyrins connected via the metal corners display efficient porphyrin-porphyrin energy transfer. The photophysical properties of the assemblies have been studied by both steady-state and time-resolved fluorescence techniques, yielding approximate rates and efficiencies for porphyrin-porphyrin energy transfer.