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This paper describes the design, characterization, and metal-binding properties of a 32-residue polypeptide called AQ-C16C19. The sequence of this peptide is composed of four repeats of the seven residue sequence Ile-Ala-Ala-Leu-Glu-Gln-Lys but with a Cys-X-X-Cys metal-binding motif substituted at positions 16-19. Size exclusion chromatography with multiangle light scattering detection (SEC-MALS) and circular dichroism (CD) spectroscopy studies showed that the apo peptide exhibits a pH-dependent oligomerization state in which a three-stranded α-helical coiled coil is dominant between pH5.4 and 8.5. The Cd(2+)-binding properties of the AQ-C16C19 peptide were studied by ultraviolet-visible spectroscopy (UV-vis), electrospray ionization mass spectrometry (ESI MS), and (113)Cd NMR techniques. The holoprotein was found to contain a polynuclear cadmium-thiolate center formed within the hydrophobic core of the triple-stranded α-helical coiled-coil structure. The X-ray crystal structure of the Cd-loaded peptide, resolved at 1.85A resolution, revealed an adamantane-like configuration of the polynuclear metal center consisting of four cadmium ions, six thiolate sulfur ligands from cysteine residues and four oxygen-donor ligands. Three of these are from glutamic acid residues and one is from an exogenous water molecule. Thus, each cadmium ion is coordinated in a distorted tetrahedral S(3)O geometry. The metal cluster was found to form cooperatively at pH5.4 but in a stepwise fashion at pH>7. The results demonstrate that synthetic coiled-coils can be designed to incorporate multinuclear metal clusters, a proof-of-concept for their potential use in developing synthetic metalloenzymes and multi-electron redox agents.

The Cu(I) binding properties of the designed peptide C16C19-GGY are reported. This peptide was designed to form an alpha-helical coiled-coil but modified to incorporate a Cys-X-X-Cys metal-binding motif along its hydrophobic face. Absorption, emission, electrospray ionization mass spectrometry (ESI-MS), and circular dichroism (CD) experiments show that a 1:1 Cu-peptide complex is formed when Cu(I) is initially added to a solution of the monomeric peptide. This is consistent with our earlier study in which the emissive 1:1 complex was shown to exist as a peptide tetramer containing a tetranuclear copper cluster Kharenko et al. (2005) [11]. The presence of the tetranuclear copper center is now confirmed by ESI-MS which along with UV data show that this cluster is formed in a cooperative manner. However, spectroscopic titrations show that continued addition of Cu(I) results in the occupation of a second, lower affinity metal-binding site in the metallopeptide. This occupancy does not significantly affect the conformation of the metallopeptide but does result in a quenching of the 600nm emission. It was further found that the exogenous reductant tris(2-carboxyethyl)phosphine (TCEP) can competitively inhibit the binding of Cu(I) to the low affinity site of the peptide, but does not interact with Cu(I) clusters.

The polypeptide H21(30-mer) folds into a two-stranded coiled-coil in which two solvent-exposed histidine residues reside on opposite sides of its surface. This peptide was allowed to react with cobalt(III) protoporphyrin IX, Co(ppIX), to produce [Co(ppIX){(H21(30-mer)}2], as determined by UV-Vis spectroscopy. This bis-axial ligation thus positions a potential coiled-coil oligomerization domain onto each face of the cobalt porphyrin ring. Circular dichroism spectroscopy and high performance size exclusion chromatography provide evidence for the solution-phase self-assembly of these porphyrin-peptide units. Evaporation of the porphyrin-peptide solution on a solid support results in the formation of long rod-like materials having millimeter-scale lengths and micron-scale diameters. The presence of Co(ppIX) in these materials was confirmed by Raman microscopy. However, they were formed only from phosphate buffer, and not from organic buffers or pure water, indicating that their formation might involve a more complicated process than originally anticipated.

Two amphipathic polypeptides were coordinated to the cis positions of a square planar Pt(II) complex in order to provide the metal center with two noncovalent oligomerization domains. This resulted in the formation of new metal-peptide nanoassemblies which are shown to exist as nanometer-sized spheres and fibrils. Construction of these assemblies was based on the 30-residue polypeptide AQ-Pal14 which was designed for its ability to self-assemble into the common protein oligomerization motif of a noncovalent coiled-coil, and modified to contain a metal-binding 4-pyridylalanine residue at its surface. When AQ-Pal14 was reacted with Pt(en)(NO 3) 2, a new metal-peptide complex was formed in which two AQ-Pal14 peptides were coordinated to a single metal center as determined by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electrospray ionization mass spectrometry (ESI-MS). When the reaction mixture was analyzed under nondenaturing conditions by high performance size exclusion chromatography (HPSEC), it was found that all species present eluted at the column void volume, indicating the formation of very large metal-peptide assemblies. This was verified by multiangle light scattering (MALS) which showed that the metal-peptide assemblies have a weight-averaged molecular mass and z-average root-mean-square radius of Mw = (7 +/- 4) x 10 (6) g/mol and Rz = 18 +/- 4 nm, respectively. The presence of such nanometer scale assemblies was confirmed by transmission electron microscopy and atomic force microscopy which showed the existence of both spherical and fibrillar nanostructures.

The emerging field of metalloprotein design seeks to prepare artificial proteins whose properties can mimic, enhance, and perhaps improve upon many features found in natural metalloenzymes. This review summarizes our recent efforts to prepare synthetic metalloproteins built from alpha-helical coiled-coils and to incorporate electron-transfer functionality within these systems. We have recently designed a cysteine-containing random-coil peptide which forms a alpha-helical coiled-coil upon binding various metals. The CuI adduct can serve as photoinduced electron-transfer agent to exogenous acceptors and undergoes collisional electron-transfer in the inverted Marcus region to various ruthenium ammine acceptors. It is speculated that this unexpected result might be due to the positioning of the CuI cofactor within the hydrophobic core of the protein which prohibits close approach between the donor and acceptor to slow the high driving force reaction rates below the diffusion limit. O campo emergente da engenharia molecular de metaloproteínas visa preparar proteínas artificiais, cujas propriedades podem imitar e talvez até mesmo melhorar várias características encontradas nas metaloenzimas naturais. Este artigo de revisão resume nossos esforços recentes na preparação de metaloproteínas sintéticas, construídas a partir de "coiled-coils" alfa-hélices, e na incorporação de grupos de transferência de elétrons nesses sistemas. Recentemente, concebemos uma cisteína contendo um peptídeo com hélice randômica, o qual forma uma estrutura "coiled-coil" alfa-helicoidal ao se ligar a vários metais. O aduto de CuI pode atuar como agente fotoindutor de transferência de elétrons para receptores exógenos, e transfere elétrons por colisão na região invertida de Marcus para várias aminas de rutênio, as quais atuam como receptores. Especula-se que este resultado inesperado advenha do posicionamento do cofator de CuI no interior da porção hidrofóbica da proteína, o qual proíbe a aproximação entre o doador e o receptor, diminuindo a velocidade de transferência eletrônica daquelas reações termodinamicamente muito favorecidas, para velocidades inferiores à do limite difusional.

The metal-induced assembly of a designed peptide-based rubredoxin model is described. The C16C19-GGY peptide has the sequence Ac-K(IEALEGK)2(CEACEGK)(IEALEGK)GGY-amide in which the presence of the Cys–X–X–Cys metal binding domain of rubredoxin was used to place cysteine residues at the hydrophobic “a” and “d” positions upon formation of a homodimeric α-helical coiled-coil. Circular dichroism spectroscopy shows that the apopeptide exists as a random coil and assembles into a coiled-coil in the presence of Cd2+. Metal binding is monitored by the appearance of a new LMCT band at 238 nm. UV–Vis titrations and SDS–PAGE experiments are used to show that this designed metalloprotein exists as a metal-bridged coiled-coil dimer.

Photoinduced electron-transfer (ET) occurs between a negatively charged metallopeptide, [Ru(bpy)(2)(phen-am)-Cys-(Glu)(5)-Gly](3-) = RuCE(5)G, and ferricytochrome c = Cyt c. In the presence of Cyt c, the triplet state lifetime of the ruthenium metallopeptide is shortened, and the emission decays via biexponential kinetics, which indicates the existence of two excited-state populations of ruthenium peptides. The faster decay component displays concentration-independent kinetics demonstrating the presence of a preformed peptide-protein complex that undergoes intra-complex electron-transfer. Values of K(b) = (3.5 +/- 0.2) x 10(4) M(-1) and k(obs)(ET)= (2.7 +/- 0.4) x 10(6) s(-1) were observed at ambient temperatures. The magnitude of k(obs)(ET) decreases with increasing solvent viscosity, and the behavior can be fit to the expression k(obs)(ET) proportional to eta(-alpha) to give alpha = 0.59 +/- 0.05. The electron-transfer process occurring in the preformed complex is therefore gated by a rate-limiting configurational change of the complex. The slower decay component displays concentration-dependent kinetics that saturate at high concentrations of Cyt c. Analysis according to rapid equilibrium formation of an encounter complex that undergoes unimolecular electron-transfer yields K(b)' = (2.5 +/- 0.7) x 10(4) M(-1) and k(obs')(ET)= (7 +/- 3) x 10(5) s(-1). The different values of k(obs)(ET) and k(obs')(ET) suggest that the peptide lies farther from the heme when in the encounter complex. The value of k(obs')(ET) is viscosity dependent indicating that the reaction occurring within the encounter complex is also configurationally gated. A value of alpha = 0.98 +/- 0.14 is observed for k(obs')(ET), which suggests that the rate-limiting gating processes in the encounter complex is different from that in the preformed complex.

A 30-residue polypeptide [H21(30-mer)] with the sequence Ac-K(IEALEGK)2(IEALEHK)(IEALEGK)G-NH2 was synthesized. The circular dichroism (CD) spectrum of the peptide shows minima at 208 and 222 nm and θ222/θ208 = 1.06, which indicates the formation of a self-assembled coiled-coil when dissolved in aqueous solution. The concentration dependence of the CD data can be fit to an expression that describes a two-state monomer−dimer equilibrium for the apopeptide (Kd = 1.5 ± 0.4 μM and θmax = −23 800 ± 130 deg cm2 dmol-1), showing that it has a maximum helicity of 69%. A [MTSL-C21(30-mer)] dimer was also prepared in which MTSL is the thiol-specific nitroxide spin label 1-oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl-methanethiosulfonate attached to C21 of the 30-mer. Fourier deconvolution analysis of the dipolar line broadening of the electron paramagnetic resonance (EPR) spectrum yields a measure of the interchain CαCα distance of 13.5 ± 0.9 A at position 21 of the coiled-coil, which is nearly identical to those...

A chimeric metallo-bZIP protein, (GBR-CC)Ru, was prepared that contains the native DNA-binding domain of the GCN-4 transcription factor (GBR), a synthetic α-helical coiled-coil dimerization site (CC), and a ruthenium polypyridyl complex (Ru) attached to a surface-exposed cysteine residue. The photophysical properties of the peptide-bound ruthenium complex include a long-lived emission that is shortened when the peptide is dissolved in air-saturated water. Electrophoretic mobility shift assays show that the chimeric metalloprotein also retains the essential DNA recognition properties of the native GCN-4 transcriptional activator. Peptide–DNA complexes are formed with the related AP1 and CRE sequences, but not the divergent Sp1 sequence. Peptide titration studies indicate that the affinities of (GBR-CC)Ru for the AP1 and CRE sites are comparable. Steady-state photolysis experiments show that (GBR-CC)Ru does not produce photoinduced DNA damage, probably due to the separation distance between the ruthenium sites and the DNA bases.

Metal coordination is used to alter the oligomerization state of a designed peptide structure. The 30-residue polypeptide AQ-Pal14Pal21contains two metal-binding 4-pyridylalanine (Pal) residues on its solvent-exposed surface and exists as a very stable two-stranded alpha-helical coiled-coil. Upon the addition of Pt(en)(NO3)2, a significant conformational change to a metal-bridged, four-helix bundle is seen.

Photoinduced electron-transfer rate constants were measured for a series of binuclear metallopeptides consisting of a [Ru(bpy)2(cmbpy)]2+ electron donor tethered to a CoIII(NH3)5 electron acceptor ...

The metal-binding properties of rationally designed, synthetic proteins were used to prepare a series of emissive silver nanoclusters having predictable sizes and emission energies. Metal-binding α-helical coiled coils were designed to exist as peptide trimers, tetramers, and hexamers and found to uniquely bind 6, 8, and 12 Ag(+) ions, respectively. Subsequent treatment with a chemical reducing agent produced a series of peptide-bound Ag(0) nanoclusters that display a strong visible fluorescence whose emission energies depend on the number of bound metal ions in excellent agreement with theory.

A new mixed-ligand polypyridylruthenium(II) complex, [Ru(bpy)2L]Cl2, has been prepared where bpy = 2,2‘-bipyridine and L = 3,5-dicarboxy-2,2‘-bipyridine. The ligand L is a non-symmetrically-substituted 2,2‘-bipyridine having two hydrophilic carboxylate groups located at the 3- and 5-positions of only one of its two pyridyl rings. In acetonitrile, the photophysical properties of the metal complex include a long-lived excited state (λem = 637 nm, τ = 846 ± 11 ns, φ = 0.046 at 295 K) whose decay involves an activated crossing to higher energy ligand field states (Ea = 4170 ± 200 cm-1). This behavior is similar to that observed for other ruthenium tris(bipyridyl) compounds. In contrast, the title compound displays several unusual photophysical properties in aqueous solution. These include a strongly red-shifted emission (λem = 685 nm) having a short, pH-dependent lifetime which is quenched by an excited-state proton transfer from solvent. The completely deprotonated form of the molecule is the dominant emissi...

Intramolecular electron-transfer studies across a series of peptides ranging from dipeptides to longer peptides with secondary structure (such as polyproline II and a 17-amino acid α helix) have been carried out. Metal ammine and bipyridine complexes have been used as donors and acceptors in these studies. These studies show that the rate of electron transfer is sensitive to the peptide structure and conformation, even for dipeptide bridges. For peptides with secondary structure, the connectivity of the donor and acceptor to the peptide is also important for the observation of long-range electron transfer. For example, for (bpy)2RuIIL(Pro)n-apyRuIII(NH3)5 (n = 9) (bpy2,2′ bipyridine, L4-carboxy-4′-methyl-2,2′-bipyridine, apy-4-aminopyridine), an electron transfer rate 2 × 104 s−1 was observed, while intramolecular electron transfer could not be observed for the α helix bridge in (bpy)2RuIIL[α-helical peptide]-(His)2RuIII(NH3)4 (α-helical peptide  Ala-Glu-(Ala)3Lys-Glu(Ala) 3Lys-His(Ala)3His-Ala). Comparative intramolecular electron-transfer experiments were also conducted with two cytochrome c derivatives: one modified at His 33 by [-Ru(NH3)4isn] and one modified at Met 65 by [-Fe(CN)5]. Although the His 33 and Met 65 sites are located at similar distances from the heme, and the two metal complexes possess similar reorganization energies and driving force, different rates of electron transfer varying by about 1000 were observed for the electron transfer from the heme to the metal complex. The experiments presented show that the shortest through-space distance is not always the most important determinent of the rate of electron transfer, and other factors such as the peptide structure and conformation and the connectivity of the donor and acceptor to the peptide bridge are very important.

A series of complexes of the type [(bpy)[sub 2]Ru[sup II]L-(Pro)[sub n]-Co[sup III](NH[sub 3])[sub 5]][sup 4+], n = 1-6, where L = 4-carboxy-4[prime]-4-methyl-2,2[prime]-bipyridine, bpy = 4,4[prime]-bipyridine, and Pro = l-proline, have been synthesized from the corresponding [(bpy)[sup 2]Ru[sup II]L] and [(NH[sub 3])[sub 5]Co[sup III](Pro)[sub n]] components. The compounds were characterized by metal analyses, electrochemical measurements, and absorption spectroscopy. For n = 4-6 prolines, the CD spectra of the complexes show a proproline II helical structure. Intramolecular electron transfer within these complexes was studied by generating the [(bpy)[sub 2]Ru[sup II]L[sup [sm bullet]]-(Pro)[sub n]-Co[sup III](NH[sub 3])[sub 5]] intermediate by the reaction of e[sub aq] (generated by pulse radiolysis) with the [(bpy)[sub 2]Ru[sup II]L-(Pro)[sub n]-Co[sup III](NH[sub 3])[sub 5]] molecules. The driving force for this reaction is estimated to be [vert bar][Delta]G[sup [circ]][vert bar] [approximately] [minus]1.1 V. The rates of intramolecular electron transfer decrease rapidly with distance for n = 1-3 prolines but show a surprisingly weak decrease with distance for the n = 4, 5, and 6 prolines, which exhibit the polyproline II helical structure. 42 refs., 6 figs., 3 tabs.

A series of binuclear donor-acceptor complexes with helical polyproline bridges [(bpy) 2 Ru II L-(Pro) n -apy-Ru III (NH 3 ) 5 ] 5+ , n = 6, 7, 9, where L = 4-carboxy-4′-methyl-2,2′-bipyridine, bpy = 4,4′-bipyridine, and apy = 4-aminopyridine, were synthesized and characterized by absorption spectra, electrochemistry and HPLC. The CD spectra of the complexes confirm that they exist in the helical polyproline II structure. Intramolecular electron transfer within these complexes was studied by generating the [(bpy) 2 Ru I L · -(Pro) n -apy-Ru III (NH 3 ) 5 ] intermediate from the reaction of e aq (from pulse radiolysis) with the [(bpy) 2 Ru II L-(Pro) n -apy-Ru III (NH 3 ) 5 ] species in aqueous solution. The driving force for this reaction is estimated to be |Δ G 0 | ≈ 1.5 V. The rates ( k , 25°C) and activation parameters (Δ H ‡ (kcal/mol), Δ S ‡ (eu)) for the intra molecular electron transfer were found to be: 1.08×10 5 s −1 , 5.6, −17; 6.40 × 10 4 s −1 , 5.1, −19; 1.91 × 10 4 s −1 , 4.0, and −26 for n = 6, 7, 9 respectively. The rate ( k , 25°C) and activation parameters (Δ H ‡ (kcal/mol), Δ S ‡ (eu)) for the inter molecular reaction between [(bpy) 2 Ru I L · ] and [(NH 3 ) 5 Ru III -apy-Pro] were found to be 2.1 × 10 9 M −1 s −1 , 3.3 and −5. This series extends our studies of the distance dependence of rate versus the number of helical prolines bridging a donor and acceptor ruthenium site to a metal-to-metal distance ≈ 40 A. The weak dependence of rate versus the number of prolines observed for n = 6, 7, and 9 is very similar to that observed earlier for [(bpy) 2 Ru II L-(Pro) n -Co III (NH 3 ) 5 ], n = 4–6. The rapid rates observed at these long distances show that long range electron transfer can be observed between an appropriate donor and acceptor directly connected to the proline bridge via peptide bonds at distances similar to the diameter of a small protein.

The temperature dependence of the emission lifetimes of two structural isomers of an osmium polypyridyl complex are reported. The compounds [Os(bpy) 2 (4,4 ′ -dcbpy)] ( 1 ) and [Os(bpy) 2 (3,5-dcbpy)] ( 2 ), where 4,4 ′ -dcbpy=4,4 ′ -dicarboxy-2,2 ′ -bipyridine and 3,5-dcbpy=3,5-dicarboxy-2,2 ′ -bipyridine, differ only in the placement of their two hydrophilic carboxylate groups within the unique bipyridine ligand. In argon-saturated water, the emission life-time of 1 decreases with increasing temperature and can be fit to the Arrhenius expression (k obs =k 0 exp (−E a /RT)) to give values of k 0 =(2.6±0.3)×10 8 s −1 and E a =+330±20 cm −1 . In contrast the emission of 2 measured in ethanol displays longer lifetimes at higher temperatures to give k 0 =(2.9±0.3)×10 6 s −1 and E a =−590±20 cm −1 . Thus, the specific localization of the hydrophilic carboxylate groups in these molecules significantly alters their photophysical properties and can produce negatively activated emission lifetimes.

AQ-Pal14 is a 30-residue polypeptide that was designed to form an α-helical coiled coil which contains a metal-binding 4-pyridylalanine residue on its solvent-exposed surface. However, characterization of this peptide shows that it exists as a three-stranded coiled coil, and not a two-stranded one as predicted from its design. Reaction with cobalt(III) protoporphyrin IX (Co-PPIX) produces a six-coordinate Co-PPIX(AQ-Pal14)2 species which creates two coiled-coil oligomerization domains coordinated to opposite faces of the porphyrin ring. It is found that this species undergoes a buffer-dependent self-assembly process: nanometer-scale globular materials were formed when these components were reacted in unbuffered H2O, while millimeter-scale rod-like materials were prepared when the reaction was performed in phosphate buffer (20 mM, pH 7). It is suggested that assembly of the globular material is dictated by the conformational properties of the coiled-coil forming AQ-Pal14 peptide, whereas that of the rod-like material involves interactions between Co-PPIX and phosphate ion.

Electron transfer is one of the fundamental reactions in biological photosynthesis, respiration, and redox-mediated enzyme catalysis. Understanding the role of peptides and proteins in mediating long-range electron transfer has important physical, chemical, and biological implications. Intramolecular electron-transfer reactions, where electron transfer occurs between donor and acceptor sites separated by a synthetic peptide or protein fragment, have so far provided the most insight into the role of the peptide and protein in mediating electron transfer. This article reviews the work the authors' group and others on small molecule systems that have been designed and studied to explore long-range electron-transfer pathways available between a donor and an acceptor separated by a rigid peptide framework. The role of the secondary structure of polyproline II in facilitating these mediations is explored. 87 refs., 3 figs, 5 tabs.

Electron transfer is one of the fundamental reactions in biological photosynthesis, respiration, and redox-mediated enzyme catalysis. Understanding the role of peptides and proteins in mediating long-range electron transfer has important physical, chemical, and biological implications. Intramolecular electron-transfer reactions, where electron transfer occurs between donor and acceptor sites separated by a synthetic peptide or protein fragment, have so far provided the most insight into the role of the peptide and protein in mediating electron transfer. This article reviews the work the authors' group and others on small molecule systems that have been designed and studied to explore long-range electron-transfer pathways available between a donor and an acceptor separated by a rigid peptide framework. The role of the secondary structure of polyproline II in facilitating these mediations is explored. 87 refs., 3 figs, 5 tabs.

A two-stranded alpha-helical coiled coil was prepared having a Cys 4 metal-binding site within its hydrophobic interior. The addition of Cd2+ results in the incorporation of 2 equiv of metal ion, which is accompanied by a conformational change of the peptide, as observed by circular dichroism (CD) spectroscopy. Isothermal titration calorimetry (ITC) shows that the addition of Cd2+ is accompanied by two thermodynamic events. A comparison of the time dependence of the ITC behavior with those of the UV absorption and CD behavior allows the assignment of these events to a preliminary endothermic metal-binding step followed by a slower exothermic conformational change.

The α-helical coiled-coil motif serves as a robust scaffold for incorporating electron-transfer functionality into synthetic metalloproteins. These structures consist of a right-handed supercoiling of two or more α-helices that are formed by the self-assembly of individual polypeptide chains whose sequences contain a repeating pattern of hydrophobic and hydrophilic residues. Early work from our group attached abiotic ruthenium-based redox sites to the most surface-exposed positions of two stranded coiled-coils and used electron-pulse radiolysis to study both intra and inter-molecular electron-transfer reactions in these systems. Later work used smaller metallopeptides to investigate the effects of conformational gating within electrostatic peptide-protein complexes. We have recently designed the C16C19-GGY peptide which contains cysteine residues located at both the “a” and “d” positions of its third heptad repeat in order to construct a native-like metal-binding domain within its hydrophobic core. It was shown that the binding of both Cd(II) and Cu(I) ions induces the peptide to undergo a conformational change from a disordered random-coil to a metal-bridged coiled-coil. However, whereas the Cd(II) protein exists as a two-stranded coiled-coil, the Cu(I) derivative exists as a four-stranded coiled-coil. Upon the incorporation of other metal ions, metal-bridged peptide dimers, tetramers, and hexamers are formed. The Cu(I)-protein is of particular interest as it exhibits a long-lived (microsecond) room-temperature luminescence at 600 nm. The luminophore in this protein is thought to be a multinuclear CuI4Cys4(N/O)4 cage complex which can be quenched by exogenous electron-acceptors in solution, as shown by emission lifetime and transient absorption experiments. It is anticipated that further investigation into these systems will contribute to the expanding effort of bioinorganic chemists to prepare new kinds of functionally-active synthetic metalloproteins.

A new class of metal-peptide nanoassemblies has been prepared by combining the principles of supramolecular coordination chemistry with those of de novo protein design.

Gated electron-transfer measurements are used to probe the configurational dynamics of complexes formed between small metallopeptides and cytochrome c. The results show that that an apparently subtle chemical alteration of the metallopeptide produces significant changes to the dynamics of the peptide-protein complex.

A peptide-based electron-transfer system has been designed in which the specific positions of redox-active metal complexes appended to either an alpha-helix, or an alpha-helical coiled-coil, can be reversed to test the effect of the helix dipole in controlling photoinduced electron-transfer rates. Two 30-residue apopeptides were prepared having the following sequences: (I) Ac-K-(IEALEGK)(ICALEGK)(IEALEHK)(IEALEGK)-G-amide, and (II) Ac-K-(IEALEGK)(IHALEGK)-(IEALECK)(IEALEGK)-G-amide. Each apopeptide was reacted first with [Ru(bpy)2(phen-ClAc)]2+, where bpy = 2,2'-bipyridine and phen-ClAc = 5-chloroacetamido-1,10-phenanthroline, to attach the ruthenium polypyridyl center to the cysteine side-chain of the polypeptide. The isolated products were then reacted with [Ru(NH3)5(H2O)]2+ to yield the binuclear electron-transfer metallopeptides ET-I and ET-II. In these systems, electron-transfer occurred from the photoexcited ruthenium polypyridyl donor to the pentammine ruthenium (III) acceptor such that the electron-transfer occurred toward the negative end of the helix dipole in ET-I, and toward the positive end in ET-II. Circular dichroism spectroscopy showed that both peptides exist as dimeric alpha-helical coiled-coils in 100 mM phosphate buffer at pH 7, and as monomeric alpha-helices in the lower dielectric solvents 2,2,2-trifluoroethanol, and a 1:1 (v/v) mixture of CH2Cl2 and 2,2,2-trifluoroethanol. The peptides were predominately (i.e., 65-72%) alpha-helical in these solvents. The emission lifetime behavior of ET-I was seen to be identical to that of ET-II in each of the three solvents: no evidence for directional electron-transfer rates was observed. Possible reasons for this behavior are discussed.

A stepwise procedure for preparing of site-specific binuclear metallopeptides is described. The modification procedure involves the alkylation of a cysteine side chain by reaction with [Ru(bpy)(2)(phen-ClA)](2+), where bpy = 2,2'-bipyridine and phen-ClA = 5-chloroacetamido-1,10-phenanthroline, followed by the coordination of a ruthenium pentammine complex to a histidine residue located elsewhere along the sequence. The apo and metalated versions of the peptides C10H21(30-mer) and H10C21(30-mer) display circular dichroism spectra having minima at 208 and 222 nm, with theta(222)/theta(208) = 1.04 to indicate that these peptides exist as alpha-helical coiled-coils in aqueous solution. When the ruthenium polypyridyl complex is attached to C10H21(30-mer), the Delta-l and Lambda-l diastereomers of the resulting metallopeptide can be readily separated from each other by reversed-phase HPLC. However, in the case of the related H10C21(30-mer) metallopeptide, the two diastereomers cannot be chromatographically resolved. These results indicate how the subtle interplay between peptide conformation/sequence and metal complex geometry may alter some of the physical characteristics of metallopeptides.

A de novo designed coiled-coil metalloprotein was prepared that uses electrostatic interactions to control both its conformational and bimolecular electron-transfer properties. The title protein exists as a coiled-coil heterodimer of the [Ru(trpy)(bpy)-KK(37-mer)] and [Ru(NH(3))(5)-EE(37-mer)] polypeptides which is formed by interhelix electrostatic attractions. Circular dichroism studies show that the electrostatic heterodimer has K(d) = 0.19 +/- 0.03 microM and is 96% helical at high concentrations. Intercomplex electron-transfer reactions were studied that involve the [Ru(NH(3))(5)-H21](2+) electron-donor and the [Ru(trpy)(bpy)-H21](3+) electron-acceptor belonging to different electrostatic dimers. An important feature of the designed metalloprotein is its two cationic redox centers embedded within protein surfaces having opposite charge. Thus, the Ru(II)(NH(3))(5)-H21 site was placed on the surface of one chain of the coiled-coil which was made to be positively charged, and the Ru(III)(trpy)(bpy)-H21 site was placed on the surface of the other chain which was negatively charged. The rates of intermolecular electron-transfer increased from (1.9 +/- 0.4) x 10(7) M(-1) s(-1) to (3.7 +/- 0.5) x 10(7) M(-1) s(-1) as the ionic strength was increased from 0.01 to 0.20 M. This indicates that the electrostatic repulsion between the ruthenium centers dominates the kinetics of these reactions. However, the presence of the oppositely charged protein surfaces in the coiled-coils creates an electrostatic recognition domain that substantially ameliorates the effects of this repulsion.

The addition of Cu(I) to the random-coil peptide, C16C19-GGY, produces a self-organized, metal-bridged 4-helix bundle which displays an intense room-temperature luminescence at 600 nm. Emission, UV, and CD titrations along with X-ray absorption studies indicate that the luminescent cofactor is likely a Cu4S4 cluster in which each Cu atom is bridged by the side chains of two cysteine residues and has terminal N/O ligation.

Temperature variation causes a switching between excited-state decay paths to give a U-shaped Arrhenius plot.

Developing axons of the corpus callosum of mice are guided across the cerebral midline by a slinglike glial structure that forms transiently between the hemispheres. If the "sling" is cut at precallosal stages, the would-be callosal fibers whirl into paired neuromas adjacent to the longitudinal cerebral fissure. In experiments on such surgically acallosal animals, the aberrant commissural axons maintained a potential to regrow across the hemispheres at prenatal and early postnatal stages if they were presented with a properly aligned, glia-covered scaffold spanning the hemispheres.