Molecular Catalysts with Diphosphine Ligands Containing Pendant Amines.

Pendant amines play an invaluable role in chemical reactivity, especially for molecular catalysts based on earth-abundant metals. As inspired by [FeFe]-hydrogenases, which contain a pendant amine positioned for cooperative bifunctionality, synthetic catalysts have been developed to emulate this multifunctionality through incorporation of a pendant amine in the second coordination sphere. Cyclic diphosphine ligands containing two amines serve as the basis for a class of catalysts that have been extensively studied and used to demonstrate the impact of a pendant base. These 1,5-diaza-3,7-diphosphacyclooctanes, now often referred to as "P2N2" ligands, have profound effects on the reactivity of many catalysts. The resulting [Ni(PR2NR'2)2]2+ complexes are electrocatalysts for both the oxidation and production of H2. Achieving the optimal benefit of the pendant amine requires that it has suitable basicity and is properly positioned relative to the metal center. In addition to the catalytic efficacy demonstrated with [Ni(PR2NR'2)2]2+ complexes for the oxidation and production of H2, catalysts with diphosphine ligands containing pendant amines have also been demonstrated for several metals for many different reactions, both in solution and immobilized on surfaces. The impact of pendant amines in catalyst design continues to expand.

Residue-Specific Insights into the Intermolecular Protein-Protein Interfaces Driving Amelogenin Self-Assembly in Solution.

Amelogenin, the dominant organic component (>90%) in the early stages of amelogenesis, orchestrates the mineralization of apatite crystals into enamel. The self-association properties of amelogenin as a function of pH and protein concentration have been suggested to play a central role in this process; however, the large molecular weight of the self-assembled quaternary structures has largely prevented structural studies of the protein in solution under physiological conditions using conventional approaches. Here, using perdeuterated murine amelogenin (0.25 mM, 5 mg/mL) and TROSY-based NMR experiments to improve spectral resolution, we assigned the 1H-15N spectra of murine amelogenin over a pH range (5.5 to 8.0) where amelogenin is reported to exist as oligomers (pH ≤∼6.8) and nanospheres (pH ≥∼7.2). The disappearance or intensity reduction of amide resonances in the 1H-15N HSQC spectra was interpreted to reflect changes in dynamics (intermediate millisecond-to-microsecond motion) and/or heterogenous interfaces of amide nuclei at protein-protein interfaces. The intermolecular interfaces were concentrated toward the N-terminus of amelogenin (L3-G8, V19-G38, L46-Q49, and Q57-L70) at pH 6.6 (oligomers) and at pH 7.2 (nanospheres) including the entire N-terminus up to Q76 and regions distributed through the central hydrophobic region (Q82-Q101, S125-Q139, and F151-Q154). At all pH levels, the C-terminus appeared disordered, highly mobile, and not involved in self-assembly, suggesting nanosphere structures with solvent-exposed C-termini. These findings present unique, residue-specific insights into the intermolecular protein-protein interfaces driving amelogenin quaternary structure formation and suggest that nanospheres in solution predominantly contain disordered, solvent-exposed C-termini.

The energetic basis for hydroxyapatite mineralization by amelogenin variants provides insights into the origin of amelogenesis imperfecta.

Small variations in the primary amino acid sequence of extracellular matrix proteins can have profound effects on the biomineralization of hard tissues. For example, a change in one amino acid within the amelogenin protein can lead to drastic changes in enamel phenotype, resulting in amelogenesis imperfecta, enamel that is defective and easily damaged. Despite the importance of these undesirable phenotypes, there is very little understanding of how single amino acid variation in amelogenins can lead to malformed enamel. Here, we aim to develop a thermodynamic understanding of how protein variants can affect steps of the biomineralization process. High-resolution, in situ atomic force microscopy (AFM) showed that altering one amino acid within the murine amelogenin sequence (natural variants T21 and P41T, and experimental variant P71T) resulted in an increase in the quantity of protein adsorbed onto hydroxyapatite (HAP) and the formation of multiple protein layers. Quantitative analysis of the equilibrium adsorbate amounts revealed that the protein variants had higher oligomer-oligomer binding energies. MMP20 enzyme degradation and HAP mineralization studies showed that the amino acid variants slowed the degradation of amelogenin by MMP20 and inhibited the growth and phase transformation of HAP. We propose that the protein variants cause malformed enamel because they bind excessively to HAP and disrupt the normal HAP growth and enzymatic degradation processes. The in situ methods applied to determine the energetics of molecular level processes are powerful tools toward understanding the mechanisms of biomineralization.

Controls of nature: Secondary, tertiary, and quaternary structure of the enamel protein amelogenin in solution and on hydroxyapatite.

Amelogenin, a protein critical to enamel formation, is presented as a model for understanding how the structure of biomineralization proteins orchestrate biomineral formation. Amelogenin is the predominant biomineralization protein in the early stages of enamel formation and contributes to the controlled formation of hydroxyapatite (HAP) enamel crystals. The resulting enamel mineral is one of the hardest tissues in the human body and one of the hardest biominerals in nature. Structural studies have been hindered by the lack of techniques to evaluate surface adsorbed proteins and by amelogenin's disposition to self-assemble. Recent advancements in solution and solid state nuclear magnetic resonance (NMR) spectroscopy, atomic force microscopy (AFM), and recombinant isotope labeling strategies are now enabling detailed structural studies. These recent studies, coupled with insights from techniques such as CD and IR spectroscopy and computational methodologies, are contributing to important advancements in our structural understanding of amelogenesis. In this review we focus on recent advances in solution and solid state NMR spectroscopy and in situ AFM that reveal new insights into the secondary, tertiary, and quaternary structure of amelogenin by itself and in contact with HAP. These studies have increased our understanding of the interface between amelogenin and HAP and how amelogenin controls enamel formation.

Understanding and Design of Bidirectional and Reversible Catalysts of Multielectron, Multistep Reactions.

Some enzymes, including those that are involved in the activation of small molecules such as H2 or CO2, can be wired to electrodes and function in either direction of the reaction depending on the electrochemical driving force and display a significant rate at very small deviations from the equilibrium potential. We call the former property "bidirectionality" and the latter "reversibility". This performance sets very high standards for chemists who aim at designing synthetic electrocatalysts. Only recently, in the particular case of the hydrogen production/evolution reaction, has it been possible to produce inorganic catalysts that function bidirectionally, with an even smaller number that also function reversibly. This raises the question of how to engineer such desirable properties in other synthetic catalysts. Here we introduce the kinetic modeling of bidirectional two-electron-redox reactions in the case of molecular catalysts and enzymes that are either attached to an electrode or diffusing in solution in the vicinity of an electrode. We emphasize that trying to discuss bidirectionality and reversibility in relation to a single redox potential leads to an impasse: the catalyst undergoes two redox transitions, and therefore two catalytic potentials must be defined, which may depart from the two potentials measured in the absence of catalysis. The difference between the two catalytic potentials defines the reversibility; the difference between their average value and the equilibrium potential defines the directionality (also called "preference", or "bias"). We describe how the sequence of events in the bidirectional catalytic cycle can be elucidated on the basis of the voltammetric responses. Further, we discuss the design principles of bidirectionality and reversibility in terms of thermodynamics and kinetics and conclude that neither bidirectionality nor reversibility requires that the catalytic energy landscape be flat. These theoretical findings are illustrated by previous results obtained with nickel diphosphine molecular catalysts and hydrogenases. In particular, analysis of the nickel catalysts highlights the fact that reversible catalysis can be achieved by catalysts that follow complex mechanisms with branched reaction pathways.

Evaluating the impacts of amino acids in the second and outer coordination spheres of Rh-bis(diphosphine) complexes for CO2 hydrogenation.

To explore the influence of a biologically inspired second and outer coordination sphere on Rh-bis(diphosphine) CO2 hydrogenation catalysts, a series of five complexes were prepared by varying the substituents on the pendant amine in the P(Et)2CH2NRCH2P(Et)2 ligands (PEtNRPEt), where R consists of methyl ester modified amino acids, including three neutral (glycine methyl ester (GlyOMe), leucine methyl ester (LeuOMe), and phenylalanine methyl ester (PheOMe)), one acidic (aspartic acid dimethyl ester (AspOMe)) and one basic (histidine methyl ester (MeHisOMe)) amino acid esters. The turnover frequencies (TOFs) for CO2 hydrogenation for each of these complexes were compared to those of the non-amino acid containing [Rh(depp)2]+ (depp) and [Rh(PEtNMePEt)2]+ (NMe) complexes. Each complex is catalytically active for CO2 hydrogenation to formate under mild conditions in THF. Catalytic activity spanned a factor of four, with the most active species being the NMe catalyst, while the slowest were the GlyOMe and the AspOMe complexes. When compared to a similar set of catalysts with phenyl-substituted phosphorous groups, a clear contribution of the outer coordination sphere is seen for this family of CO2 hydrogenation catalysts.

Solid-State NMR Identification of Intermolecular Interactions in Amelogenin Bound to Hydroxyapatite.

Biomineralization processes govern the formation of hierarchical hard tissues such as bone and teeth in living organisms, and mimicking these processes could lead to the design of new materials with specialized properties. However, such advances require structural characterization of the proteins guiding biomineral formation to understand and mimic their impact. In their "active" form, biomineralization proteins are bound to a solid surface, severely limiting our ability to use many conventional structure characterization techniques. Here, solid-state NMR spectroscopy was applied to study the intermolecular interactions of amelogenin, the most abundant protein present during the early stages of enamel formation, in self-assembled oligomers bound to hydroxyapatite. Intermolecular dipolar couplings were identified that support amelogenin dimer formation stabilized by residues toward the C-termini. These dipolar interactions were corroborated by molecular dynamics simulations. A ß-sheet structure was identified in multiple regions of the protein, which is otherwise intrinsically disordered in the absence of hydroxyapatite. To our knowledge, this is the first intermolecular protein-protein interaction reported for a biomineralization protein, representing an advancement in understanding enamel development and a new general strategy toward investigating biomineralization proteins.

Secondary structure and dynamics study of the intrinsically disordered silica-mineralizing peptide P5 S3 during silicic acid condensation and silica decondensation.

The silica forming repeat R5 of sil1 from Cylindrotheca fusiformis was the blueprint for the design of P5 S3 , a 50-residue peptide which can be produced in large amounts by recombinant bacterial expression. It contains 5 protein kinase A target sites and is highly cationic due to 10 lysine and 10 arginine residues. In the presence of supersaturated orthosilicic acid P5 S3 enhances silica-formation whereas it retards the dissolution of amorphous silica (SiO2 ) at globally undersaturated concentrations. The secondary structure of P5 S3 during these 2 processes was studied by circular dichroism (CD) spectroscopy, complemented by nuclear magnetic resonance (NMR) spectroscopy of the peptide in the absence of silicate. The NMR studies of dual-labeled (13 C, 15 N) P5 S3 revealed a disordered structure at pH 2.8 and 4.5. Within the pH range of 4.5-9.5 in the absence of silicic acid, the CD data showed a disordered structure with the suggestion of some polyproline II character. Upon silicic acid polymerization and during dissolution of preformed silica, the CD spectrum of P5 S3 indicated partial transition into an α-helical conformation which was transient during silica-dissolution. The secondary structural changes observed for P5 S3 correlate with the presence of oligomeric/polymeric silicic acid, presumably due to P5 S3 -silica interactions. These P5 S3 -silica interactions appear, at least in part, ionic in nature since negatively charged dodecylsulfate caused similar perturbations to the P5 S3 CD spectrum as observed with silica, while uncharged ß-d-dodecyl maltoside did not affect the CD spectrum of P5 S3 . Thus, with an associated increase in α-helical character, P5 S3 influences both the condensation of silicic acid into silica and its decondensation back to silicic acid.

Amino acid modified Ni catalyst exhibits reversible H2 oxidation/production over a broad pH range at elevated temperatures.

Hydrogenases interconvert H2 and protons at high rates and with high energy efficiencies, providing inspiration for the development of molecular catalysts. Studies designed to determine how the protein scaffold can influence a catalytically active site have led to the synthesis of amino acid derivatives of [Ni(P2(R)N2(R'))2](2+) complexes, [Ni(P2(Cy)N2(Amino acid))2](2+) (CyAA). It is shown that these CyAA derivatives can catalyze fully reversible H2 production/oxidation at rates approaching those of hydrogenase enzymes. The reversibility is achieved in acidic aqueous solutions (pH = 0-6), 1 atm 25% H2/Ar, and elevated temperatures (tested from 298 to 348 K) for the glycine (CyGly), arginine (CyArg), and arginine methyl ester (CyArgOMe) derivatives. As expected for a reversible process, the catalytic activity is dependent upon H2 and proton concentrations. CyArg is significantly faster in both directions (∼300 s(-1) H2 production and 20 s(-1) H2 oxidation; pH = 1, 348 K, 1 atm 25% H2/Ar) than the other two derivatives. The slower turnover frequencies for CyArgOMe (35 s(-1) production and 7 s(-1) oxidation under the same conditions) compared with CyArg suggests an important role for the COOH group during catalysis. That CyArg is faster than CyGly (3 s(-1) production and 4 s(-1) oxidation) suggests that the additional structural features imparted by the guanidinium groups facilitate fast and reversible H2 addition/release. These observations demonstrate that outer coordination sphere amino acids work in synergy with the active site and can play an important role for synthetic molecular electrocatalysts, as has been observed for the protein scaffold of redox active enzymes.

Carbon-Nanotube-Supported Bio-Inspired Nickel Catalyst and Its Integration in Hybrid Hydrogen/Air Fuel Cells.

A biomimetic nickel bis-diphosphine complex incorporating the amino acid arginine in the outer coordination sphere was immobilized on modified carbon nanotubes (CNTs) through electrostatic interactions. The functionalized redox nanomaterial exhibits reversible electrocatalytic activity for the H2 /2 H+ interconversion from pH 0 to 9, with catalytic preference for H2 oxidation at all pH values. The high activity of the complex over a wide pH range allows us to integrate this bio-inspired nanomaterial either in an enzymatic fuel cell together with a multicopper oxidase at the cathode, or in a proton exchange membrane fuel cell (PEMFC) using Pt/C at the cathode. The Ni-based PEMFC reaches 14 mW cm-2 , only six-times-less as compared to full-Pt conventional PEMFC. The Pt-free enzyme-based fuel cell delivers ≈2 mW cm-2 , a new efficiency record for a hydrogen biofuel cell with base metal catalysts.

Single-Amino Acid Modifications Reveal Additional Controls on the Proton Pathway of [FeFe]-Hydrogenase.

The proton pathway of [FeFe]-hydrogenase is essential for enzymatic H2 production and oxidation and is composed of four residues and a water molecule. A computational analysis of this pathway in the [FeFe]-hydrogenase from Clostridium pasteurianum revealed that the solvent-exposed residue of the pathway (Glu282) forms hydrogen bonds to two residues outside of the pathway (Arg286 and Ser320), implying that these residues could function in regulating proton transfer. In this study, we show that substituting Arg286 with leucine eliminates hydrogen bonding with Glu282 and results in an ∼3-fold enhancement of H2 production activity when methyl viologen is used as an electron donor, suggesting that Arg286 may help control the rate of proton delivery. In contrast, substitution of Ser320 with alanine reduces the rate ∼5-fold, implying that it either acts as a member of the pathway or influences Glu282 to permit proton transfer. Interestingly, quantum mechanics/molecular mechanics and molecular dynamics calculations indicate that Ser320 does not play a structural role or indirectly influence the barrier for proton movement at the entrance of the channel. Rather, it may act as an additional proton acceptor for the pathway or serve in a regulatory role. While further studies are needed to elucidate the role of Ser320, collectively these data provide insights into the complex proton transport process.

Investigating the role of chain and linker length on the catalytic activity of an H2 production catalyst containing a ß-hairpin peptide.

Building on our recent report of an active H2 production catalyst [Ni(PPh 2NProp-peptide)2]2+ (Prop = para-phenylpropionic acid, peptide (R10) = WIpPRWTGPR-NH2, p = D-proline and P2N = 1-aza-3,6-diphosphacycloheptane) that contains structured ß-hairpin peptides, here we investigate how H2 production is effected by: (1) the length of the hairpin (eight or ten residues) and (2) limiting the flexibility between the peptide and the core complex by altering the length of the linker: para-phenylpropionic acid (three carbons) or para-benzoic acid (one carbon). Reduction of the peptide chain length from ten to eight residues increases or maintains the catalytic current for H2 production for all complexes, suggesting a non-productive steric interaction at longer peptide lengths. While the structure of the hairpin appears largely intact for the complexes, NMR data are consistent with differences in dynamic behavior which may contribute to the observed differences in catalytic activity. Molecular dynamics simulations demonstrate that complexes with a one-carbon linker have the desired effect of restricting the motion of the hairpin relative to the complex; however, the catalytic currents are significantly reduced compared to complexes containing a three-carbon linker as a result of the electron withdrawing nature of the -COOH group. These results demonstrate the complexity and interrelated nature of the outer coordination sphere on catalysis.

The leucine-rich amelogenin protein (LRAP) is primarily monomeric and unstructured in physiological solution.

Amelogenin proteins are critical to the formation of enamel in teeth and may have roles in controlling growth and regulating microstructures of the intricately woven hydroxyapatite (HAP). Leucine-rich amelogenin protein (LRAP) is a 59-residue splice variant of amelogenin and contains the N- and C-terminal charged regions of the full-length protein thought to control crystal growth. Although the quaternary structure of full-length amelogenin in solution has been well studied and can consist of self-assemblies of monomers called nanospheres, there is limited information on the quaternary structure of LRAP. Here, sedimentation velocity analytical ultracentrifugation (SV) and small angle neutron scattering (SANS) were used to study the tertiary and quaternary structure of LRAP at various pH values, ionic strengths, and concentrations. We found that the monomer is the dominant species of phosphorylated LRAP (LRAP(+P)) over a range of solution conditions (pH 2.7-4.1, pH 4.5-8, 50 mmol/L(mM) to 200 mM NaCl, 0.065-2 mg/mL). The monomer is also the dominant species for unphosphorylated LRAP (LRAP(-P)) at pH 7.4 and for LRAP(+P) in the presence of 2.5 mM calcium at pH 7.4. LRAP aggregates in a narrow pH range near the isoelectric point of pH 4.1. SV and SANS show that the LRAP monomer has a radius of ∼2.0 nm and an asymmetric structure, and solution NMR studies indicate that the monomer is largely unstructured. This work provides new insights into the secondary, tertiary, and quaternary structure of LRAP in solution and provides evidence that the monomeric species may be an important functional form of some amelogenins.

Molecular dynamics study of the proposed proton transport pathways in [FeFe]-hydrogenase.

Possible proton transport pathways in Clostridium pasteurianum (CpI) [FeFe]-hydrogenase were investigated with molecular dynamics simulations. This study was undertaken to evaluate the functional pathway and provide insight into the hydrogen bonding features defining an active proton transport pathway. Three pathways were evaluated, two of which consist of water wires and one of predominantly amino acid residues. Our simulations suggest that protons are not transported through water wires. Instead, the five-residue motif (Glu282, Ser319, Glu279, H2O, Cys299) was found to be the likely pathway, consistent with previously made experimental observations. The pathway was found to have a persistent hydrogen bonded core (residues Cys299 to Ser319), with less persistent hydrogen bonds at the ends of the pathway for both H2 release and H2 uptake. Single site mutations of the four residues have been shown experimentally to deactivate the enzyme. The theoretical evaluation of these mutations demonstrates redistribution of the hydrogen bonds in the pathway, resulting in enzyme deactivation. Finally, coupling between the protein dynamics near the proton transport pathway and the redox partner binding regions was also found as a function of H2 uptake and H2 release states, which may be indicative of a correlation between proton and electron movement within the enzyme.

Beyond the active site: the impact of the outer coordination sphere on electrocatalysts for hydrogen production and oxidation.

Redox active metalloenzymes play a major role in energy transformation reactions in biological systems. Examples include formate dehydrogenases, nitrogenases, CO dehydrogenase, and hydrogenases. Many of these reactions are also of interest to humans as potential energy storage or utilization reactions for photoelectrochemical, electrolytic, and fuel cell applications. These metalloenzymes consist of redox active metal centers where substrates are activated and undergo transformation to products accompanied by electron and proton transfer to or from the substrate. These active sites are typically buried deep within a protein matrix of the enzyme with channels for proton transport, electron transport, and substrate/product transport between the active site and the surface of the protein. In addition, there are amino acid residues that lie in close proximity to the active site that are thought to play important roles in regulating and enhancing enzyme activity. Directly studying the outer coordination sphere of enzymes can be challenging due to their complexity, and the use of modified molecular catalysts may allow us to provide some insight. There are two fundamentally different approaches to understand these important interactions. The "bottom-up" approach involves building an amino acid or peptide containing outer coordination sphere around a functional molecular catalyst, and the "top-down" approach involves attaching molecular catalyst to a structured protein. Both of these approaches have been undertaken for hydrogenase mimics and are the emphasis of this Account. Our focus has been to utilize amino acid or peptide based scaffolds on an active functional enzyme mimic for H2 oxidation and production, [Ni(P(R)2N(R('))2)2](2+). This "bottom-up" approach has allowed us to evaluate individual functional group and structural contributions to electrocatalysts for H2 oxidation and production. For instance, using amine, ether, and carboxylic acid functionalities in the outer coordination sphere enhances proton movement and results in lower catalytic overpotentials for H2 oxidation, while achieving water solubility in some cases. Amino acids with acidic and basic side chains concentrate substrate around catalysts for H2 production, resulting in up to 5-fold enhancements in rate. The addition of a structured peptide in an H2 production catalyst limited the structural freedom of the amino acids nearest the active site, while enhancing the overall rate. Enhanced stability to oxygen or extreme conditions such as strongly acidic or basic conditions has also resulted from an amino acid based outer coordination sphere. From the "top-down" approach, others have achieved water solubility and photocatalytic activity by associating this core complex with photosystem-I. Collectively, by use of this well understood core, the role of individual and combined features of the outer coordination sphere are starting to be understood at a mechanistic level. Common mechanisms have yet to be defined to predictably control these processes, but our growing knowledge in this area is essential for the eventual mimicry of enzymes by efficient molecular catalysts for practical use.

Sequence-Defined Energetic Shifts Control the Disassembly Kinetics and Microstructure of Amelogenin Adsorbed onto Hydroxyapatite (100).

The interactions between proteins and surfaces are critical to a number of important processes including biomineralization, the biocompatibility of biomaterials, and the function of biosensors. Although many proteins exist as monomers or small oligomers, amelogenin is a unique protein that self-assembles into supramolecular structures called "nanospheres," aggregates of hundreds of monomers that are 20-60 nm in diameter. The nanosphere quaternary structure is observed in solution; however, the quaternary structure of amelogenin adsorbed onto hydroxyapatite (HAP) surfaces is not known even though it may be important to amelogenin's function in forming highly elongated and intricately assembled HAP crystallites during enamel formation. We report studies of the interactions of the enamel protein, amelogenin (rpM179), with a well-defined (100) face prepared by the synthesis of large crystals of HAP. High-resolution in situ atomic force microscopy (AFM) was used to directly observe protein adsorption onto HAP at the molecular level within an aqueous solution environment. Our study shows that the amelogenin nanospheres disassemble onto the HAP surface, breaking down into oligomeric (25-mer) subunits of the larger nanosphere. In some cases, the disassembly event is directly observed by in situ imaging for the first time. Quantification of the adsorbate amounts by size analysis led to the determination of a protein binding energy (17.1k(b)T) to a specific face of HAP (100). The kinetics of disassembly are greatly slowed in aged solutions, indicating that there are time-dependent increases in oligomer-oligomer binding interactions within the nanosphere. A small change in the sequence of amelogenin by the attachment of a histidine tag to the N-terminus of rpM179 to form rp(H)M180 results in the adsorption of a complete second layer on top of the underlying first layer. Our research elucidates how supramolecular protein structures interact and break down at surfaces and how small changes in the primary sequence of amelogenin can affect the disassembly process.

Improved protocol to purify untagged amelogenin - Application to murine amelogenin containing the equivalent P70→T point mutation observed in human amelogenesis imperfecta.

Amelogenin is the predominant extracellular protein responsible for converting carbonated hydroxyapatite into dental enamel, the hardest and most heavily mineralized tissue in vertebrates. Despite much effort, the precise mechanism by which amelogenin regulates enamel formation is not fully understood. To assist efforts aimed at understanding the biochemical mechanism of enamel formation, more facile protocols to purify recombinantly expressed amelogenin, ideally without any tag to assist affinity purification, are advantageous. Here we describe an improved method to purify milligram quantities of amelogenin that exploits its high solubility in 2% glacial acetic acid under conditions of low ionic strength. The method involves heating the frozen cell pellet for two 15min periods at ∼70°C with 2min of sonication in between, dialysis twice in 2% acetic acid (1:250 v/v), and reverse phase chromatography. A further improvement in yield is obtained by resuspending the frozen cell pellet in 6M guanidine hydrochloride in the first step. The acetic acid heating method is illustrated with a murine amelogenin containing the corresponding P70→T point mutation observed in an human amelogenin associated with amelogenesis imperfecta (P71T), while the guanidine hydrochloride heating method is illustrated with wild type murine amelogenin (M180). The self-assembly properties of P71T were probed by NMR chemical shift perturbation studies as a function of protein (0.1-1.8mM) and NaCl (0-367mM) concentration. Relative to similar studies with wild type murine amelogenin, P71T self-associates at lower protein or salt concentrations with the interactions initiated near the N-terminus.

Solid-state NMR studies of proteins immobilized on inorganic surfaces.

Solid state NMR is the primary tool for studying the quantitative, site-specific structure, orientation, and dynamics of biomineralization proteins under biologically relevant conditions. Two calcium phosphate proteins, statherin (43 amino acids) and leucine rich amelogenin protein (LRAP; 59 amino acids), have been studied in depth and have different dynamic properties and 2D- and 3D-structural features. These differences make it difficult to extract design principles used in nature for building materials with properties such as high strength, unusual morphologies, or uncommon phases. Consequently, design principles needed for developing synthetic materials controlled by proteins are not clear. Many biomineralization proteins are much larger than statherin and LRAP, necessitating the study of larger biomineralization proteins. More recent studies of the significantly larger full-length amelogenin (180 residues) represent a significant step forward to ultimately investigate the full diversity of biomineralization proteins. Interactions of amino acids, a silaffin derived peptide, and the model LK peptide with silica are also being studied, along with qualitative studies of the organic matrices interacting with calcium carbonate. Dipolar recoupling techniques have formed the core of the quantitative studies, yet the need for isolated spin pairs makes this approach costly and time intensive. The use of multi-dimensional techniques to study biomineralization proteins is becoming more common, methodology which, despite its challenges with these difficult-to-study proteins, will continue to drive future advancements in this area.

Acidic ionic liquid/water solution as both medium and proton source for electrocatalytic H2 evolution by [Ni(P2N2)2]2+ complexes.

The electrocatalytic reduction of protons to H(2) by [Ni((P(Ph)(2)N(C6H4-hex))(2)(2)]((BF(4))(2) (where P(Ph)(2)N(C6H4-hex)(2) = 1,5-di(4-n-hexylphenyl)-3,7-diphenyl-1,5-diaza-3,7-diphosphacyclooctane) in the highly acidic ionic liquid dibutylformamidium bis(trifluoromethanesulfonyl)amide shows a strong dependence on added water. A turnover frequency of 43,000-53,000 s(-1) has been measured for hydrogen production at 25 °C when the mole fraction of water (χ(H(2)O)) is 0.72. The same catalyst in acetonitrile with added dimethylformamidium trifluoromethanesulfonate and water has a turnover frequency of 720 s(-1). Thus, the use of an ionic liquid/aqueous solution enhances the observed catalytic rate by more than a factor of 50, compared to a similar acid in a traditional organic solvent. Complexes [Ni((P(Ph)(2)N(C6H4X))(2)(2)]((BF(4))(2) (X = H, OMe,CH(2)P(O)(OEt)(2), Br) are also catalysts in the ionic liquid/water mixture, and the observed catalytic rates correlate with the hydrophobicity of X.

Direct comparison of the performance of a bio-inspired synthetic nickel catalyst and a [NiFe]-hydrogenase, both covalently attached to electrodes.

The active site of hydrogenases has been a source of inspiration for the development of molecular catalysts. However, direct comparisons between molecular catalysts and enzymes have not been possible because different techniques are used to evaluate both types of catalysts, minimizing our ability to determine how far we have come in mimicking the enzymatic performance. The catalytic properties of the [Ni(P(Cy) 2 N(Gly) 2 )2 ](2+) complex with the [NiFe]-hydrogenase from Desulfovibrio vulgaris immobilized on a functionalized electrode were compared under identical conditions. At pH 7, the enzyme shows higher activity and lower overpotential with better stability, while at low pH, the molecular catalyst outperforms the enzyme in all respects. This is the first direct comparison of enzymes and molecular complexes, enabling a unique understanding of the benefits and detriments of both systems, and advancing our understanding of the utilization of these bio-inspired complexes in fuel cells.