Michael R. Duff

DNA-based supramolecular artificial light harvesting complexes.

Solar radiation reaching this planet is distributed over a wide range of wavelengths, and efficient collection and conversion of solar energy requires light harvesting over multiple wavelengths. Yet, the design, synthesis, and testing of novel, efficient, inexpensive light harvesting complexes are lacking. Engineered protein-DNA complexes are used here to self-assemble donor and acceptor molecules into artificial light harvesting units with an association constant of 3.3 +/- 1.2 muM(-1). Excitation of the DNA-bound donors resulted in a 540% increase in emission from the protein-bound acceptors, and the presence of one acceptor for each pair of donors was sufficient to quench approximately 50% of donor emission. Successful self-assembly of DNA-based light harvesting units is expected to facilitate economic/efficient conversion of solar energy, and model systems to achieve this goal are demonstrated here. We anticipate that success along these lines would facilitate more efficient approaches for solar energy capture.

Spectroscopic identification of binding modes of anthracene probes and DNA sequence recognition.

Abstract The binding properties of two anthracene derivatives with calf thymus DNA (CT DNA), poly(dA-dT), and poly(dG)·poly(dC) are reported. One contained bulky, cyclic cationic substituents at the 9 and 10 positions, and the other carried acylic, branched, cationic substituents. Binding of the probes to the DNA was examined by calorimetry, spectroscopy and helix melting studies. The cyclic derivative indicated exothermic binding, strong hypochromism, bathochromism, positive induced circular dichroism (CD, 300–400 nm), significant unwinding of the helix, large increases in the helix melting temperature, strong but negative linear dichroism (LD, 300–400 nm) and considerable stabilization of the helix. In contrast, the acyclic analog indicated thermoneutral binding, smaller hypochromism, no bathochromism, very weak induced CD, and no change in the helix melting temperature with any of the DNA polymers. A sharp distinction between the binding properties of the two probes is indicated, and both have intrinsic binding constants of ∼106 M−1 for the three polymers. However, when the ionic strength of the medium was lowered (10 mM NaCl), the absorption as well as CD spectral changes associated with the binding of the acyclic derivative corresponded with those of the cyclic derivative. The acyclic derivative showed large preference (10-fold) for poly(dG)·poly(dC) over poly(dA-dT), whereas the cyclic analog showed no preference. The characteristic spectroscopic signatures of the two distinct binding modes of these probes will be helpful in deciphering the interaction of other anthracene derivatives with DNA.

Rational Design of Anthracene-Based DNA Binders

Achieving the goal of rational design of DNA-binding ligands is important, and many inroads have been made in this direction. Toward that goal, we report a simple, systematic, and quantitative approach to design DNA-binding anthracene derivatives. Current data show that the binding free energies (DeltaG degrees) as well as enthalpies (DeltaH degrees) are related to specific structural features of the binders. Systematic design of anthracene probes, for example, indicated that the affinity can be enhanced via the introduction of methylene groups. Each methylene group contributed, on an average, -0.08+/-0.002 kcal/mol (at 1 M ionic strength, 293 K) toward the total binding free energy. Binding of the probes to DNA depended on ionic strength, and ionic strength studies were used to factor out to parse free-energy contributions due to specific interactions. The intrinsic free-energy contributions (DeltaGMol) of the probes are obtained by factoring out contributions from ionic interactions, hydration, conformational changes, polyelectrolyte effect, and the loss of rotational/translational motion. A strong, linear correlation was noted between DeltaGMol and the number of methylene groups present in the probe, and the correlation indicated free-energy contributions of -1.49 kcal/mol per methylene (at 50 mM NaCl, 293 K). This important observation provides a convenient handle to systematically fine-tune the intrinsic affinities of DNA binders. DeltaH values also showed clear trends, and each methylene contributed +0.28 kcal/mol toward the overall binding enthalpy (at 50 mM NaCl, 293 K), and this aspect is useful to fine-tune DeltaH contributions to binding. These important physical insights, derived from systematic modifications of the side chains of the DNA binders, are useful in the rational design of novel DNA binders.

Protein-Solid Interactions: Important Role of Solvent, Ions, Temperature, and Buffer in Protein Binding to α-Zr(IV) Phosphate

The interaction of proteins with a solid surface involves a complex set of interactions, and elucidating the details of these interactions is essential in the rational design of solid surfaces for applications in biosensors, biocatalysis, and biomedical applications. We examined the enthalpy changes accompanying the binding of met-hemoglobin, met-myoglobin, and lysozyme to layered alpha-Zr(IV)phosphate (20 mM NaPipes, 1 mM TBA, pH 7.2, 298 K) by titration calorimetry, under specific conditions. The corresponding binding enthalpies for the three proteins are -24.2 +/- 2.2, -10.6 +/- 2, and 6.2 +/- 0.2 kcal/mol, respectively. The binding enthalpy depended on the charge of the protein where the binding of positively charged proteins to the negatively charged solid surface was endothermic while the binding of negatively charged proteins to the negatively charged solid was exothermic. These observations are contrary to a simple electrostatic model where binding to the oppositely charged surface is expected to be exothermic. The binding enthalpy depended on the net charge on the protein, ionic strength of the medium, the type of buffer ions present, and temperature. The temperature dependence studies of binding enthalpies resulted in the estimation of heat capacity changes accompanying the binding. The heat capacity changes observed with Hb, Mb, and lysozyme are 1.4 +/- 0.3, 0.89 +/- 0.2, and 0.74 +/- 0.1 kcal/(mol.K), respectively, and these values depended on the net charge of the protein. The enthalpy changes also depended linearly on the enthalpy of ionization of the buffer, and the numbers of protons released per protein estimated from this data are 12.6 +/- 2, 6.0 +/- 1.2, and 1.2 +/- 0.5 for Hb, Mb, and lysozyme, respectively. Binding enthalpies, independent of buffer ionization, are also estimated from these data. Entropy changes are related to the loss in the degrees of freedom when the protein binds to the solid and the displacement of solvent molecules/protons/ions from the protein-solid interface. Proton coupled protein binding is one of the major processes in these systems, which is novel, and the binding enthalpies can be predicted from the net charge of the protein, enthalpy of buffer ionization, ionic strength, and temperature.

Molecular Signatures of Enzyme-Solid Interactions: Thermodynamics of Protein Binding to α-Zr(IV) Phosphate Nanoplates

Isothermal titration calorimetry (ITC) was used to determine the thermodynamics of protein binding to the nanoplates of alpha-Zr(HPO4)2.H2O (alpha-ZrP). The binding constants (K(b)) and DeltaG, DeltaH, and DeltaS have been evaluated for a small set of proteins, and K(b) values are in the range of 2-760 x 10(5) M(-1). The binding of positively charged proteins to the negatively charged alpha-ZrP was endothermic, while the binding of negatively charged proteins was exothermic, and these are contrary to expectations based on a simple electrostatic model. The binding enthalpies of the proteins varied over a range of -24 to +25 kcal/mol, and these correlated roughly with the net charge on the protein (R2 = 0.964) but not with other properties such as the number of basic residues, polar residues, isoelectric point, surface area, or molecular mass. Linear fits to the enthalpy plots indicated that each charge on the protein contributes 1.18 kcal/mol toward the binding enthalpy. Binding entropies of positively charged proteins were favorable (>0) while the binding entropies of negatively charged proteins were unfavorable (<0). The DeltaS values varied over a range of -51 to +98 cal/mol x K, and these correlated very well with the net charge on the protein (R2 = 0.999), but DeltaS is in the opposite direction of DeltaH. The binding or release of cations to/from the protein-solid interface can account for these observations. There was no correlation between the binding free energy (DeltaG(obs)) and any specific molecular properties, but it is likely to be a sum of several opposing interactions of large magnitudes. For the first time, the binding enthalpies and entropies are connected to specific molecular properties. The model suggests that the thermodynamic parameters can be controlled by choosing appropriate cations or by adjusting the net charge on the protein. We hope that physical insights such as these will be useful in understanding the complex behavior of proteins at biological interfaces.

The metallomics approach: use of Fe( ii ) and Cu( ii ) footprinting to examine metal binding sites on serum albumins

Metal binding to serum albumins is examined by oxidative protein-cleavage chemistry, and relative affinities of multiple metal ions to particular sites on these proteins were identified using a fast and reliable chemical footprinting approach. Fe(ii) and Cu(ii), for example, mediate protein cleavage at their respective binding sites on serum albumins, in the presence of hydrogen peroxide and ascorbate. This metal-mediated protein-cleavge reaction is used to evaluate the binding of metal ions, Na(+), Mg(2+), Ca(2+), Al(3+), Cr(3+), Mn(2+), Co(2+), Ni(2+), Zn(2+), Cd(2+), Hg(2+), Pb(2+), and Ce(3+) to albumins, and the relative affinities (selectivities) of the metal ions are rapidly evaluated by examining the extent of inhibition of protein cleavage. Four distinct systems Fe(II)/BSA, Cu(II)/BSA, Fe(II)/HSA and Cu(II)/HSA are examined using the above strategy. This metallomics approach is novel, even though the cleavage of serum albumins by Fe(II)/Cu(II) has been reported previously by this laboratory and many others. The protein cleavage products were analyzed by SDS PAGE, and the intensities of the product bands quantified to evaluate the extent of inhibition of the cleavage and thereby evaluate the relative binding affinities of specific metal ions to particular sites on albumins. The data show that Co(II) and Cr(III) showed the highest degree of inhibition, across the table, followed by Mn(II) and Ce(III). Alakali metal ions and alkaline earth metal ions showed very poor affinity for these metal sites on albumins. Thus, metal binding profiles for particular sites on proteins can be obtained quickly and accurately, using the metallomics approach.

Towards building artificial light harvesting complexes: enhanced singlet–singlet energy transfer between donor and acceptor pairs bound to albumins

Specific donor and acceptor pairs have been assembled in bovine serum albumin (BSA), at neutral pH and room temperature, and these dye-protein complexes indicated efficient donor to acceptor singlet-singlet energy transfer. For example, pyrene-1-butyric acid served as the donor and Coumarin 540A served as the acceptor. Both the donor and the acceptor bind to BSA with affinity constants in excess of 2x10(5) M(-1), as measured in absorption and circular dichroism (CD) spectral titrations. Simultaneous binding of both the donor and the acceptor chromophores was supported by CD spectra and one chromophore did not displace the other from the protein host, even when limited concentrations of the host were used. For example, a 1:1:1 complex between the donor, acceptor and the host can be readily formed, and spectral data clearly show that the binding sites are mutually exclusive. The ternary complexes (two different ligands bound to the same protein molecule) provided opportunities to examine singlet-singlet energy transfer between the protein-bound chromophores. Donor emission was quenched by the addition of the acceptor, in the presence of limited amounts of BSA, while no energy transfer was observed in the absence of the protein host, under the same conditions. The excitation spectra of the donor-acceptor-host complexes clearly show the sensitization of acceptor emission by the donor. Protein denaturation, as induced by the addition of urea or increasing the temperature to 360 K, inhibited energy transfer, which indicate that protein structure plays an important role. Sensitization also proceeded at low temperature (77 K) and diffusion of the donor or the acceptor is not required for energy transfer. Stern-Volmer quenching plots show that the quenching constant is (3.1+/-0.2)x10(4) M(-1), at low acceptor concentrations (<35 microM). Other albumins such as human and porcine proteins also served as good hosts for the above experiments. For the first time, non-natural systems have been self-assembled which can capture donor-acceptor pairs and facilitate singlet-singlet energy transfer. Such systems may form a basis for the design and construction of protein-based multi-chromophore self-assemblies for solar light harvesting, conversion and storage.

Novel, Simple, Versatile and General Synthesis of Nanoparticles Made from Proteins, Nucleic Acids and other Materials

A new, simple, and versatile method was developed to prepare protein nanoparticles, for the first time, and the approach was extended to prepare organic, inorganic, and biological nanomaterials. For example, nanoparticles of met-hemoglobin and glucose oxidase are readily prepared by contacting a fine spray of aqueous solutions of the proteins to an organic solvent such as methanol or acetonitrile. The protein nanoparticles suspended in organic solvents retained their secondary structure and biological activities to a significant extent. Using this approach, we also successfully prepared nanoparticles of transition metal complexes, organic molecules, nucleic acids, inorganic polymers, and organic polymers. Particle size depended on reagent concentrations, pH and the solvent used, and particle sizes have been controlled from 20 to 200 nm by adjusting these parameters. In each case, particle sizes and size distributions were determined by dynamic light scattering and the data have been confirmed by electron microscopy. Addition of appropriate electrolytes to the nanoparticle supensions stabilized them against aggregation or crystallization, and particles were stable over months of storage at 4°C. Nanoparticles of met-hemoglobin, glucose oxidase, and calf thymus DNA indicated retention of their native-like structures, as evidenced from their respective circular dichroism spectra. Enzyme nanoparticles retained their catalytic activities to a significant extent. For example, peroxidase-like activity of met-hemoglobin nanoparticles suspended in methanol was 0.3 M-1 s-1, which is comparable to the activity of met-hmoglobin in aqueous buffer (1.0 M-1 s-1) even though the former has been measured in methanol. This activity is far greater than the activity of free heme in methanol. Thus, the nanobiocatalysts retained substantial activity in organic solvents. Nanoparticles of anthracene indicated extensive excitonic coupling due to inter-chromophore interactions. The current method of nanoparticle synthesis is rapid, simple, versatile, reproducible and resulted in the formation of nanoparticles from a variety of materials, many of them for the first time.