INTELLECTUAL MERIT: This proposal develops a strategy for attaching enzymes to solid substrates for use in various applications where their high chemo-, regio-, stereo- and chiral selectivities can be exploited. Because enzymes bound to solids are often less active than the corresponding free enzymes, the PI will initially examine the mechanism of enzyme binding to layered solids such as alpha-Zr(IV)phosphate/phosphonates (alpha-ZrRPs). The surface functions (R) of alpha-ZrRP will be varied systematically to control these interactions, and the PI will quantify the nature and magnitudes of the enthalpy and entropy contributions of specific processes that accompany the binding event, in order to elucidate the molecular signatures of enzyme-solid interactions. Using these insights, he will prepare rationally designed enzyme-DNA-ZrRP conjugates, under reagent-less conditions, via DNA-mediated self-assembly. The proposed approach consists of: (1) covalent attachment of single stranded DNA to specific ZrRP nanoparticles via a long tether, (2) linking of complementary single-stranded DNA to surface COOH groups of enzymes, and (3) hybridization of enzyme-DNA with DNA-ZrRP to produce enzyme-DNA-nanomaterials. Tethers will consist of oligoethyleneglycol (OEG) spacers as these are known to have little or no interaction with proteins or DNA. DNA hybridization is well known and it should result in enzyme- DNA-nanoparticles under regent-less conditions. DNA provides a robust, reversible, indirect link between the solid and the protein. DNA-labeled enzymes and nanoparticles will provide numerous opportunities to construct higher order assemblies for a variety of practical applications.
BROADER IMPACTS: At the graduate level, the PI will continue to develop two new courses (Biological Chemistry I, and II, Chem360/361, 3 credits each) designed to train students in the application of chemical principles to important biological systems. Biological electron transfer, biomaterials, biocalorimetry, and DNA/protein structure are some of the topics contributed by the PI to these courses. The PI is developing a third graduate course in Technical Communications, Writing, and Ethics. At the undergraduate level, the PI will provide research training in biomaterials, protein-inorganic materials, and DNA-inorganic materials. Nearly 30 undergraduate students have been given such research experience in the PI's laboratory in the last 9 years as a part of the Chemistry Department Internship Program. Participating undergraduate students are often included as co-authors on resulting publications. At the high school level, the PI participates in the university-wide Mentor Connection program for outstanding high school students, sponsored by the University of Connecticut. He also hosts middle school students who participate in a day-long hands-on lab experience for students from throughout Connecticut.
I. Intellectual Merit: Detailed understanding of how proteins interact with solid surfaces is important in protein arrays, biosensing, biocatalysis and biomaterial design. Toward this goal, we examined the interactions of a small set of proteins with α-ZrIVphosphate (α-Zr(HPO4)2.H2O, abbreviated as α-ZrP, Fig. 1A, B) as model systems. Previously, we proposed ion-coupled protein binding (ICPB) model (Fig. 1C) to explain protein binding thermodynamics. During the current grant period, this model was tested by quantifying the role of metal ions, protein charge and nature of the binding surface on these interactions. 1.1 Role of metal ions in the mechanism: We chose two anionic proteins, hemoglobin (Hb) and glucose oxidase (GO), and negatively charged α-ZrP nanosheets as model systems. The addition of either 1 mM Zr(IV) or 1 mM Ca(II) increased the binding of GO and Hb to α-ZrP by ~380-fold and 43-fold (Fig. 1D), respectively. Zeta potential studies clearly showed binding of metal ions to α-ZrP, and TEMs showed highly ordered structures of the protein/metal/α-ZrP intercalates, confirmed by powder XRD data. Protein binding affinities depended on metal ion charge and its phosphophilicity. These data confirm proposed role of metal ions in the ICPB model. 1.2 Protein charge ladders: The ICPB model predicts that binding affinity is related to protein charge, and protein charge was continuously tuned by controlled amidation of the COOH groups of proteins with polyamines (Fig. 2A, RNH2 where R= H, (NH3Cl); (CH2CH2-NH)n-CH2CH2-NH2: n=2, TETA; and n=3, TEPA. Cationized proteins retained their structure and activities to a significant extent (60-200%), and chemical modification provided functional protein charge ladders (Fig. 2B). 1.3 Effect of protein charge in the mechanism: These charge ladders were used to study protein binding as a function of protein charge. Zeta potential data showed that binding of the charge ladders resulted in charge neutralization of α-ZrP nanosheets, and protein affinity increased with charge (Fig. 2C). The binding affinity of GO-TEPA (charge=+37), for example, increased (Kb=8+2x106 M-1) 180-fold when compared to that of GO. Activity of α-ZrP/GO-TEPA was 2.5-fold that of GO/α-ZrP (Fig. 3A). A plot of binding free energy vs protein charge was linear with a slope of -0.22+0.05 kcal/mol of charge. Thus, chemical modification provided a powerful, benign, predictable tool to control protein binding, as predicted by the ICPB model. 1.4 Protein binding to DNA: We tested the validity of ICPB model with DNA as a 1-dimensional analog of anionic α-ZrP. BSA-TETA charge ladder, for example, bound to calf thymus DNA and self assembled into micron scale structures (Fig. 3B). A remarkable feature of these assemblies is that they retained >89% of secondary structure, even after heating to 80 oC for >56 days. Chemical modification, thus, provided a convenient handle to induce binding of anionic proteins to DNA, with high control over affinity, stability and self assembly. Novel DNA-based biomaterials were produced by a rational design. 1.5 Protein binding to poly(acrylic acid): The ICPB model was tested further by using anionic polymers. In collaboration with the Kasi group at UCONN, we examined protein binding to linear poly(acrylic acid) (PAA), as a one-dimensional, flexible analog of α-ZrP (Fig. 3C). Strong binding of Hb-TETA charge ladder to PAA is indicated, and ΔH vs charge plot had a slope of -3.8 kcal per mol of charge (Fig. 3C). Binding became more exothermic with increase in charge, due to contributions from TETA side chains. This explanation was supported by the observation that ΔH vs charge plot for the Hb-NH3Cl charge ladder had a slope of only -1.1 kcal/mol of charge. Thus, chemical modification provided a predictable tool to control protein binding to PAA and produced novel protein-polymer biomaterials. 1.6. Ultra-stable Protein nanoparticles: EDC coupling of Hb with PAA produced discrete, completely soluble, nanoparticles which are denoted as Hb-PAA(X)-Y where X is the stoichiometric ratio Hb:PAA, and Y is the pH of synthesis. TEMs clearly indicated discrete, negatively charged nanobodies (80-100 nm in diameter, Fig. 3B). After steam sterilization (121oC, 40 minutes, 17-21 psi), Hb-PAA(100)-6/7/8 and Hb-PAA(100)-6/7/8 retained nearly 75-85% of activities of Hb (Fig. 3C), while Hb denatured irreversibly. These are the very first examples of steam-sterilizable advanced biomaterials. These resulted in 15 publications appeared, four submitted, three under preparation; and out of these, three are only partly supported by NSF. II. Broader Impacts: Each year, we trained several (>10 per year) undergraduate students during the academic year, as well as in summer, in the area of bionanomaterials. We trained 3-4 high school students for three weeks, each summer, with hands-on training in bionanomaterials. We have been developing a writing course for advanced graduate students. The PI has visited Fudan and Shanghai Jiatong Universities in China, during January 2012, and working with two other faculty members, initiated an undergradaute research program with UCONN. The PI will be hosting one student from Shanghai Jiatong University for 9 weeks, during summer 2013.
