Trevor D. Rapson

Micromolar biosensing of nitric oxide using myoglobin immobilized in a synthetic silk film.

In this work we investigate the use of coiled-coil silk proteins, produced in recombinant Escherichia coli, as a new material for immobilizing biosensors. Myoglobin was embedded in transparent honeybee silk protein films. Immobilized myoglobin maintained a high affinity for nitric oxide (KD NO=52 µM) and good sensitivity with a limit of detection of 5 µM. The immobilized myoglobin-silk protein film was stable and could be stored as a dry film at room temperature for at least 60 days. The effect of immobilization on the structure of myoglobin was fully investigated using UV/visible, Fourier Transform Infrared and Raman spectroscopy, which indicated a weakening in the strength of the iron-histidine bond. This study demonstrates that recombinant coiled-coil silk proteins provide a safe and environmentally friendly alternative to sol-gels for stabilizing heme proteins for use as optical biosensors.

Fluorescent nitric oxide detection using cobalt substituted myoglobin

We report two advances in optical biosensing of nitric oxide (NO). Firstly, we developed an improved biomolecular gated system for fluorescent transduction of heme–NO binding. Secondly, through a cobalt substitution, the detection limit for NO was decreased an order of magnitude lower than that of native myoglobin.

Silk provides a new avenue for third generation biosensors: Sensitive, selective and stable electrochemical detection of nitric oxide

Using heme entrapped in recombinant silk films, we have produced 3rd generation biosensors, which allow direct electron transfer from the heme center to an electrode avoiding the need for electron mediators. Here, we demonstrate the use of these heme-silk films for the detection of nitric oxide (NO) at nanomolar levels in the presence and absence of oxygen. The sensor was prepared by drop-casting a silk solution on a glassy carbon electrode modified with multiwalled carbon nanotubes (MWCNT) followed by infusion with heme. The sensor was characterized by cyclic voltammetry and showed well defined and reversible Fe+/ Fe3+ redox couple activity, with NO detection by oxidation at potentials above +0.45V or reduction at potentials below - 0.7V. Evaluation of the effect of pH on the sensor response to NO reduction indicated a maximum response at pH 3. The sensor showed good linearity in the concentration range from 19 to 190nM (R2 = 0.99) with a detection limit of 2nM. The sensor had excellent selectivity towards NO with no or negligible interference from oxygen, nitrite, nitrate, dopamine and ascorbic acid and retained 86% of response after 2 months of operation and storage at room temperature.

Phosphorescent oxygen-sensing and singlet oxygen production by a biosynthetic silk

A recombinant coiled-coil silk was utilised to immobilise heavy-metal-macrocycles which are known to undergo efficient intersystem crossing from the singlet state to the triplet state following excitation with visible light. This spin-forbidden transition leads to phosphorescence and the production of cytotoxic oxygen species. We explore the requirements for specific binding of these macrocycles and demonstrate that immobilisation does not adversely affect their photochemical properties. The biocompatible materials developed here have potential biomedical applications in photodynamic therapy (PDT) and dynamic oxygen-sensing.

Recombinant Structural Proteins and Their Use in Future Materials

Recombinant proteins are polymers that offer the materials engineer absolute control over chain length and composition: key attributes required for design of advanced polymeric materials. Through this control, these polymers can be encoded to contain information that enables them to respond as the environment changes. However, despite their promise, protein-based materials are under-represented in materials science. In this chapter we investigate why this is and describe recent efforts to address this. We discuss constraints limiting rational design of structural proteins for advanced materials; advantages and disadvantages of different recombinant expression platforms; and, methods to fabricate proteins into solid-state materials. Finally, we describe the silk proteins used in our laboratory as templates for information-containing polymers.

Bioinspired electrocatalysts for oxygen reduction using recombinant silk films

Fuel cells are a promising avenue for renewable energy production. While oxygen remains the preferred oxidant, its slow reduction kinetics has limited fuel cell performance and it currently requires the use of platinum as the cathode catalyst. In the search for non-platinum cathodes, inspiration has been sought from biological oxygen reduction processes which use heme proteins for respiration. Here, we describe the use of recombinant honeybee silk protein, which can be produced at high scale in E. coli, to generate a heme–protein material. In these solid-state silk materials, a tyrosine residue coordinates directly to the heme iron center. This axial coordination promotes heterolytic O–O bond cleavage, rather than homolytic cleavage, avoiding the generation of destructive hydroxyl radicals. The heme–silk materials can fully reduce oxygen to water with 3.7 electrons transferred to oxygen and only 14% hydrogen peroxide produced. Importantly, the films demonstrate remarkable stability. The films retained activity when used under continuous operation for over 16 hours and retained 85% of their catalytic activity when used at pH 3 for two hours.

Conversion of nitrous oxide to nitrogen by cobalt-substituted myoglobin

Developing technology to decrease greenhouse gas emissions is one of the greatest challenges we face in the 21st century. Nitrous oxide (N2O) is an important greenhouse gas, which is estimated to contribute 6% of the overall global warming effect. Herein we report the use of cobalt substituted heme proteins to reduce N2O to nitrogen (N2). This catalysis was electrochemically driven using methyl viologen or benzyl viologen as electron transfer partners for cobalt myoglobin. Using bulk electrolysis we demonstrated the production of 15N2 from 15N2. This catalysis, however, was noted to be poor, most likely due to oxidative damage to the protein scaffold.

Rational design of new materials using recombinant structural proteins: Current state and future challenges

Sequence-definable polymers are seen as a prerequisite for design of future materials, with many polymer scientists regarding such polymers as the holy grail of polymer science. Recombinant proteins are sequence-defined polymers. Proteins are dictated by DNA templates and therefore the sequence of amino acids in a protein is defined, and molecular biology provides tools that allow redesign of the DNA as required. Despite this advantage, proteins are underrepresented in materials science. In this publication we investigate the advantages and limitations of using proteins as templates for rational design of new materials.

Design of silk proteins with increased heme binding capacity and fabrication of silk-heme materials

In our previous studies, heme was bound into honeybee silk to generate materials that could function as nitric oxide sensors or as recoverable heterogeneous biocatalysts. In this study, we sought to increase the heme-binding capacity of the silk protein by firstly redesigning the heme binding site to contain histidine as the coordinating residue and secondly, by adding multiple histidine residues within the core of the coiled coil core region of the modified silk protein. We used detergent and a protein denaturant to confirm the importance of the helical structure of the silk for heme coordination. Aqueous methanol treatment, which was used to stabilize the materials, transformed the low-spin, six-coordinate heme to a five-coordinate high-spin complex, thus providing a vacant site for ligand binding. The optimal aqueous methanol treatment time that simultaneously maintains the helical protein structure and stabilizes the silk material without substantial leaching of heme from the system was determined.

Modification of Honeybee Silk by the Addition of Antimicrobial Agents

Honeybee silk proteins can be produced at high levels in recombinant systems, fabricated into materials, and are tolerant of amino acid modifications: properties that make them exciting templates for designing new functional materials. Here, we explore the properties of materials either made from silk-antimicrobial peptide (AMP) fusion proteins or silk containing entrapped AMPs or silver nanoparticles. Inclusion of AMP within the silk protein sequence did not affect our ability to express the proteins or process them into films. When AMP-silk proteins and Escherichia coli cells were coincubated in solution, a reduction in cell numbers was observed after degradation of the chimeric protein to release a truncated version of the AMP. In films, the AMP was retained in the silk with leaching rates of <1% per day. Films containing silver nanoparticles were antimicrobial, with the silk preventing aggregation of nanoparticles and slowing the rate of dissolution of the particles.