Molten Proteins: synthesis and design of novel biomolecule-based liquid nanomaterials and their application in bionanochemistry

Making new materials that have small-scale structures and multiple components is expected to be of great importance in a wide range of applications such as sensing, data storage, electronics and catalysis. One new area where small-scale structures could make a significant breakthrough is in the use of proteins, which are large biological molecules with a wide range of properties. There is therefore a growing interest in preparing nanomaterials that include biological components because molecules such as proteins and enzymes have finely tuned activities not readily available in synthetic counterparts. Proteins are similar to many other forms of nanoscale objects in that they have persistent 3-D structures that can be prepared in the form of dry powders, or more usually, as dispersions in aqueous solutions. However, it is interesting to note that proteins in the pure liquid state are not known; they simply do not exist at ambient temperature and pressure. As a consequence there is a missing state of biomolecular matter that remains to be discovered and explored.The absence of a liquid protein phase in the absence of solvent is a problem that is encountered with nanoparticles in general, and raises fundamental questions concerning the potential existence of this state of matter in nanoscale objects. The problem arises because the liquid state is stabilized by inter-molecular forces that extend considerably in range compared with the size of the individual molecules, but this relationship breaks down for proteins, which are generally larger than the range of the force field. So, whilst heating a conventional solid under atmospheric pressure usually produces the liquid state because the increased thermal energy is dissipated by correlated motions between the molecules, heating a dried protein powder results in thermal degradation. That is, the protein molecules are so firmly held together at a very short range and hardly interact at a longer distance that the increase in thermal energy destroys the molecular structure, or when under very low pressure, drives the molecules directly into the gas phase (sublimation), where the intermolecular forces are very weak or non-existent.The proposed research aims to address this missing state of biomolecular matter by producing the first examples of liquid proteins. We intend to do this by modifying the surface properties of several different types of proteins such that the molecules will continue to interact at longer distances. Effectively what we will do is chemically attach groups to the protein surface that behave as a fluidization layer in the absence of a solvent. These groups need to be designed carefully so that the modified proteins behave as a single component so that true liquids can be prepared. In our preliminary studies we have achieved this by first making the protein surface highly positively charged, and then adding a negatively charged polymer surfactant that electrostatically binds to the cationic sites. We then meticulously remove all the water by freeze drying techniques to give a soft solid that melts at around 27 degress to produce a liquid protein. Our proposed work intends to develop this new approach to discover a wide range of liquid proteins with different functions. In each case we will investigate the internal structure of the liquids, as well as their composition and properties such as viscosity. We will also determine if the natural properties of the proteins are still active in the liquid state. Finally, once we understand how these systems work, then it should be possible to use the results to start to develop new types of materials based on liquid proteins. For example, we are interested in exploring the protein melts as smart liquids, biosensors and as new types of materials for use as wound dressings.

Research in nanoscience continues to develop rapidly, and in many cases new functional materials in the form of inorganic, organic or hybrid nanoparticles are being exploited as bulk powders, thin films, 2D superlattices, 3D crystals, or as colloidal suspensions in aqueous or non-aqueous solvents. However, the absence of solvent-free nanoparticle liquids has generally gone unnoticed and unquestioned, even though such materials could be of great practical interest. Thus our proposed work, which aims to extend the phase behaviour of proteins to include the liquid state and liquid crystalline fluids, will represent a significant advance in the properties and potential applications of proteins in diverse fields such as bio-nanotechnology, diagnostics, and food and health care. Our work will therefore have a significant impact on promoting new possibilities that could increase the competitiveness and economic performance of UK-based sectors of applied nanoscience. In particular, we expect significant interest in our work from small-scale companies involved in commercialization of new nanoscale-based technologies. As we expect the strategies described in this paper to contribute significantly to the general preparation of a new class of substances in the form of solvent-free protein melts with zero vapour pressure, we envisage several key areas of impact in the wider economic environment concerning near-future technologies. Liquid melts comprise exceedingly high mass percentages of protein (650 mg per 1g of melt for ferritin), and should find applications as smart fluids, for example in enzymatic catalysis and sensing, with the additional advantage of solvent-free storage. Liquid proteins could also offer new opportunities as novel solvents, and be employed as functional components of multiphase systems prepared from mixtures of solvent-free melts. For example, smart fluids that exhibit magnetic or electrically-conductive properties could be produced by preparing blends with inorganic nanoparticles, or green processing of functional devices achieved from nanostructured films of the protein melts. These technologies could have general utility; for instance, protein fluids with high thermal mass and conductivity might show promise as new coolants or lubricants, or as novel reaction media with zero vapour pressures. Solvent-less liquid proteins could also provide a unique opportunity to blend the structure-function relationships of proteins (catalysis), with the structure-driven rheological properties of polymers and polymer surfactants. As solvent-less liquid proteins have a mass fraction well in excess of the aqueous solubility limit, they may have a significant impact in the development of new health care technologies. For example, they could be developed as high-potency topical enzymatic treatments, and as pharmaceutical transport and storage agents. An area that could benefit significantly from these materials is the development of new wound dressing treatments, where large protein concentrations could be applied locally in the form of barrier films consisting of melts with controllable properties. For example, oxygen delivery to a wound might be accomplished using a glucose oxidase liquid protein coupled with a glucose-containing alginate or chitin gel. Alternatively, solvent-less liquids of proteolytic enzymes, could be developed as barrier films to promote the degradation of necrotic debris associated with cell breakdown upon healing. Finally, the concept of solvent-less liquid proteins can be communicated in a straightforward manner without the need for jargon or esoteric technical know-how. The concept behind our work can therefore be readily understood within the wider non-scientifically trained public arena. Moreover, as it addresses fundamental questions such as what is a liquid , why do liquids exist or not exist? , it is a topic that would engage the public interest in science education in general.