Kelly Velonia

Well-defined protein–polymer conjugates—synthesis and potential applications

During the last decades, numerous studies have focused on combining the unique catalytic/functional properties and structural characteristics of proteins and enzymes with those of synthetic molecules and macromolecules. The aim of such multidisciplinary studies is to improve the properties of the natural component, combine them with those of the synthetic, and create novel biomaterials in the nanometer scale. The specific coupling of polymers onto the protein structures has proved to be one of the most straightforward and applicable approaches in that sense. In this article, we focus on the synthetic pathways that have or can be utilized to specifically couple proteins to polymers. The different categories of well-defined protein–polymer conjugates and the effect of the polymer on the protein function are discussed. Studies have shown that the specific conjugation of a synthetic polymer to a protein conveys its physico-chemical properties and, therefore, modifies the biodistribution and solubility of the protein, making it in certain cases soluble and active in organic solvents. An overview of the applications derived from such bioconjugates in the pharmaceutical industry, biocatalysis, and supramolecular nanobiotechnology is presented at the final part of the article.

In situ ATRP-mediated hierarchical formation of giant amphiphile bionanoreactors.

Amphiphilic bioconjugates can be synthesized in situ by grafting polystyrene from a protein. The resulting giant amphiphiles display low polydispersities and the characteristic aggregation properties of amphiphilic biomacromolecules. A second, catalytically active guest protein can also be included within the superstructures.

Formation of giant amphiphiles by post-functionalization of hydrophilic protein–polymer conjugates

A novel, generic method for the synthesis of families of tri-block protein–polymer giant amphiphiles was designed and developed. We have synthesized a hydrophilic α-maleimido poly-1-alkyne with Mn = 9.5 kDa (1H-NMR) and narrow PDi (1.15 as measured by SEC) via ATRP (Atom Transfer Radical Polymerization). This polymer was succesfully coupled to BSA to afford a hydrophilic multifunctional bioconjugate which was isolated using protein purification techniques and fully characterized. Following the post-functionalization approach, we introduced hydrophobicity to the resulting hydrophilic biohybrid by a straightforward, high yield “click”-chemistry cycloaddition step. The resulting tri-block protein–polymer amphiphiles were isolated and showed interesting aggregation patterns (TEM, confocal microscopy).

Protein-polymer amphiphilic chimeras: recent advances and future challenges

During the past decades numerous studies have focused on the bridging of components derived from both the natural and the synthetic world with the aim of creating new biomaterials with improved properties and functions. This review illustrates the range of well-defined protein-polymer amphiphiles—Giant Amphiphiles—prepared in very recent years. Giant Amphiphiles are classified in terms of the conceptually different approaches developed to tackle their synthesis. The variety of high turnover methodologies, the expression of the intrinsic properties of the synthetic and/or the biological component and the aggregation/activity profile of each individual system are discussed with the aim of providing a basis for the rational design of new generations of biomacromolecular chimeras aimed for high-impact applications.

LCD-based detection of enzymatic action

By incorporating an ester-containing substrate in a self-assembled alignment layer for liquid crystal cells, the presence of a lipase (CALB) can be directly detected through its enzymatic action on the alignment layer, without the need for fluorescent labelling or enzyme assays.

Stereospecificity of hydrogen transfer by the NAD+-linked alcohol dehydrogenase from the Antarctic psychrophile Moraxella sp. TAE123

Investigation of the stereochemistry of the hydride transfer in reactions catalyzed by the recently isolated NAD(+)-linked alcohol dehydrogenase from the Antarctic psychrophile Moraxella sp. TAE123 was accomplished by using 1H NMR spectroscopy of the deuterated coenzyme. It was found that this new psychrophilic enzyme is a type A dehydrogenase. Moraxella sp. ADH reduces stereospecifically 2-butanone to produce (S)-2-butanol.

Amphiphilic poly-N-vinylpyrrolidone nanoparticles as carriers for non-steroidal, anti-inflammatory drugs: In vitro cytotoxicity and in vivo acute toxicity study

Polymeric nanoparticles were prepared from self-assembled amphiphilic N-vinylpyrrolidone polymers in aqueous media and evaluated as novel carriers of indomethacin, a non-steroidal, anti-inflammatory drug. It was determined that these nanoparticles could be created in spherical morphologies with sizes less than 100nm, narrow size distributions and high indomethacin contents(up to 35%) combined with high drug loading efficiencies(up to 95%). In cytotoxicity tests using the human embryonic stem cell derived fibroblasts (EBF-H9) and hepatocellular carcinoma cells (HepG2), the indomethacin-loaded polymeric nanoparticles showed higher cell viability compared to that of free indomethacin at the same concentration. The median LD50 values, determined by the Litchfield-Wilcoxon method, were 55-70mg/kg body weight depending on the polymer molecular design in both mice and rats. Based on the acquired results, these novel amphiphilic poly-N-vinylpyrrolidone nanoparticles can be considered as potential carriers for new, highly efficient, injectable drug delivery systems for hydrophobic drugs such as indomethacin.

Correctly validating results from single molecule data: The case of stretched exponential decay in the catalytic activity of single lipase B molecules

The question of how to validate and interpret correctly the waiting time probability density functions (WT-PDFs) from single molecule data is addressed. It is shown by simulation that when a stretched exponential WT-PDF, /off ðt Þ¼ /0e �ð t=sÞ a , generates the off periods of a two-state trajectory, a reliable recovery of the input /off(t) from the trajectory is obtained even when the bin size used to define the trajectory, dt, is much larger than the parameter s. This holds true as long as the first moment of the WT-PDF is much larger than dt. Our results validate the results in an earlier study of the activity of single lipase B molecules and disprove recent related critique.

Dismutation of aldehydes catalyzed by alcohol dehydrogenases

The dismutation of aldehydes with the following three alcohol dehydrogenases, the mesophilic Saccharomyces cerevisiae ADH; the thermophilic Thermoanaerobium brockii ADH; and the recently isolated psychrophilic Moraxella sp. TAE123 ADH, was studied with high-resolution 1H NMR spectroscopy. All three ADHs catalyzed the rapid dismutation of aldehydes to the corresponding alcohols and carboxylic acids.

Stereospecificity of hydride transfer during the dismutation of aldehydes catalyzed by alcohol dehydrogenases

The stereochemistry of the oxidation of aldehydes to acids with alcohol dehydrogenases was studied with respect to the selectivity towards the cofactor.