Synthesis of Anthracene Conjugates of Truncated Antifreeze Protein Sequences: Effect of the End Group and Photocontrolled Dimerization on Ice Recrystallization Inhibition Activity.

Biomacromolecular antifreezes distinguish ice from water, function by binding to specific planes of ice, and could have many applications from cryobiology to aerospace where ice is a problem. In biology, antifreeze protein (AFP) activity is regulated by protein expression levels via temperature and light-regulated expression systems, but in the laboratory (or applications), the antifreeze activity is "always on" without any spatial or temporal control, and hence methods to enable this switching represent an exciting synthetic challenge. Introduction of an abiotic functionality into short peptides (e.g., from solid-phase synthesis) to enable switching is also desirable rather than on full-length recombinant proteins. Here, truncated peptide sequences based on the consensus repeat sequence from type-I AFPs (TAANAAAAAAA) were conjugated to an anthracene unit to explore their photocontrolled dimerization. Optimization of the synthesis to ensure solubility of the hydrophobic peptide included the addition of a dilysine solubilizing linker. It was shown that UV-light exposure triggered reversible dimerization of the AFP sequence, leading to an increase in molecular weight. Assessment of the ice recrystallization inhibition activity of the peptides before and after dimerization revealed only small effects on activity. However, it is reported here for the first time that addition of the anthracene unit to a 22-amino-acid truncated peptide significantly enhanced ice recrystallization inhibition compared to the free peptide, suggesting an accessible synthetic route to allow AFP activity using shorter, synthetically accessible peptides with a photoreactive functionality.

Mimicking the Ice Recrystallization Activity of Biological Antifreezes. When is a New Polymer "Active"?

Antifreeze proteins and ice-binding proteins have been discovered in a diverse range of extremophiles and have the ability to modulate the growth and formation of ice crystals. Considering the importance of cryoscience across transport, biomedicine, and climate science, there is significant interest in developing synthetic macromolecular mimics of antifreeze proteins, in particular to reproduce their property of ice recrystallization inhibition (IRI). This activity is a continuum rather than an "on/off" property and there may be multiple molecular mechanisms which give rise to differences in this observable property; the limiting concentrations for ice growth vary by more than a thousand between an antifreeze glycoprotein and poly(vinyl alcohol), for example. The aim of this article is to provide a concise comparison of a range of natural and synthetic materials that are known to have IRI, thus providing a guide to see if a new synthetic mimic is active or not, including emerging materials which are comparatively weak compared to antifreeze proteins, but may have technological importance. The link between activity and the mechanisms involving either ice binding or amphiphilicity is discussed and known materials assigned into classes based on this.

Structural and functional determination of homologs of the Mycobacterium tuberculosis N-acetylglucosamine-6-phosphate deacetylase (NagA).

The Mycobacterium tuberculosis (Mtb) pathogen encodes a GlcNAc-6-phosphate deacetylase enzyme, NagA (Rv3332), that belongs to the amidohydrolase superfamily. NagA enzymes catalyze the deacetylation of GlcNAc-6-phosphate (GlcNAc6P) to glucosamine-6-phosphate (GlcN6P). NagA is a potential antitubercular drug target because it represents the key enzymatic step in the generation of essential amino-sugar precursors required for Mtb cell wall biosynthesis and also influences recycling of cell wall peptidoglycan fragments. Here, we report the structural and functional characterization of NagA from Mycobacterium smegmatis (MSNagA) and Mycobacterium marinum (MMNagA), close relatives of Mtb Using a combination of X-ray crystallography, site-directed mutagenesis, and biochemical and biophysical assays, we show that these mycobacterial NagA enzymes are selective for GlcNAc6P. Site-directed mutagenesis studies revealed crucial roles of conserved residues in the active site that underpin stereoselective recognition, binding, and catalysis of substrates. Moreover, we report the crystal structure of MSNagA in both ligand-free form and in complex with the GlcNAc6P substrate at 2.6 and 2.0 Å resolutions, respectively. The GlcNAc6P complex structure disclosed the precise mode of GlcNAc6P binding and the structural framework of the active site, including two divalent metals located in the α/ß binuclear site. Furthermore, we observed a cysteine residue located on a flexible loop region that occludes the active site. This cysteine is unique to mycobacteria and may represent a unique subsite for targeting mycobacterial NagA enzymes. Our results provide critical insights into the structural and mechanistic properties of mycobacterial NagA enzymes having an essential role in amino-sugar and nucleotide metabolism in mycobacteria.

Facially Amphipathic Glycopolymers Inhibit Ice Recrystallization.

Antifreeze glycoproteins (AFGPs) from polar fish are the most potent ice recrystallization (growth) inhibitors known, and synthetic mimics are required for low-temperature applications such as cell cryopreservation. Here we introduce facially amphipathic glycopolymers that mimic the three-dimensional structure of AFGPs. Glycopolymers featuring segregated hydrophilic and hydrophobic faces were prepared by ring-opening metathesis polymerization, and their rigid conformation was confirmed by small-angle neutron scattering. Ice recrystallization inhibition (IRI) activity was reduced when a hydrophilic oxo-ether was installed on the glycan-opposing face, but significant activity was restored by incorporating a hydrophobic dimethylfulvene residue. This biomimetic strategy demonstrates that segregated domains of distinct hydrophilicity/hydrophobicity are a crucial motif to introduce IRI activity, which increases our understanding of the complex ice crystal inhibition processes.

Polyproline as a Minimal Antifreeze Protein Mimic That Enhances the Cryopreservation of Cell Monolayers.

Tissue engineering, gene therapy, drug screening, and emerging regenerative medicine therapies are fundamentally reliant on high-quality adherent cell culture, but current methods to cryopreserve cells in this format can give low cell yields and require large volumes of solvent "antifreezes". Herein, we report polyproline as a minimum (bio)synthetic mimic of antifreeze proteins that is accessible by solution, solid-phase, and recombinant methods. We demonstrate that polyproline has ice recrystallisation inhibition activity linked to its amphipathic helix and that it enhances the DMSO cryopreservation of adherent cell lines. Polyproline may be a versatile additive in the emerging field of macromolecular cryoprotectants.