Developing Novel Biointerfaces: Using Chlorhexidine Surface Attachment as a Method for Creating Anti-Fungal Surfaces.

There is an increasing focus in healthcare environments on combatting antimicrobial resistant infections. While bacterial infections are well reported, infections caused by fungi receive less attention, yet have a broad impact on society and can be deadly. Fungi are eukaryotes with considerable shared biology with humans, therefore limited technologies exist to combat fungal infections and hospital infrastructure is rarely designed for reducing microbial load. In this study, a novel antimicrobial surface (AMS) that is modified with the broad-spectrum biocide chlorhexidine is reported. The surfaces are shown to kill the opportunistic fungal pathogens Candida albicans and Cryptococcus neoformans very rapidly (<15 min) and are significantly more effective than current technologies available on the commercial market, such as silver and copper.

Location-Dependent Lanthanide Selectivity Engineered into Structurally Characterized Designed Coiled Coils.

Herein we report unprecedented location-dependent, size-selective binding to designed lanthanide (Ln3+ ) sites within miniature protein coiled coil scaffolds. Not only do these engineered sites display unusual Ln3+ selectivity for moderately large Ln3+ ions (Nd to Tb), for the first time we demonstrate that selectivity can be location-dependent and can be programmed into the sequence. A 1 nm linear translation of the binding site towards the N-terminus can convert a selective site into a highly promiscuous one. An X-ray crystal structure, the first of a lanthanide binding site within a coiled coil to be reported, coupled with CD studies, reveal the existence of an optimal radius that likely stems from the structural constraints of the coiled coil scaffold. To the best of our knowledge this is the first report of location-dependent metal selectivity within a coiled coil scaffold, as well as the first report of location-dependent Ln3+ selectivity within a protein.

Stability of Cell-Penetrating Peptide anti-VEGF Formulations for the Treatment of Age-Related Macular Degeneration.

AIM: The development of a polyarginine cell-penetrating peptide (CPP) could enable the treatment of age-related macular degeneration, with drugs like bevacizumab, to be administered using eye drops instead of intravitreal injections. Topical formulations have a vast potential impact on healthcare by increasing patient compliance while reducing the financial burden. However, as the ocular preparations may contain several doses, it is essential to understand the stability of the bevacizumab+CPP conjugate produced. MATERIALS AND METHODS: In this work, we examine the stability of a bevacizumab solution with and without cell-penetrating peptide using dynamic light scattering and circular dichroism to assess the physical stability. We use HPLC to assess the chemical stability and ELISA to assess its biological activity. We also examine the potential of the CPP to be used as an antimicrobial agent in place of preservatives in the eye drop. RESULTS: The structural stability of bevacizumab with and without the CPP was found not to be affected by temperature: samples stored at either 20°C or 4°C were identical in behavior. However, physical instability was observed after five weeks, leading to aggregation and precipitation. Further investigation revealed that the addition of the polypeptide led to increased aggregation, as revealed through dynamic light scattering and concentration analysis of the peptide through HPLC. Complexing the bevacizumab with CPP had no effect on biological stability or degradation. CONCLUSIONS: Our findings suggest that the shelf life of CPP+bevacizumab complexes is at least 38 days from its initial formulation. Currently, the mechanism for aggregation is not fully understood but does not appear to occur through chemical degradation.

Tuning coordination chemistry through the second sphere in designed metallocoiled coils.

The metal hydration state within a designed coiled coil can be progressively tuned across the full integer range (3 → 0 aqua ligands), by careful choice of a second sphere terminal residue, including the lesser used Trp. Potential implications include a four-fold change in MRI relaxivity when applied to lanthanide coiled coils.

De Novo Design of Xeno-Metallo Coiled Coils.

Bioinorganic chemists aspire to achieve the same exquisite and highly controlled inorganic chemistry featured in biology. An exciting mimetic approach involves the use of miniature artificial protein scaffolds designed de novo (often based on the coiled coil (CC) scaffold), for reproducing native metal ion sites and their function. Recently, there is increased interest, instead, in the design of xeno-metal sites within CC assemblies. This involves incorporating either non-biological metal ions, cofactors or non-proteinogenic amino acid ligands for metal ion coordination, whilst retaining a minimal CC protein scaffold. Using this approach, one should be able to create functional designs with unique and unusual properties, which combine the advantages of both biology and 'traditional' non-biological inorganic chemistry. It is the recent progress with respect to the design of xeno-metallo CCs which will be discussed in this Focus Review.

Location dependent coordination chemistry and MRI relaxivity, in de novo designed lanthanide coiled coils.

Herein, we establish for the first time the design principles for lanthanide coordination within coiled coils, and the important consequences of binding site translation. By interrogating design requirements and by systematically translating binding site residues, one can influence coiled coil stability and more importantly, the lanthanide coordination chemistry. A 10 Å binding site translation along a coiled coil, transforms a coordinatively saturated Tb(Asp)3(Asn)3 site into one in which three exogenous water molecules are coordinated, and in which the Asn layer is no longer essential for binding, Tb(Asp)3(H2O)3. This has a profound impact on the relaxivity of the analogous Gd(iii) coiled coil, with more than a four-fold increase in the transverse relaxivity (21 to 89 mM-1 s-1), by bringing into play, in addition to the outer sphere mechanism present for all Gd(iii) coiled coils, an inner sphere mechanism. Not only do these findings warrant further investigation for possible exploitation as MRI contrast agents, but understanding the impact of binding site translation on coordination chemistry has important repercussions for metal binding site design, taking us an important step closer to the predictable and truly de novo design of metal binding sites, for new functional applications.