Drug resistance is a public health concern that threatens to undermine decades of medical progress. ESKAPE pathogens cause most nosocomial infections, and are frequently resistant to carbapenem antibiotics, usually leaving tigecycline and colistin as the last treatment options. However, increasing tigecycline resistance and colistin’s nephrotoxicity severely restrict use of these antibiotics. We have designed antimicrobial peptides using a maximum common subgraph approach. Our best peptide (Ω76) displayed high efficacy against carbapenem and tigecycline-resistant Acinetobacter baumannii in mice. Mice treated with repeated sublethal doses of Ω76 displayed no signs of chronic toxicity. Sublethal Ω76 doses co-administered alongside sublethal colistin doses displayed no additive toxicity. These results indicate that Ω76 can potentially supplement or replace colistin, especially where nephrotoxicity is a concern. To our knowledge, no other existing antibiotics occupy this clinical niche. Mechanistically, Ω76 adopts an α-helical structure in membranes, causing rapid membrane disruption, leakage, and bacterial death.

Natural molecular machines contain protein components that undergo motion relative to each other. Designing such mechanically constrained nanoscale protein architectures with internal degrees of freedom is an outstanding challenge for computational protein design. Here we explore the de novo construction of protein machinery from designed axle and rotor components with internal cyclic or dihedral symmetry. We find that the axle-rotor systems assemble in vitro and in vivo as designed. Using cryo–electron microscopy, we find that these systems populate conformationally variable relative orientations reflecting the symmetry of the coupled components and the computationally designed interface energy landscape. These mechanical systems with internal degrees of freedom are a step toward the design of genetically encodable nanomachines.

Computational protein design is a rapidly maturing field within structural biology, with the goal of designing proteins with custom structures and functions. Such proteins could find widespread medical and industrial applications. Here, we have adapted algorithms from the Rosetta software suite to design much larger proteins, based on ideal geometric and topological criteria. Furthermore, we have developed techniques to incorporate symmetry into designed structures. For our first design attempt, we targeted the (α/β)8 TIM barrel scaffold. We gained novel insights into TIM barrel folding mechanisms from studying natural TIM barrel structures, and from analyzing previous TIM barrel design attempts. Computational protein design and analysis was performed using the Rosetta software suite and custom scripts. Genes encoding all designed proteins were synthesized and cloned on the pET20-b vector. Standard circular dichroism and gel chromatographic experiments were performed to determine protein biophysical characteristics. 1D NMR and 2D HSQC experiments were performed to determine protein structural characteristics. Extensive protein design simulations coupled with ab initio modeling yielded several all-atom models of ideal, 4-fold symmetric TIM barrels. Four such models were experimentally characterized. The best designed structure (Symmetrin-1) contained a polar, histidine-rich pore, forming an extensive hydrogen bonding network. Symmetrin-1 was easily expressed and readily soluble. It showed circular dichroism spectra characteristic of well-folded alpha/beta proteins. Temperature melting experiments revealed cooperative and reversible unfolding, with a Tm of 44 °C and a Gibbs free energy of unfolding (ΔG°) of 8.0 kJ/mol. Urea denaturing experiments confirmed these observations, revealing a Cm of 1.6 M and a ΔG° of 8.3 kJ/mol. Symmetrin-1 adopted a monomeric conformation, with an apparent molecular weight of 32.12 kDa, and displayed well resolved 1D-NMR spectra. However, the HSQC spectrum revealed somewhat molten characteristics. Despite the detection of molten characteristics, the creation of a soluble, cooperatively folding protein represents an advancement over previous attempts at TIM barrel design. Strategies to further improve Symmetrin-1 are elaborated. Our techniques may be used to create other large, internally symmetric proteins.

Antimicrobial peptides are ubiquitous molecules that form the innate immune system of organisms across all kingdoms of life. Despite their prevalence and early origins, they continue to remain potent natural antimicrobial agents. Antimicrobial peptides are therefore promising drug candidates in the face of overwhelming multi-drug resistance to conventional antibiotics. Over the past few decades, thousands of antimicrobial peptides have been characterized in vitro, and their efficacy data are now available in a multitude of public databases. Computational antimicrobial peptide design attempts typically use such data. However, utilizing heterogenous data aggregated from different sources presents significant drawbacks. In this report, we present a uniform dataset containing 20 antimicrobial peptides assayed against 30 organisms of Gram-negative, Gram-positive, mycobacterial, and fungal origin. We also present circular dichroism spectra for all antimicrobial peptides. We draw simple inferences from this data, and we discuss what characteristics are essential for antimicrobial peptide efficacy. We expect our uniform dataset to be useful for future projects involving computational antimicrobial peptide design.

Aspergillus niger strain melanoliber was isolated from the coastal region of Mumbai, Maharashtra, India. This strain secretes water-soluble melanin attached to peptides possessing metal-chelation properties. Here, we report the whole-genome sequence of Aspergillus niger strain melanoliber. The genome was sequenced using Illumina Novaseq6000 giving a total size of 3.86 Mb.

Melanin is an amorphous, highly heterogeneous polymer found across all kingdoms of life. Although the properties of melanin can greatly vary, most forms are insoluble and strongly absorb light, appearing dark brown to black. Here, we describe a water-soluble form of melanin (peptidomelanin) secreted by the spores of Aspergillus niger (strain: melanoliber) during germination. Peptidomelanin is composed of an insoluble L-DOPA core polymer that is solubilized via short, copolymerized heterogeneous peptide chains forming a “corona” with a mean amino acid length of 2.6 ± 2.3. Based on in vitro experiments, we propose a biochemical copolymerization mechanism involving the hydroxylation of tyrosynylated peptides. Peptidomelanin is capable of chelating heavy metals such as lead, mercury, and uranium (as uranyl) in large quantities. Preliminary data indicates that peptidomelanin may have applications for the remediation of heavy metals in situ, including in agricultural settings.