Protecting Proteins from Desiccation Stress Using Molecular Glasses and Gels.

Faced with desiccation stress, many organisms deploy strategies to maintain the integrity of their cellular components. Amorphous glassy media composed of small molecular solutes or protein gels present general strategies for protecting against drying. We review these strategies and the proposed molecular mechanisms to explain protein protection in a vitreous matrix under conditions of low hydration. We also describe efforts to exploit similar strategies in technological applications for protecting proteins in dry or highly desiccated states. Finally, we outline open questions and possibilities for future explorations.

Properties of a tardigrade desiccation-tolerance protein aerogel.

Lyophilization is promising for tackling degradation during the drying and storage of protein-based drugs. Tardigrade cytosolically abundant heat soluble (CAHS) proteins are necessary and sufficient for desiccation-tolerance in vivo and protein protection in vitro. Hydrated CAHS proteins form coiled-coil-based fine-stranded, cold-setting hydrogels, but the dried protein remains largely uncharacterized. Here, we show that dried CAHS D gels (i.e., aerogels) retain the structural units of their hydrogels, but the details depend on prelyophilization CAHS concentrations. Low concentration samples (<10 g/L) form thin (<0.2 µm) tangled fibrils lacking regular structure on the micron scale. Upon increasing the concentration, the fibers thicken and form slabs comprising the walls of the aerogel pores. These changes in morphology are associated with a loss in disorder and an increase in large ß sheets and a decrease in α helices and random coils. This disorder-to-order transition is also seen in hydrated gels as a function of concentration. These results suggest a mechanism for pore formation and indicate that using CAHS proteins as excipients will require attention to initial conditions because the starting concentration impacts the lyophilized product.

Macromolecular Crowding by Polyethylene Glycol Reduces Protein Breathing.

Most efforts to understand macromolecular crowding focus on global (i.e., complete) unfolding, but smaller excursions, often called breathing, promote aggregation, which is associated with several diseases and the bane of pharmaceutical and commercial protein production. We used NMR to assess the effects of ethylene glycol (EG) and polyethylene glycols (PEGs) on the structure and stability of the B1 domain of protein G (GB1). Our data show that EG and PEGs stabilize GB1 differently. EG interacts with GB1 more strongly than PEGs, but neither affects the structure of the folded state. EG and 12000 g/mol PEG stabilize GB1 more than PEGs of intermediate size, but EG and smaller PEGs stabilize GB1 enthalpically while the largest PEG acts entropically. Our key finding is that PEGs turn local unfolding into global unfolding, and meta-analysis of published data supports this conclusion. These efforts provide knowledge that can be applied to improve biological drugs and commercial enzymes.

Lists of Chemical Warfare Agents and Precursors from International Nonproliferation Frameworks: Structural Annotation and Chemical Fingerprint Analysis.

To support efforts to stem the proliferation of chemical weapons (CWs), we have curated and structurally annotated CW-control lists from three key international nonproliferation frameworks: the Chemical Weapons Convention (CWC), the Australia Group (AG), and the Wassenaar Arrangement. The curated lists are available as web tables at the Costanzi Research website (https://costanziresearch.com/cw-control-lists/). The annotations include manually curated 2D structural images, which provide a means to appreciate at a glance the similarities and differences between different entries, as well as downloadable 2D structures, in two different formats and three different structural identifiers, namely, simplified molecular-input line-entry system, standard InChI, and standard InChIKey, which are intended to provide a platform for cheminformatics analyses. The tables also include links to National Center for Biotechnology Information's PubChem and National Institute of Standards and Technology's Chemistry WebBook cards, hence providing prompt access to a wealth of physicochemical, analytical chemistry, and toxicological information. To showcase the importance of structural annotations, we discuss a discrepancy in a CW-control list covering the defoliant Agent Orange, which we identified through our curation process, and propose a solution to address it. Moreover, we present the results of chemical fingerprinting analyses, through which we clustered the entries of the three CW-control lists under study into structurally related groups and studied the overlaps between the three lists. As an application of this study, we examine the recent updates of CWC Schedule 1 and the AG precursors list, highlighting the relationships between the two amendments and proposing the possible addition of further chemicals. Our research is intended to facilitate the communication between scientific advisors and policymakers as well as the work of chemists and cheminformaticians involved in the CW nonproliferation field. Ultimately, we seek to provide tools to bolster the control of CWs and support the global efforts to rid the world of this category of weapons.