The human microbiome encodes vast numbers of uncharacterized enzymes, limiting our functional understanding of this community and its effects on host health and disease. By incorporating information about enzymatic chemistry into quantitative metagenomics, we determined the abundance and distribution of individual members of the glycyl radical enzyme superfamily among the microbiomes of healthy humans. We identified many uncharacterized family members, including a universally distributed enzyme that enables commensal gut microbes and human pathogens to dehydrate trans-4-hydroxy-L-proline, the product of the most abundant human posttranslational modification. This ‘chemically-guided functional profiling’ workflow can therefore use ecological context to facilitate the discovery of enzymes in microbial communities.

Fluorine ((19)F) NMR is a valuable tool for studying dynamic biological processes. However, increasing the sensitivity of fluorinated reporter molecules is a key to reducing acquisition times and accessing transient biological interactions. Here, we evaluate the utility a novel amino acid, L-O-(perfluoro-t-butyl)-homoserine (pFtBSer), that can easily be synthesized and incorporated into peptides and provides greatly enhanced sensitivity over currently used (19)F biomolecular NMR probes. Incorporation of pFtBSer into the potent antimicrobial peptide MSI-78 results in a sharp (19)F NMR singlet that can be readily detected at concentrations of 5 µm and lower. We demonstrate that pFtBSer incorporation into MSI-78 provides a sensitive tool to study binding through (19)F NMR chemical shift and nuclear relaxation changes. These results establish future potential for pFtBSer to be incorporated into various proteins where NMR signal sensitivity is paramount, such as in-cell investigations.

Propanediol dehydratase (PD), a recently characterized member of the glycyl radical enzyme (GRE) family, uses protein-based radicals to catalyze the chemically challenging dehydration of ( S)-1,2-propanediol. This transformation is also performed by the well-studied enzyme B-dependent propanediol dehydratase (B-PD) using an adenosylcobalamin cofactor. Despite the prominence of PD in anaerobic microorganisms, it remains unclear if the mechanism of this enzyme is similar to that of B-PD. Here we report O labeling experiments that suggest PD and B-PD employ distinct mechanisms. Unlike B-PD, PD appears to catalyze the direct elimination of a hydroxyl group from an initially formed substrate-based radical, avoiding the generation of a 1,1- gem diol intermediate. Our studies provide further insights into how GREs perform elimination chemistry and highlight how nature has evolved diverse strategies for catalyzing challenging reactions.

The introduction of highly fluorinated analogues of hydrophobic amino acid residues into proteins has proved an effective and general strategy for increasing protein stability toward both chemical denaturants and heat. However, the thermodynamic basis for this stabilizing effect, whether enthalpic or entropic in nature, has not been extensively investigated. Here we describe studies in which the values of ΔH°, ΔS°, and ΔCp° have been determined for the unfolding of a series of 12 small, de novo-designed proteins in which the hydrophobic core is packed with various combinations of fluorinated and non-fluorinated amino acid residues. The increase in the free energy of unfolding with increasing fluorine content is associated with increasingly unfavorable entropies of unfolding and correlates well with calculated changes in apolar solvent-accessible surface area. ΔCp° for unfolding is positive for all the proteins and, similarly, correlates with changes in apolar solvent-accessible surface area. ΔH° for unfolding shows no correlation with either fluorine content or changes in apolar solvent-accessible surface area. We conclude that conventional hydrophobic effects adequately explain the enhanced stabilities of most highly fluorinated proteins. The extremely high thermal stability of these proteins results, in part, from their very low per-residue ΔCp°, as has been observed for natural thermostable proteins.

Surfers are a heterogeneous population with a common interest in riding waves. Surfers qualitatively describe the surfing sensation as a hybrid of meditative and athletic experience. Numerous empirical studies link both meditative experience and exercise with reduced incidence of depression and anxiety; this potentially suggests that surfers may endorse fewer symptoms of either disorder. One hundred surfers (N= 100) were administered the Beck Depression Inventory-II, the Beck Anxiety Inventory, the Coping Inventory for Stressful Situations, and a demographics questionnaire. Results indicate that surfers reported significantly fewer symptoms of depression and anxiety, and employed emotion-based coping responses to stressful situations significantly less than the general populace. Surfers also employed avoidance-based coping strategies more frequently than the general populace. Future study should evaluate causal relationships between surfing and incidence of depression and anxiety.