Radical-Mediated Covalent Azidylation of Hydrophobic Microdomains in Water-Soluble Proteins.

Hydrophobic microdomains, also known as hydrophobic patches, are essential for many important biological functions of water-soluble proteins. These include ligand or substrate binding, protein-protein interactions, proper folding after translation, and aggregation during denaturation. Unlike transmembrane domains, which are easily recognized from stretches of contiguous hydrophobic sidechains in amino acids via primary protein sequence, these three-dimensional hydrophobic patches cannot be easily predicted. The lack of experimental strategies for directly determining their locations hinders further understanding of their structure and function. Here, we posit that the small triatomic anion N3- (azide) is attracted to these patches and, in the presence of an oxidant, forms a radical that covalently modifies C-H bonds of nearby amino acids. Using two model proteins (BSA and lysozyme) and a cell-free lysate from the model higher plant Arabidopsis thaliana, we find that radical-mediated covalent azidylation occurs within buried catalytic active sites and ligand binding sites and exhibits similar behavior to established hydrophobic probes. The results herein suggest a model in which the azido radical is acting as an "affinity reagent" for nonaqueous three-dimensional protein microenvironments and is consistent with both the nonlocalized electron density of the azide moiety and the known high reactivity of azido radicals widely used in organic chemistry syntheses. We propose that the azido radical is a facile means of identifying hydrophobic microenvironments in soluble proteins and, in addition, provides a simple new method for attaching chemical handles to proteins without the need for genetic manipulation or specialized reagents.

Mass spectrometric based analysis of whole eggs dissolved in formic acid.

We have developed a method for complete dissolution of whole eggs in formic acid that provides a new approach to analyzing egg biomolecules. As expected from prior work with extracted lipids, phosphatidylcholine represents the most abundant 31P NMR signal. A simplified methanol/chloroform partitioning method for separating the dissolved egg solution into metabolites, lipids and protein was performed and after ultra-high mass resolution and tandem MS fragmentation analyses several phosphatidylcholine molecules containing different fatty acid chain lengths as well as number and position of double bonds was detected. The MS based proteomic analysis further revealed 6 Gallus sequences annotated as 'uncharacterized' because they show no sequence homology with any other protein found in nature and thus, may represent proteins uniquely evolved to perform functions specific to chickens. Overall, this procedure is a rapid and facile means of characterizing in a high throughput and comprehensive manner, the molecular components of whole eggs.