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.

Expression of a translationally fused TAP-tagged plasma membrane proton pump in Arabidopsis thaliana.

The Arabidopsis thaliana plasma membrane proton ATPase genes, AHA1 and AHA2, are the two most highly expressed isoforms of an 11 gene family and are collectively essential for embryo development. We report the translational fusion of a tandem affinity-purification tag to the 5' end of the AHA1 open reading frame in a genomic clone. Stable expression of TAP-tagged AHA1 in Arabidopsis rescues the embryonic lethal phenotype of endogenous double aha1/aha2 knockdowns. Western blots of SDS-PAGE and Blue Native gels show enrichment of AHA1 in plasma membrane fractions and indicate a hexameric quaternary structure. TAP-tagged AHA1 rescue lines exhibited reduced vertical root growth. Analysis of the plasma membrane and soluble proteomes identified several plasma membrane-localized proteins with alterred abundance in TAP-tagged AHA1 rescue lines compared to wild type. Using affinity-purification mass spectrometry, we uniquely identified two additional AHA isoforms, AHA9 and AHA11, which copurified with TAP-tagged AHA1. In conclusion, we have generated transgenic Arabidopsis lines in which a TAP-tagged AHA1 transgene has complemented all essential endogenous AHA1 and AHA2 functions and have shown that these plants can be used to purify AHA1 protein and to identify in planta interacting proteins by mass spectrometry.

A pipeline for 15N metabolic labeling and phosphoproteome analysis in Arabidopsis thaliana.

Within the past two decades, the biological application of mass spectrometric technology has seen great advances in terms of innovations in hardware, software, and reagents. Concurrently, the burgeoning field of proteomics has followed closely (Yates et al., Annu Rev Biomed Eng 11:49-79, 2009)-and with it, importantly, the ability to globally assay altered levels of posttranslational modifications in response to a variety of stimuli. Though many posttranslational modifications have been described, a major focus of these efforts has been protein-level phosphorylation of serine, threonine, and tyrosine residues (Schreiber et al., Proteomics 8:4416-4432, 2008). The desire to examine changes across signal transduction cascades and networks in their entirety using a single mass spectrometric analysis accounts for this push-namely, preservation and enrichment of the transient yet informative phosphoryl side group. Analyzing global changes in phosphorylation allows inferences surrounding cascades/networks as a whole to be made. Towards this same end, much work has explored ways to permit quantitation and combine experimental samples such that more than one replicate or experimental condition can be identically processed and analyzed, cutting down on experimental and instrument variability, in addition to instrument run time. One such technique that has emerged is metabolic labeling (Gouw et al., Mol Cell Proteomics 9:11-24, 2010), wherein biological samples are labeled in living cells with nonradioactive heavy isotopes such as (15)N or (13)C. Since metabolic labeling in living organisms allows one to combine the material to be processed at the earliest possible step, before the tissue is homogenized, it provides a unique and excellent method for comparing experimental samples in a high-throughput, reproducible fashion with minimal technical variability. This chapter describes a pipeline used for labeling living Arabidopsis thaliana plants with nitrogen-15 ((15)N) and how this can be used, in conjunction with a technique for enrichment of phosphorylated peptides (phosphopeptides), to determine changes in A. thaliana's phosphoproteome on an untargeted, global scale.

Molecular characterization of mutant Arabidopsis plants with reduced plasma membrane proton pump activity.

Arabidopsis mutants containing gene disruptions in AHA1 and AHA2, the two most highly expressed isoforms of the Arabidopsis plasma membrane H(+)-ATPase family, have been isolated and characterized. Plants containing homozygous loss-of-function mutations in either gene grew normally under laboratory conditions. Transcriptome and mass spectrometric measurements demonstrate that lack of lethality in the single gene mutations is not associated with compensation by increases in RNA or protein levels. Selected reaction monitoring using synthetic heavy isotope-labeled C-terminal tryptic peptides as spiked standards with a triple quadrupole mass spectrometer revealed increased levels of phosphorylation of a regulatory threonine residue in both isoforms in the mutants. Using an extracellular pH assay as a measure of in vivo ATPase activity in roots, less proton secreting activity was found in the aha2 mutant. Among 100 different growth conditions, those that decrease the membrane potential (high external potassium) or pH gradient (high external pH) caused a reduction in growth of the aha2 mutant compared with wild type. Despite the normal appearance of single mutants under ideal laboratory growth conditions, embryos containing homozygous double mutations are lethal, demonstrating that, as expected, this protein is absolutely essential for plant cell function. In conclusion, our results demonstrate that the two genes together perform an essential function and that the effects of their single mutations are mostly masked by overlapping patterns of expression and redundant function as well as by compensation at the post-translational level.