Biology subjects
Publications and source records attributed to Taylor, M. T..
We report here a method for the chemical synthesis of Fmocprotected -hydroxyglyine (-OH-Gly) dipeptides. Our method features operational simplicity and is compatible with protecting groups for peptide synthesis. Utility is then demonstrated through substitution at the -OH-Gly position to yield myriad non-natural amino acid-containing dipeptide fragments.
We report here a photosensitized strategy for protein labelling in which N-substituted pyridinium salts are activated using a 2,4-diaryl-N-methyl quinolinium scaffold. Structure-reactivity relationships were performed to optimize the sensitizer structure, and ultimately generated a system that gives protein labelling in minutes at micromolar reagent concentrations. Mechanistic studies suggest a photo-induced electron transfer-based sensitization mechanism. The mildness of this system enabled us to assay sensitization both on individual biomolecules and in complex proteomes and demonstrated excellent compatibility with lysate- and the live-cell-based systems. Imaging of photolabelled HeLa cells were performed and revealed that catalysis occurs in multiple cellular compartments. Chemical proteomics performed at the lysate level resulted in the enrichment of 319 proteins with a 93% selectivity to Tryptophan residues. Live cell labelling resulted in 101 enriched proteins, primarily from the nucleus.
Photo-induced protein labelling strategies have become essential tools in chemical biology, but most strategies require high energy wavelengths of light as input to drive reactivity. Recently, we reported a biocompatible method for engaging photo-induced electron transfer to drive protein labelling using biaryl pyridinium salts and, here, we report the design of a series of aromatic cation salts that trigger this process using longer wavelengths of light while maintaining a sterically minimal profile. We achieved this through the systematic study of structure-reactivity relationships of various donor-acceptor pyridinium salts possessing extended conjugation, and these studies revealed the need of a constrained trans-stilbene relationship between the probes donor and acceptor substituents in order to achieve protein labelling. Probes with chromene-based donor groups in particular showed either robust protein labelling, significant fluorescence quantum yields, or state-dependent photophysical properties; in turn enabling the same probes to be used for both photo-induced protein labelling and wash-free live-cell imaging. We also demonstrate that these enhanced probes possess robust reactivity in complex biological environments through green light-triggered intracellular labelling in live HeLa cells, resulting in the identification of 659 enriched proteins. This series of experiments not only demonstrates the ability of this latest generation of probes to engage in photo-induced labelling using lower energy light in complex proteomes, but also reveals new capabilities for photophysical state-dependent reactivity and measurements. Table of Contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/681063v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1be9a0eorg.highwire.dtl.DTLVardef@741221org.highwire.dtl.DTLVardef@525596org.highwire.dtl.DTLVardef@170ed11_HPS_FORMAT_FIGEXP M_FIG C_FIG
The rationale for the use of metformin as a treatment to slow aging was largely based on data collected from metabolically unhealthy individuals. For healthspan extension metformin will also be used in periods of good health. To understand potential context specificity of metformin treatment on skeletal muscle, we used a rat model (HCR/LCR) with a divide in intrinsic aerobic capacity. Outcomes of metformin treatment differed based on baseline intrinsic mitochondrial function, oxidative capacity of the muscle (gastroc vs soleus), and the mitochondrial population (IMF vs SS). Metformin caused lower ADP-stimulated respiration in LCRs, with less of a change in HCRs. However, a washout of metformin resulted in an unexpected doubling of respiratory capacity in HCRs. These improvements in respiratory capacity were accompanied by mitochondrial remodeling that included increases in protein synthesis and changes in morphology. Our findings raise questions about whether the positive findings of metformin treatment are broadly applicable.
Viroporins constitute a class of viral membrane proteins with diverse roles in the viral life cycle. They can self-assemble and form pores within the bilayer that transport substrates, such as ions and genetic material, that are critical to the viral infection cycle. However, there is little known about the oligomeric state of most viroporins. Here, we use native mass spectrometry (MS) in detergent micelles to uncover the patterns of oligomerization of the full-length SARS-CoV-2 envelope (E) protein, poliovirus VP4, and HIV Vpu. Our data suggest that the E protein is a specific dimer, VP4 is exclusively monomeric, and Vpu assembles into a polydisperse mixture of oligomers under these conditions. Overall, these results revealed the diversity in the oligomerization of viroporins, which has implications for mechanisms of their biological functions as well as their potential as therapeutic targets.