Peer-reviewed research and public-facing science writing, spanning biophysical chemistry and science policy.
Published in the Albany Times Union, translating the public-health and scientific consequences of federal research defunding for a general audience.
Read the op-ed →Peer-Reviewed Publications
Abstract
Non-canonical amino acids (ncAAs) are valuable tools in chemical biology and biochemistry for labeling, probing, and tracking biomolecules. ncAAs that can be recombinantly incorporated using native E. coli machinery are particularly useful because they allow for global protein incorporation and avoid complex genetic code expansion. Here, we demonstrate successful incorporation of a methionine analog, L-cyanohomoalanine (Cha), by the methionyl-tRNA synthetase of E. coli into mutant superfolder GFP (sfGFP) expressed in methionine auxotroph bacterial cultures. We compare to methionine auxotroph bacterial cultures supplemented with L-methionine (Met) or L-azidohomoalanine (Aha). In control prototrophic E. coli, bacterial growth rates are inhibited with high concentrations of Aha but not Cha. However, less sfGFP is produced in auxotrophic cells supplemented with Cha compared to Aha and Met. Thus, while Cha is non-toxic to E. coli it is incorporated less efficiently into proteins than Aha or Met. Mass spectrometry confirmed that N-terminal Cha, Aha, and Met are cleaved, as expected for the sfGFP mutants. Other sites of Cha and Aha incorporation were confirmed by mass spectrometry, with labeling efficiency varying by position. Thermal melts of purified sfGFPs demonstrate that Cha and Aha labeling does not significantly perturb the protein stability. In the future, Cha may be useful for proteome labeling by wild-type methionyl-tRNA synthetase and could be implemented in metabolic pulse-labeling of newly synthesized proteins with other methionine analogs. Additionally, the nitrile moiety of Cha may be used to perform reactions orthogonal to azide/alkyne click chemistry or could serve as a vibrational reporter of the environment.
Abstract
Infrared spectroscopy is a powerful tool for identifying biomolecules. In biological systems, infrared spectra provide information on structure, reaction mechanisms, and conformational change of biomolecules. However, the promise of applying infrared imaging to biological systems has been hampered by low spatial resolution and the overwhelming water background arising from the aqueous nature of in cell and in vivo work. Recently, optical photothermal infrared microscopy (OPTIR) has overcome these barriers and achieved both spatially and spectrally resolved images of live cells and organisms. Here, we determine the most effective modes of collection on a commercial OPTIR microscope for work in biological samples. We examine three cell lines (Huh-7, differentiated 3T3-L1, and U2OS) and three organisms (E. coli, tardigrades, and zebrafish). Our results suggest that the information provided by multifrequency imaging is comparable to hyperspectral imaging while reducing imaging times twenty-fold. We also explore the utility of IR active probes for OPTIR using global and site-specific noncanonical azide containing amino acid probes of proteins. We find that photoreactive IR probes are not compatible with OPTIR. We demonstrate live imaging of cells in buffers with water. 13C glucose metabolism monitored in live fat cells and E. coli highlights that the same probe may be used in different pathways. Further we demonstrate that some drugs (e.g. neratinib) have IR active moieties that can be imaged by OPTIR. Our findings illustrate the versatility of OPTIR, and together, provide a direction for future dynamic imaging of living cells and organisms.
Abstract
Lipogenesis is a vital but often dysregulated metabolic pathway. Here we use optical photothermal infrared imaging to quantify lipogenesis rates of isotopically labelled oleic acid and glucose concomitantly in live cells. In hepatocytes, but not adipocytes, we find that oleic acid feeding at 60 µM increases the number and size of lipid droplets (LDs) while simultaneously inhibiting storage of de novo synthesized lipids in LDs. Our results demonstrate alternate regulation of lipogenesis between cell types.
Abstract
De novo lipogenesis (DNL) is a critical metabolic process that provides the majority of lipids for adipocyte and liver tissue. In cancer, obesity, type II diabetes, and nonalcoholic fatty liver disease DNL becomes dysregulated. A deeper understanding of the rates and of sub-cellular organization of DNL is necessary for identifying how this dysregulation occurs and varies across individuals and diseases. However, DNL is difficult to study inside the cell because labeling lipids and their precursors is not trivial. Existing techniques either can only measure parts of DNL, like glucose uptake, or do not provide spatiotemporal resolution. Here, we track DNL in space and time as isotopically labeled glucose is converted to lipids in adipocytes using optical photothermal infrared microscopy (OPTIR). OPTIR provides sub-micron resolution infrared imaging of the glucose metabolism in both living and fixed cells while also reporting on the identity of lipids and other biomolecules. We show significant incorporation of the labeled carbons into triglycerides in lipid droplets over the course of 72 hours. Live cells had better preservation of lipid droplet morphology but both showed similar DNL rates. Rates of DNL, as measured by the ratio of 13C labeled lipid to 12C labeled lipid, were heterogenous, with differences within and between lipid droplets and from cell to cell. The high rates of DNL measured in adipocyte cells match upregulated rates of DNL previously reported in PANC1 pancreatic cancer cells. Taken together, our findings support a model where DNL is locally regulated to meet energy needs within cells.
Abstract
Liquid–liquid phase separation (LLPS) is a biological phenomenon wherein a metastable and concentrated droplet phase of biomolecules spontaneously forms. A link may exist between LLPS of proteins and the disease-related process of amyloid fibril formation; however, this connection is not fully understood. Here, we investigated the relationship between LLPS and aggregation of the C-terminal domain of TAR DNA binding protein 43, an amyotrophic lateral sclerosis–related protein known to both phase separate and form amyloids, by monitoring conformational changes during droplet aging using Raman spectroscopy. We found that the earliest aggregation events occurred within droplets as indicated by the development of β-sheet structure and increased thioflavin-T emission. Interestingly, filamentous aggregates appeared outside the solidified droplets at a later time, suggestive that amyloid formation is a heterogeneous process under LLPS solution conditions. Furthermore, the secondary structure content of aggregated structures inside droplets is distinct from that in de novo fibrils, implying that fibril polymorphism develops as a result of different environments (LLPS versus bulk solution), which may have pathological significance.
Abstract
Aggregated TAR DNA-binding protein 43 (TDP-43) forms the cytoplasmic hallmarks associated with patients suffering from amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin. Under normal conditions, TDP-43 is a 414-amino acid protein; however, aggregates are enriched with N-terminal truncations which contain residues 267–414, known as the C-terminal domain of TDP-43 (TDP-43CTD). To gain residue-specific information on the aggregation process of TDP-43CTD, we created three single-Trp containing mutants (W385F/W412F, W334F/W412F, and W334F/W385F) by substituting two of the three native Trp residues with Phe, yielding fluorescent probes at W334, W385, and W412, respectively. Aggregation kinetics, secondary structure, and fibril morphology were compared to the wild-type protein using thioflavin-T fluorescence, Raman spectroscopy, and transmission electron microscopy, respectively. While only W334 is determined to be in the proteinase-K resistant core, all three sites are sensitive reporters of aggregation, revealing site-specific differences. Interestingly, W334 exhibited unusual multistep Trp kinetics, pinpointing a distinctive role for W334 and its nearby region during aggregation. This behavior is retained even upon seeding, suggesting the observed spectral change is related to fibril growth. This work provides new insights into the aggregation mechanism of TDP-43CTD and exemplifies the advantages of Trp as a site-specific environmentally sensitive fluorescent probe.
Full publication list: Google Scholar