Abstract: The vast majority of chemistry and biology occurs in solution, and new label-free analytical techniques that can help resolve solution-phase complexity at the single-molecule level can provide new microscopic perspectives of unprecedented detail. Here, we use the increased light-molecule interactions in high-finesse fiber Fabry-Pérot microcavities to detect individual biomolecules as small as 1.2 kDa (10 amino acids) with signal-to-noise ratios >100, even as the molecules are freely diffusing in solution.  Our method delivers 2D intensity and temporal profiles, enabling the distinction of sub-populations in mixed samples. Strikingly, we observe a linear relationship between passage time and molecular radius, unlocking the potential to gather crucial information about diffusion and solution-phase conformation. Furthermore, mixtures of biomolecule isomers of the same molecular weight can also be resolved. Detection is based on a novel molecular velocity filtering and dynamic thermal priming mechanism leveraging both photo-thermal bistability and Pound-Drever-Hall cavity locking. This technology holds broad potential for applications in life and chemical sciences and represents a major advancement in label-free in vitro single-molecule techniques.

Methanogens are a diverse group of archaea with ancient evolutionary origins. They are found in a wide range of anoxic environments where they carry out a form of anaerobic respiration known as methanogenesis. This process reduces simple oxidized carbon compounds to generate methane as an end product. Another group of archaea related to methanogens carry out the anaerobic oxidation of methane (AOM) and are known as anaerobic methanotrophs (ANME).  Methanogens and ANME are both key components in the global carbon cycle and play a central role in controlling atmospheric methane concentrations. Consistent with their anaerobic lifestyles and ancient evolutionary origins, methanogens and ANME contain an abundance of Fe-S cluster proteins. Radical S-adenosylmethionine (SAM) enzymes are [4Fe-4S]-cluster containing enzymes that catalyze a wide variety of difficult biochemical reactions through the generation of a highly reactive 5’-deoxyadenosyl radical. Here, we discuss our recent progress towards uncovering the functions of novel radical SAM enzymes in methanogens and ANME. We identified the missing glutamate 2,3-aminomutase important for salt tolerance in marine organisms as well as characterized the first archaeal methylthiotransferase involved in tRNA modification. 

The structure of plant cell wall carbohydrates creates the strength and flexibility of the plant cell wall, which shapes plants’ overall agronomic fitness in the field. Differences in cell wall carbohydrates and associated compounds are also influential post-harvest, since carbohydrate structural characteristics can influence a material’s food processing characteristics, feed value for livestock, and biofuel production potential.

The core areas of Dr. Schendel’s research program at the University of Kentucky are plant cell wall characterization (especially detailed structural analysis of cell wall carbohydrates) and analysis and application of phenolics and other secondary plant metabolites. Strategic collaborations have allowed us to explore applied questions such as ruminant microbe fermentation of cell wall carbohydrates. This seminar will share results from several projects, including our in-depth characterizations of the cell walls of cool-season forages and hempseeds and exploration of their seasonal and species/cultivar variation. 

Chamikara Karunasena

Graduate Student Profile

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Abstract: The immense synthetic design space and material versatility have driven the exploration and development of organic semiconductors (OSC) over several decades. While many OSC designs focus on the chemistries of the molecular or polymer building blocks, a priori, multiscale control over the solid-state morphology is required for effective application of the active layer in a given technology. However, molecular assembly during solid-state formation is a complex function interconnecting the building block chemistry and the processing environment. Insufficient knowledge as to the how these aspects engage, especially at the atomistic and molecular scales, have so far limited the ability to predict OSC solid-state morphology, leaving Edisonian approaches as the stalwart methods. Therefore, through multiscale simulations combining atomistic quantum scale modeling and modern advanced sampling molecular dynamics (MD), we aim to establish first principles understanding required to synthetically regulate solid-state morphology of organic semiconductors (OSC) as a function of molecular chemistry and processing. In turn we try to understand the deceivingly simple yet complex mechanisms behind molecular aggregation and crystallization of OSC. Simultaneously, we develop semi-to-fully automated high-throughput schemes to automate the complex and labor-intensive analyses to generate data based on various crystal structures in different crystallization environments. Ultimately, we aim to bridge molecular-scale information revealed on solid-state physical organization, understood in the context of chromophore chemistry and the molecular environment, with the macro scale properties to uncover useful guidelines for rational design and morphology regulation of OSC systems.

Graduate Student Profile

Abstract: Plant-derived compounds have the potential to produce value-added compounds with a variety of applications. For example, the lignin part of the lignocellulosic biomass, produced in large quantities as waste from the paper and pulp industries, is a rich source of phenolics with potential applications in the renewable energy sector, pharmaceutical, and chemical industries. On the other hand, plant alkaloids are the primary source for developing plant-derived therapeutics. Unfortunately, the recalcitrant nature of plant cell walls, low extraction yields of small secondary metabolites, and the lack of effective analytical methods for a rapid and accurate identification of plant-based compounds and plant’s degradation products are the major limitations in plant-based valorization efforts.

In order to address some of these challenges, this dissertation focuses on utilizing different mass spectrometry-based techniques such as UHPLC-MS, GC-MS, and direct infusion high-resolution accurate orbitrap and ion trap mass spectrometry for the detection and structure elucidation of plant-based phenolics and alkaloids in order to contribute to ongoing efforts toward valorization of plant-based compounds. Mass spectrometry-based techniques are widely used in pharmaceutical and chemical industries, and have been emerged as one of the most promising analytical techniques for the analysis of plant-based compounds.

In the second chapter of this dissertation, a mass spectrometric method based on lithium cationization was developed to sequence lignin model oligomers with mixed bonding motifs, with a potential application in facilitating the structure elucidation of lignin degradation end products with β-β and β-O-4 linkages. In the third chapter, an important lignan, syringaresinol, was characterized in bourbon whiskey. The origin of syringaresinol was investigated using a model aging experiment to further our understanding of bourbon’s chemical composition. In chapter four, the development of a mild ethanosolv treatment combined with a GC-MS method enabled the detection of several different phenolic compounds in lignocellulosic biomass, which can be potentially used to rapidly compare different biomass samples for the valorization applications. Lastly, in chapter five, synthetic methods in combination with extensive mass spectrometry-based analysis were used to semi-synthesize new plant-based alkaloids with potential applications in drug discovery and development.

Overall, these studies confirm that mass spectrometry-based techniques provide a sensitive and robust analytical platform for the analysis of plant-based products.