Andreas GahlmannAssistant Professor of Chemistry and Molecular Physiology and Biological Physics
One key area in understanding bacterial cell biology is spatiotemporal phenomena: Where, when, and how do individual biomolecules act and interact to govern the overall physiology of the cell? To answer this question, we develop new high-resolution imaging methods for 3D single-molecule localization in intact bacterial cells. In particular, we combine the resolving power of the electron microscope with the single-molecule sensitivity and specificity of fluorescence-based methods. With these tools, we can localize single biomolecules in 3D space with a precision of a few nanometers, track their motion over time, and then zoom in further to visualize how specific biomolecules combine with others to produce functioning assemblies in their native environment.
Bacteria are highly relevant to important challenges of our time. For example, the looming inability to effectively combat pathogenic bacteria with current antibiotics presents a major health concern. Finding new avenues to selectively target and alter key molecular pathways can provide us with further options for effective antibiotic drug development. Because bacteria are the smallest and arguably the simplest living organisms on the planet, they are also fundamentally interesting to study the molecular-level biology of the cell. Bacteria are able to precisely regulate protein activity throughout the intracellular space through finely tuned molecular interactions. Of particular importance are scaffolding proteins that partition the cytoplasm and provide specialized subcellular compartments for specific biochemical reactions to occur. On a smaller scale, scaffolding proteins are hypothesized to spatially organize multiple enzymes into biomolecular assemblies. Parts of these assemblies can be highly dynamic and therefore the precise architectures and the resulting functional consequences remain elusive.
Rapid progress of evolution has made the bacteria an extremely diverse and widely abundant group of single-celled organisms that affects almost every aspect of life on earth. The resulting bacterial physiological traits present a biological treasure trove that remains to be investigated with molecular resolution and, where possible, exploited to our benefit. With this in mind, we continue to push the limits of cellular imaging, as well as in situ structural characterization of biomolecular assemblies.
Single-molecule tracking in live Yersinia enterocolitica reveals distinct cytosolic complexes of injectisome subunits. J. Rocha, C. Richardson, M. Zhang, C. Darch, E. Cai, A. Diepold, A. Gahlmann, Integrative Biology, 2018, 10, 502 (Cover Article)
BACT-3D: A level set segmentation approach for dense multi-layered 3D bacterial biofilms. J. Wang, R. Sarkar, A. Aziz, A. Vaccari, A. Gahlmann, S. Acton, 2017 IEEE International Conference on Image Processing (ICIP)
Bacterial Scaffold Directs Pole-Specific Centromere Segregation. J.L. Ptacin, A. Gahlmann, G.R. Bowman , A.M. Perez, A.R.S. von Diezmann, M.R. Eckart, W.E. Moerner, and L. Shapiro. Proc. Natl. Acad. Sci. USA, 2014, 111, E2046
Exploring Bacterial Cell Biology with Single-Molecule Tracking and Super-Resolution Imaging. A. Gahlmann and W.E. Moerner. Nat. Rev. Microbiol., 2013, 12, 9 (Cover Article)
Quantitative Multicolor Subdiffraction Imaging of Bacterial Protein Ultrastructures in Three Dimensions. A. Gahlmann, J.L. Ptacin, G. Grover, S. Quirin, A.R.S. von Diezmann, M.K. Lee, M.P. Backlund, L. Shapiro, R. Piestun, and W.E. Moerner. Nano Lett., 2013, 13, 987
Direct Structural Determination of Conformations of Photoswitchable Molecules by Laser Desorption-Electron Diffraction. A. Gahlmann, I-R. Lee, and A.H. Zewail. Angew. Chem. Int. Ed., 2010, 49, 6524
Michael HilinskiAssistant Professor of Chemistry
The science of organic synthesis is central to both the discovery and manufacturing of pharmaceuticals and other fine chemicals and the emergence of subdisciplines of biology that are becoming increasingly focused on phenomena at the molecular level (e.g., synthetic biology and chemical biology). Over the last half-century revolutionary advances in synthetic organic chemistry have made it possible to synthesize virtually any molecule given enough time, money, and manpower. However, this is frequently not enough since a lack of practical and cost-effective synthetic access can and does prevent promising drug leads from ever helping patients. The grand challenge for synthetic organic chemistry is therefore to advance the field of synthesis to the point where any molecule can be not only synthesized, but also synthesized in a way that minimizes the cost, time, and manpower required as well as environmental impact. Our group’s research is focused on eliminating synthetic considerations as a barrier to the discovery of new therapeutics.
Organocatalytic, Dioxirane-Mediated C-H Hydroxylation under Mild Conditions Using Oxone. W. G. Shuler, S. L. Johnson, M. K. Hilinski, Org. Lett. 2017, 19, 4790–4793. An Iminium Salt Organocatalyst for Selective Aliphatic C–H Hydroxylation. D. Wang, W. G. Shuler, C. J. Pierce, M. K. Hilinski, Org. Lett. 2016, 18, 3826–3829. Intermolecular Electrophilic Addition of Epoxides to Alenes: [3+2] Cycloadditions Catalyzed by Lewis Acids. W. G. Shuler, L. A. Combee, I. D. Falk, M. K. Hilinski, Eur. J. Org. Chem. 2016, 3335–3338. Chemoselective Hydroxylation of Aliphatic sp3 C–H Bonds Using a Ketone Catalyst and Aqueous H2O2. C. J. Pierce, M. K. Hilinski, Org. Lett. 2014, 16, 6504–6507.
Brooks H. PateWilliam R. Kenan, Jr. Professor of Chemistry
Broadband Rotational Spectroscopy for Chemical Analysis
The Pate lab develops instruments for molecular rotational spectroscopy and uses these instruments to solve challenging problems in chemical analysis. Molecular rotational spectroscopy uses low frequency light, typically in the microwave region of the electromagnetic spectrum, to excite transitions between the quantized energy levels that come from the rotational kinetic energy. The energy level patterns for the rotational kinetic energy are determined by the principal moments-of-inertia of the molecule and are, therefore, directly related to the mass distribution relative to the molecular center-of-mass. The fact that the measurement is connected to the mass distribution, and not just the total mass, makes molecular rotational spectroscopy ideally suited for isomer analysis.
Broadband Rotational Spectroscopy
The Pate lab has pioneered the technique of chirped-pulse Fourier transform rotational spectroscopy. The key advantage of this technique over previous instrument designs is the ability to measure a large spectral range in a single spectrum acquisition event. The method has similarities to Fourier transform nuclear magnetic resonance (NMR) spectroscopy: A high-power light pulse (the chirped pulse) creates a macroscopic polarization in the sample by aligning the molecular dipole moments. After the excitation pulse dissipates, the molecular rotational resonances are detected by the light waves coherently emitted by the rotating molecules. This coherent emission eventually decays through Doppler dephasing or collisions. The broadband free-induction decay (FID) from the rotational motion is collected using a high-speed digitizer. Sensitivity is enhanced by acquiring several measurements and co-adding the FIDs. The molecular rotational spectrum is produced by subsequent Fourier transform analysis of the FID. This measurement approach has been extended to low-frequency instruments (2-8 GHz) that are used for analysis of large molecules and to high-frequency instruments (mm-wave range) for smaller molecules such as those important in astrochemistry
Initial Applications of Broadband Rotational Spectroscopy
The first application of chirped-pulse Fourier transform rotational spectroscopy was in the field of intramolecular dynamics. In this application, the instrument is used to acquire the rotational spectrum of a highly vibrationally excited molecule that is prepared by laser excitation. The complex nuclear motion from intramolecular vibrational energy redistribution and conformational isomerization produce dynamic effects in the spectrum. For example, the coalescence of rotational spectra associated with different conformational geometries can be used to measure the energy-resolved unimolecular isomerization rate under collision free conditions.
A second area of application is the structure of molecular clusters. Clusters form in the pulsed jet expansion used to create the cold gas sample used for analysis. The aggregation is driven by non-covalent interactions such as hydrogen bonding and London dispersion forces. A challenge for the analysis of the clusters is that a wide range of cluster sizes are produced and there can be many isomeric structures for each cluster size. Rotational spectroscopy provides the highest spectral resolution of molecular spectroscopy techniques used for chemical analysis. As a result, the technique has no trouble resolving the spectra from all cluster geometries present in the sample. The application of broadband rotational spectroscopy to water clusters has dramatically increased the number of known structures, demonstrated the presence of multiple isomers for a single cluster size, and revealed the dynamics of quantum mechanical tunneling within the water cluster.
Chiral Analysis with Applications to Pharmaceutical Chemistry
The most recent work in the Pate lab is the application of broadband rotational spectroscopy to chiral analysis. The group is developing techniques for quantitative chiral analysis that uses the formation of clusters of an analyte and a small, enantiopure chiral molecule (called the chiral tag) to determine the enantiomeric excess of the sample. Using quantum chemistry modeling of the chiral tag complexes it is also possible to establish the absolute configuration of the dominant enantiomer. A key strength of rotational spectroscopy is that the measurement can simultaneously perform other analyses that are related to the synthesis of chiral molecules. For example, when the analyte has multiple chiral centers there are two types of stereoisomers: the enantiomers (left- and right-handed versions of a molecule) and diastereomers (isomers with different structures and mass distributions that are easily distinguished by molecular rotational spectroscopy). The technique can also be used to quantify regioisomers that are often present due to addition reactions that occur at other activated sites of the reagent in the reaction step. The high-resolution of rotational spectroscopy makes it possible to perform these analyses without extensive sample purification. Furthermore, cavity-enhanced rotational spectroscopy instruments can be used to monitor the synthesis of chiral molecules in real-time by sampling directly out of the reaction flask.
Gordon G. Brown, Brian C. Dian, Kevin O. Douglass, Scott M. Geyer, and Brooks H. Pate, “A Broadband Fourier Transform Microwave Spectrometer Based on Chirped Pulse Excitation” Rev. Sci. Instrum. 79, 053103 (2008).
Brian C. Dian, Gordon G. Brown, Kevin O. Douglass, and Brooks H. Pate, “Measuring Picosecond Isomerization Dynamics via Ultra-broadband Fourier Transform Microwave Spectroscopy”, Science 320, 924-928 (2008).
Cristóbal Pérez, Matt T. Muckle, Daniel P. Zaleski, Nathan A. Seifert, Berhane Temelso, George C. Shields, Zbigniew Kisiel, and Brooks H. Pate, “Structures of Cage, Prism, and Book Isomers of Water Hexamer from Broadband Rotational Spectroscopy”, Science 336, 897-901 (2012).
Jeremy O. Richardson, Cristóbal Pérez, Simon Lobsiger, Adam A. Reid, Berhane Temelso, George C. Shields, Zbigniew Kisiel, David J. Wales, Brooks H. Pate, and Stuart C. Althorpe, “Concerted hydrogen-bond breaking by quantum tunneling in the water hexamer prism”, Science 351, 1310-1313 (2016).
B.H. Pate, L. Evangelisti, W. Caminati, Y. Xu, J. Thomas, D. Patterson, C. Perez, and M. Schnell, “Quantitative Chiral Analysis by Molecular Rotational Spectroscopy”, in Chiral Analysis 2nd Edition Advances in Spectroscopy, Chromatography, and Emerging Methods, P.L. Polavarapu, Editor, Elsevier (2018).
Justin L. Neill, Yuan Yang, Matt T. Muckle, Roger L. Reynolds, Luca Evangelisti, Reilly E. Sonstrom, Brooks H. Pate, and B. Frank Gupton, “Online Stereochemical Process Monitoring by Molecular Rotational Resonance Spectroscopy”, Org. Process Res. Dev. 23, 1046-1051 (2019).
Lin PuProfessor of Chemistry
Organic, Polymer and Organometallic Chemistry; Asymmetric Catalysis; Chiral Sensors; Optically Active Materials
Multi-disciplinary research programs involving organic synthesis, molecular recognition, fluorescent sensing, asymmetric catalysis, and polymers are conducted in our laboratory. The 1,1′-bi-2-naphthol (BINOL) and its derivatives are chosen as the chiral building blocks to construct novel chiral molecules and macromolecules for diverse applications. We have developed a family of enantioselective fluorescent sensors for the recognition of organic molecules such as alpha-hydroxycarboxylic acids, amino acids, amino alcohols, and amines. These sensors are potentially useful for rapid assay of the enantiomeric composition of chiral compounds and for high throughput chiral catalyst screening. They are also potentially useful for biological analysis and imaging. New chiral conjugated polymers and dendrimers are prepared for applications in materials, catalysis, and sensing. We have discovered that the Lewis acid complexes of the optically active binaphthyl molecules and polymers can carry out highly enantioselective organic reactions such as organozinc additions to aldehydes, hetero-Diels-Alder reactions, 1,3-dipolar cycloadditions, reductions of ketones, Michael additions, epoxidations, and others. Interesting chiral organic molecules including those of biological functions are prepared by using these catalysts.
Enantioselective Fluorescent Sensors
Simultaneous Determination of Concentration and Enantiomeric Composition in Fluorescent Sensing. Lin Pu. Acc. Chem. Res. 2017, 50, 1032-1040.
Regiospecific Hydration of N-(Diphenylphosphinoyl)propargyl amines: Synthesis of b-Amino Ketones by Au(III) Catalysis. Ying, J.; Pu, L. J. Org. Chem. 2016, 81, 8135−8141. A Featured Article. Highlight on the cover.
Conjugated polymer-enhanced enantioselectivity in fluorescent sensing. Zhang, X. –P.; Wang, C.; Wang, P.; Du, J. –J.; Zhang, G. –Q.; Pu, L. Chem. Sci. 2016, 7, 3614–3620.
Rational Design of a Fluorescent Sensor to Simultaneously Determine Both the Enantiomeric Composition and Concentration of Chiral Functional Amines. Wen, K. –L.; Yu, S. –S.; Huang, Z.; Chen, L. –M.; Xiao, M.; Yu, X. –Q.; Pu, L. J. Am. Chem. Soc. 2015, 137, 4517-4524.
Asymmetric Functional Organozinc Additions to Aldehydes Catalyzed by BINOLs. Pu, L. Acc. Chem. Res. 2014, 47, 1523–1535.
Zn(II) Promoted Dramatic Enhancement in the Enantioselective Fluorescent Recognition of Chiral Amines by a Chiral Aldehyde. Huang, Z.; Yu, S. S.; Yu, X. Q.; Pu. L. Chem. Sci. 2014, 5, 3457-3462.
Enantioselective Fluorescent Sensors: A Tale of BINOL. Pu, L. Acc. Chem. Res. 2012, 45, 150–163.
Simultaneous Determination of Both the Enantiomeric Composition and Concentration of a Chiral Substrate with One Fluorescent Sensor. Yu, S.; Plunkett, W.; Kim, M.; Pu, L. J. Am. Chem. Soc. 2012, 134, 20282–20285. A spotlight article reported in J. Am. Chem. Soc., 2013, 135 (3), 949–950.
Kevin WelchAssistant Professor, General Faculty & Director of Undergraduate Advising
Professor Kevin Welch is interested in developing curricula for undergraduate instruction in general chemistry and organic chemistry. In particular, his focus is on updating these courses to accommodate the diverse educational background in chemistry of the students enrolling in chemistry at the University of Virginia, as well as providing a strong chemical foundation for the students as they continue on in their educational and post-educational careers in a variety of fields. In the past, he has taught undergraduate courses in general chemistry, organic chemistry, inorganic chemistry, environmental chemistry, and scientific writing.
In addition to a focus on undergraduate education, Kevin’s interests in chemistry involve research investigating metal-ligand bonding interactions and the use of transition metal complexes to address challenges in fuel generation and energy storage.
A native of northern Virginia, Kevin received his B.S. in chemistry from Gettysburg College in 2002, and his Ph.D. in chemistry from the University of Virginia in 2007, where he focused on synthetic organometallic chemistry. He spent three years in central Washington as a Department of Energy Postdoctoral Researcher at the Pacific Northwest National Laboratory, working on the development of transition metal catalysts for energy storage and fuel cell technology. From 2010 to 2016, Kevin was a visiting assistant professor at Swarthmore College outside of Philadelphia, Pennsylvania, where he taught and conducted research with undergraduate students. He returned to the University of Virginia in the fall of 2016 and currently teaches courses in general chemistry and laboratories for organic chemistry.