Megan C. Thielges

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Name: Thielges, Megan
Organization: Indiana University , USA
Department: Department of Chemistry
Title: (PhD)
Co-reporter:Amanda L. Le Sueur, Sashary Ramos, Jonathan D. Ellefsen, Silas Cook, and Megan C. Thielges
Analytical Chemistry May 16, 2017 Volume 89(Issue 10) pp:5254-5254
Publication Date(Web):April 13, 2017
DOI:10.1021/acs.analchem.6b04650
Two-dimensional infrared (2D IR) spectroscopy provides a powerful approach for the direct study of molecular dynamics with high spatial and temporal resolution. Its application for investigating specific locations in proteins requires the incorporation of IR probe groups with spectrally isolated absorptions to avoid the congestion inherent to protein spectra. This has motivated extensive efforts toward the development of new IR probes, but there remains a need for those that can extend the experimental time range, which is limited by their vibrational lifetimes. Toward this goal, isotopically labeled p-(13C15N-cyano)phenylalanine was synthesized, site-selectively incorporated into the protein plastocyanin, and evaluated for its potential as a 2D IR probe. The isotopic labeling increases the vibrational lifetime about 2-fold, which results in larger signals at longer time scales. However, isotopic labeling simultaneously shifts the absorption to a spectral region with greater water absorbance, which results in greater heating-induced signals in the background that overlap those of the nitrile probe. The study demonstrates the use of a new 2D IR probe to measure the side chain dynamics in a protein and also illustrates the multiple factors to consider in development of 2D IR probes for studying proteins.
Co-reporter:Edward J. Basom, Bryce A. Manifold, and Megan C. Thielges
Biochemistry June 27, 2017 Volume 56(Issue 25) pp:3248-3248
Publication Date(Web):June 5, 2017
DOI:10.1021/acs.biochem.7b00238
The broad and variable substrate specificity of cytochrome P450 enzymes makes them a model system for studying the determinants of protein molecular recognition. The archetypal cytochrome P450cam (P450cam) is a relatively specific P450, a feature once attributed to the high rigidity of its active site. However, increasingly studies have provided evidence of the importance of conformational changes to P450cam activity. Here we used infrared (IR) spectroscopy to investigate the molecular recognition of P450cam. Toward this goal, and to assess the influence of a hydrogen bond (H-bond) between active site residue Y96 and substrates, two variants in which Y96 is replaced by a cyanophenyl (Y96CNF) or phenyl (Y96F) group were characterized in complexes with the substrates camphor, isoborneol, and camphane. These combinations allow for a comparison of complexes in which the moieties on both the protein and substrate can serve as a H-bond donor, acceptor, or neither. The IR spectra of heme-bound CO and the site-specifically incorporated CN of Y96CNF were analyzed to characterize the number and nature of environments in each protein, both in the free and bound states. Although the IR spectra do not support the idea that protein–substrate H-bonding is central to P450cam recognition, the data altogether suggest that the differing conformational heterogeneity in the active site of the P450cam variants and changes in heterogeneity upon binding of different substrates likely contribute to their variable affinities via a conformational selection mechanism. This study further extends our understanding of the molecular recognition of archetypal P450cam and demonstrates the application of IR spectroscopy combined with selective protein modification to delineate protein–ligand interactions.
Co-reporter:S. Ramos;K. J. Scott;R. E. Horness;A. L. Le Sueur;M. C. Thielges
Physical Chemistry Chemical Physics 2017 vol. 19(Issue 15) pp:10081-10086
Publication Date(Web):2017/04/12
DOI:10.1039/C7CP00403F
The importance of dynamics to the function of proteins is well appreciated, but the difficulty in their measurement impedes investigation into their precise role(s). 2D IR spectroscopy is a developing approach for the study of dynamics and has motivated efforts to develop spectrally resolved IR probe groups that enable its application for measuring the dynamics at specific sites in a protein. A challenge with this approach is that the timescales accessible are limited by the vibrational lifetimes of the probes. Toward development of better probes for 2D IR spectroscopy of protein dynamics, we report the characterization of p-cyano-seleno-phenylalanine (CNSePhe), a derivative of the well established IR probe p-cyano-phenylalanine (CNPhe), by FT IR, pump–probe, and 2D IR spectroscopy. The incorporation of the heavy Se atom decouples the CN vibration from the rest in the molecule. Although this leads to a reduction of the transition dipole strength, and thus a reduction in signal intensity, it also dramatically increases the vibrational lifetime, enabling collection of 2D IR spectra for analysis of molecular dynamics on much longer timescales. Interestingly, we also find that the lifetime for CNSePhe shows increased sensitivity to the presence of hydrogen bonding interactions with the CN, suggesting that the probe should be useful for interpretation of CN spectra and possibly for the study of solvation.
Co-reporter:Rachel E. Horness; Edward J. Basom; John P. Mayer
Journal of the American Chemical Society 2016 Volume 138(Issue 4) pp:1130-1133
Publication Date(Web):January 19, 2016
DOI:10.1021/jacs.5b11999
Conformational heterogeneity and dynamics are increasingly evoked in models of protein molecular recognition but are challenging to experimentally characterize. Here we combine the inherent temporal resolution of infrared (IR) spectroscopy with the spatial resolution afforded by selective incorporation of carbon-deuterium (C–D) bonds, which provide frequency-resolved absorptions within a protein IR spectrum, to characterize the molecular recognition of the Src homology 3 (SH3) domain of the yeast protein Sho1 with its cognate proline-rich (PR) sequence of Pbs2. The IR absorptions of C–D bonds introduced at residues along a peptide of the Pbs2 PR sequence report on the changes in the local environments upon binding to the SH3 domain. Interestingly, upon forming the complex the IR spectra of the peptides labeled with C–D bonds at either of the two conserved prolines of the PXXP consensus recognition sequence show more absorptions than there are C–D bonds, providing evidence for the population of multiple states. In contrast, the NMR spectra of the peptides labeled with 13C at the same residues show only single resonances, indicating rapid interconversion on the NMR time scale. Thus, the data suggest that the SH3 domain recognizes its cognate peptide with a component of induced fit molecular recognition involving the adoption of multiples states, which have previously gone undetected due to interconversion between the populated states that is too fast to resolve using conventional methods.
Co-reporter:Amanda L. Le Sueur; Richard N. Schaugaard; Mu-Hyun Baik
Journal of the American Chemical Society 2016 Volume 138(Issue 22) pp:7187-7193
Publication Date(Web):May 10, 2016
DOI:10.1021/jacs.6b03916
The reactivity of metal sites in proteins is tuned by protein-based ligands. For example, in blue copper proteins such as plastocyanin (Pc), the structure imparts a highly elongated bond between the Cu and a methionine (Met) axial ligand to modulate its redox properties. Despite extensive study, a complete understanding of the contribution of the protein to redox activity is challenged by experimentally accessing both redox states of metalloproteins. Using infrared (IR) spectroscopy in combination with site-selective labeling with carbon–deuterium (C–D) vibrational probes, we characterized the localized changes at the Cu ligand Met97 in the oxidized and reduced states, as well as the Zn(II) or Co(II)-substituted, the pH-induced low-coordinate, the apoprotein, and the unfolded states. The IR absorptions of (d3-methyl)Met97 are highly sensitive to interaction of the sulfur-based orbitals with the metal center and are demonstrated to be useful reporters of its modulation in the different states. Unrestricted Kohn–Sham density functional theory calculations performed on a model of the Cu site of Pc confirm the observed dependence. IR spectroscopy was then applied to characterize the impact of binding to the physiological redox partner cytochrome (cyt) f. The spectral changes suggest a slightly stronger Cu–S(Met97) interaction in the complex with cyt f that has potential to modulate the electron transfer properties. Besides providing direct, molecular-level comparison of the oxidized and reduced states of Pc from the perspective of the axial Met ligand and evidence for perturbation of the Cu site properties by redox partner binding, this study demonstrates the localized spatial information afforded by IR spectroscopy of selectively incorporated C–D probes.
Co-reporter:Edward J. Basom, Michał Maj, Minhaeng Cho, and Megan C. Thielges
Analytical Chemistry 2016 Volume 88(Issue 12) pp:6598
Publication Date(Web):May 17, 2016
DOI:10.1021/acs.analchem.6b01520
Conformational changes are central to protein function but challenging to characterize with both high spatial and temporal precision. The inherently fast time scale and small chromophores of infrared (IR) spectroscopy are well-suited for characterization of potentially rapidly fluctuating environments, and when frequency-resolved probes are incorporated to overcome spectral congestion, enable characterization of specific sites in proteins. We selectively incorporated p-cyanophenylalanine (CNF) as a vibrational probe at five distinct locations in the enzyme cytochrome P450cam and used IR spectroscopy to characterize the environments in substrate and/or ligand complexes reflecting those in the catalytic cycle. Molecular dynamics (MD) simulations were performed to provide a structural basis for spectral interpretation. Together the experimental and simulation data suggest that the CN frequencies are sensitive to both long-range influences, resulting from the particular location of a residue within the enzyme, as well as short-range influences from hydrogen bonding and packing interactions. The IR spectra demonstrate that the environments and effects of substrate and/or ligand binding are different at each position probed and also provide evidence that a single site can experience multiple environments. This study illustrates how IR spectroscopy, when combined with the spectral decongestion and spatial selectivity afforded by CNF incorporation, provides detailed information about protein structural changes that underlie function.
Co-reporter:Amanda L. Le Sueur, Rachel E. Horness and Megan C. Thielges  
Analyst 2015 vol. 140(Issue 13) pp:4336-4349
Publication Date(Web):15 May 2015
DOI:10.1039/C5AN00558B
Two-dimensional infrared (2D IR) spectroscopy has recently emerged as a powerful tool with applications in many areas of scientific research. The inherent high time resolution coupled with bond-specific spatial resolution of IR spectroscopy enable direct characterization of rapidly interconverting species and fast processes, even in complex systems found in chemistry and biology. In this minireview, we briefly outline the fundamental principles and experimental procedures of 2D IR spectroscopy. Using illustrative example studies, we explain the important features of 2D IR spectra and their capability to elucidate molecular structure and dynamics. Primarily, this minireview aims to convey the scope and potential of 2D IR spectroscopy by highlighting select examples of recent applications including the use of innate or introduced vibrational probes for the study of nucleic acids, peptides/proteins, and materials.
Co-reporter:Rachel E. Horness, Edward J. Basom and Megan C. Thielges  
Analytical Methods 2015 vol. 7(Issue 17) pp:7234-7241
Publication Date(Web):08 Apr 2015
DOI:10.1039/C5AY00523J
Local heterogeneity of microenvironments in proteins is important in biological function, but difficult to characterize experimentally. One approach is the combination of infrared (IR) spectroscopy and site-selective incorporation of probe moieties with spectrally resolved IR absorptions that enable characterization within inherently congested protein IR spectra. We employed this method to study molecular recognition of a Src homology 3 (SH3) domain from the yeast protein Sho1 for a peptide containing the proline-rich recognition sequence of its physiological binding partner Pbs2. Nitrile IR probes were introduced at four distinct sites in the protein by selective incorporation of p-cyanophenylalanine via the amber codon suppressor method and characterized by IR spectroscopy. Variation among the IR absorption bands reports on heterogeneity in local residue environments dictated by the protein structure, as well as on residue-dependent changes upon peptide binding. The study informs on the molecular recognition of SH3Sho1 and illustrates the speed and simplicity of this approach for characterization of select microenvironments within proteins.
Co-reporter:Edward J. Basom, James W. Spearman, and Megan C. Thielges
The Journal of Physical Chemistry B 2015 Volume 119(Issue 22) pp:6620-6627
Publication Date(Web):May 8, 2015
DOI:10.1021/acs.jpcb.5b03896
Conformational heterogeneity and dynamics likely contribute to the remarkable activity of enzymes but are challenging to characterize experimentally. These features are of particular interest within the cytochrome P450 class of monooxygenases, which are of great academic, medicinal, and biotechnological interest as they recognize a broad range of substrates, such as various lipids, steroid precursors, and xenobiotics, including therapeutics. Here, we use linear and 2D IR spectroscopy to characterize the prototypical P450, cytochrome P450cam, bound to three different substrates, camphor, norcamphor, or thiocamphor, which are hydroxylated with high, low, and intermediate regioselectivity, respectively. The data suggest that specific interactions with the substrate drive the population of two different conformations, one that is associated with high regioselectivity and another associated with lower regioselectivity. Although Y96 mediates a hydrogen bond thought necessary to orient the substrate for high regioselectivity, the population and dynamics of the conformational states are largely unaltered by the Y96F mutation. This study suggests that knowledge of the conformational landscape is central to understanding P450 activity, which has important practical ramifications for the design of therapeutics with optimized pharmacokinetics, and the manipulation of P450s, and possibly other enzymes, for biotechnological applications.
Co-reporter:S. Ramos, K. J. Scott, R. E. Horness, A. L. Le Sueur and M. C. Thielges
Physical Chemistry Chemical Physics 2017 - vol. 19(Issue 15) pp:NaN10086-10086
Publication Date(Web):2017/03/24
DOI:10.1039/C7CP00403F
The importance of dynamics to the function of proteins is well appreciated, but the difficulty in their measurement impedes investigation into their precise role(s). 2D IR spectroscopy is a developing approach for the study of dynamics and has motivated efforts to develop spectrally resolved IR probe groups that enable its application for measuring the dynamics at specific sites in a protein. A challenge with this approach is that the timescales accessible are limited by the vibrational lifetimes of the probes. Toward development of better probes for 2D IR spectroscopy of protein dynamics, we report the characterization of p-cyano-seleno-phenylalanine (CNSePhe), a derivative of the well established IR probe p-cyano-phenylalanine (CNPhe), by FT IR, pump–probe, and 2D IR spectroscopy. The incorporation of the heavy Se atom decouples the CN vibration from the rest in the molecule. Although this leads to a reduction of the transition dipole strength, and thus a reduction in signal intensity, it also dramatically increases the vibrational lifetime, enabling collection of 2D IR spectra for analysis of molecular dynamics on much longer timescales. Interestingly, we also find that the lifetime for CNSePhe shows increased sensitivity to the presence of hydrogen bonding interactions with the CN, suggesting that the probe should be useful for interpretation of CN spectra and possibly for the study of solvation.
Co-reporter:
Analytical Methods (2009-Present) 2015 - vol. 7(Issue 17) pp:NaN7241-7241
Publication Date(Web):2015/04/08
DOI:10.1039/C5AY00523J
Local heterogeneity of microenvironments in proteins is important in biological function, but difficult to characterize experimentally. One approach is the combination of infrared (IR) spectroscopy and site-selective incorporation of probe moieties with spectrally resolved IR absorptions that enable characterization within inherently congested protein IR spectra. We employed this method to study molecular recognition of a Src homology 3 (SH3) domain from the yeast protein Sho1 for a peptide containing the proline-rich recognition sequence of its physiological binding partner Pbs2. Nitrile IR probes were introduced at four distinct sites in the protein by selective incorporation of p-cyanophenylalanine via the amber codon suppressor method and characterized by IR spectroscopy. Variation among the IR absorption bands reports on heterogeneity in local residue environments dictated by the protein structure, as well as on residue-dependent changes upon peptide binding. The study informs on the molecular recognition of SH3Sho1 and illustrates the speed and simplicity of this approach for characterization of select microenvironments within proteins.
Dithionate
cytochrome F from turnip
Bicyclo[2.2.1]heptane-2-thione,1,7,7-trimethyl-