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The 2018 Award winner discusses her career challenges and her research on the use of specially engineered proteins, combined with 2D IR spectroscopy, for investigating protein function dynamics.
Protein engineering may be used to insert infrared-active groups, such as CNF, at specific sites within protein molecules. When combined with 2D infrared (IR) spectroscopy, these engineered probes, such as plastocyanin, enable the study of protein microenvironments and binding properties. This specialized technique provides details on site-specific protein heterogeneity and dynamics. Megan Thielges, an associate professor of chemistry at Indiana University, is a pioneer in the development and use of such probes with 2D IR. She is also the recipient of the 2018 Emerging Leader in Molecular Spectroscopy Award presented by Spectroscopy magazine. Thielges recently spoke with us about her work demonstrating the application of site-specific 2D IR spectroscopy for investigating protein function dynamics. She also talked about her career development and the biggest challenges and rewards in her daily work.
Please tell us about some of your earliest research interests. How did you get started in science? What has kept you motivated?
Honestly, since I can remember, I have wanted to be a scientist. When we graduated from elementary school, my teacher wrote predictions for each student's career; I was to become a cancer researcher and create my own designer line of labcoats. As I was exposed to new areas, my interests evolved from microbiology to biochemistry to physical chemistry, essentially becoming more focused on smaller-scale phenomena and more fundamental descriptions. My goal always has been to have a career doing something I love, which has motivated me throughout my life.
What have been the most difficult aspects of your research to date? How have you worked to overcome these challenges?
Our work is highly interdisciplinary. It makes my work fun, but challenging. We study protein dynamics via highly technical methods, with the ultimate goal of uncovering whether and how protein dynamics are tailored by evolution for function. This endeavor requires that our group operate a femtosecond laser system and understand the fairly challenging theoretical basis of this technology, while at the same time we have to make and handle delicate protein samples and address important questions about complex systems. Juggling the disparate and difficult aspects of our work is a constant challenge.
You did postdoctoral research at Stanford University with Professor Michael D. Fayer.What was the highlight of that work?
I first demonstrated 2D IR spectroscopy of an IR probe introduced as an unnatural amino acid as an approach to protein 2D IR. Personally, the highlight of my postdoctoral studies was the self-improvement gained from working with Mike and the research group he had assembled. Interacting with such an intelligent group of people was inspiring. I consider it to be one of the best times of my life.
Of all your research papers so far, what are two of the most meaningful papers you would like to highlight for our readers?
One of our studies employed infrared (IR) spectroscopy to characterize carbon deuterium IR probes for investigating the binding of peptides with proline recognition motifs to an Src homology 3 domain. We uncovered multiple IR absorptions for single vibrational modes, indicating multiple populated states. We then prepared the same system labeled with 13C to facilitate NMR spectroscopy. The same bonds that showed multiple states via IR spectroscopy showed single resonances via NMR spectroscopy, due to their being too fast to resolve on the NMR timescale. I think this work clearly showed the power of the inherently fast timescale of IR spectroscopy to uncover all potentially important conformational states.
The second paper I would choose focused on the role of dynamics in the regioselectivity of P450 catalysis. We characterized the dynamics of the P450 and a mutant in complex with several substrates that are acted upon with varying regioselectivity and found that the spectral dynamics, but not the average frequency of the IR probe, correlated with activity. The idea that motion on fast timescales contributes to protein function is controversial. However, the environment of a chemical in solution fluctuates on picosecond timescales. I think of an enzyme active site as an evolved path, so I consider such very fast motions of protein side chains are likewise to be important to our complete understanding of protein function. I think our study contributed to experimentally demonstrating that idea.
What is the most difficult or challenging aspect of your current research and teaching role?
Balancing all my time commitments is a challenge. I am working on the art of saying "no" to requests, but I am still not very good at it. I am also constantly working to be more effective mentor and to manage and inspire a group of varying personalities.
What is the most exciting part of your work day?
Interacting with my students. Even when I feel drained, I find myself rejuvenated after talking with them.
Are the opportunities in the scientific arena changing for women? What would you say to young girls with an interest in science that may hesitate to pursue their aspirations?
There is increasing support of females in science, perhaps more so than in other career areas, and I would say that I rarely experience overt bias or harassment. I also find that younger scientists have less inherent bias from being raised from birth in a society that at least claims to view women and men as equals. As the older generations retire over time, the climate will get continually better, just as is occurring in the greater society.
Your research focuses on the use of vibrational probes, with two-dimensional IR spectroscopy (2D IR), to investigate the structures and dynamics of proteins. Why did you decide to pursue this area of research?
I am interested in the biophysics underlying protein function and think that the dynamics are a major deficient aspect of our current understanding. As a graduate student I measured protein dynamics using nonlinear visible spectroscopy. Visible chromophores are huge and not natively found in most proteins, and their spectroscopy is much more complicated to theoretically describe than IR chromophores. I considered pursing NMR spectroscopy, but the technique is so powerful because it is so well developed. I thought that a different approach might be able to uncover new aspects of protein biophysics.
What new discoveries has your work revealed in the basic understanding of biological systems?
Generally, we have contributed to experimentally uncovering rapidly interconverting states of proteins and assessing their relation to function. In the example of proline-rich motif recognition by Src homology 3 domains, a long-standing question regards the molecular basis of the typically unfavorable entropy of binding. By IR spectroscopy, we found evidence for multiple rapidly interconverting states associated with backbone configurations of proline residues that change upon complexation in a manner that helps to explain the variation in the binding entropies measured for different sequences. Another example of new information from site-specific IR spectroscopy came from our study of the metal site in the blue copper protein plastocyanin, and how the complexation with electron transfer partner cytochrome f affects it. We used carbon deuterium bonds incorporated at the axial ligand as a non-perturbative way to monitor the binding-induced changes of the metal site and found that increased ionic interaction between the axial ligand and copper ion resulted from the protein–protein interaction, providing a mechanism for the long known decrease in redox potential in the complex.
Would you briefly describe your work of detailed functional studies on cytochrome P450s? Have you been able to determine that different substrate binding configurations and the fast dynamics of conformational interchange will be key to understanding the drug-degradation products in promiscuous enzymes?
We have data that support that fast dynamics are important to the specificity of cytochrome P450 catalysis, but our work thus far is just a start. We prepared the enzyme with a carbon monoxide bound at the active site to use as a vibrational probe, and then characterized the vibrational spectra and dynamics via 1D and 2D spectroscopy when the enzyme was in complex with substrates that are acted upon with varying regioselectivity. The spectra indicated that the enzyme–substrate complexes varyingly populated two conformational states–one with fast dynamics among substates, the other with slow dynamics. The extent the complex populated one or the other states correlated with the regioselectivity of P450 on the substrate, with fast dynamics associated with lower selectivity. The activity did not correlate with the average frequency of the spectra, which is reflective of the average electrostatic environment at the IR probe, suggesting that dynamics in the conformation were the important factor. We are currently working on testing our model with different IR probes located at various sites in the enzyme, as well as with different P450 enzymes, including the human isoforms that are important to pharmaceutical development.
How is your work in advanced coherent 2D IR spectroscopy relevant to fundamental understanding of enzymology and signaling structure and functions?
A focus of our research is elucidating how motions of proteins contribute to molecular recognition; the recognition of substrates by enzymes and of linear sequence motifs by signaling domains are two examples. Due to the complexity of proteins, investigating the role of dynamics in their function requires experimental measurement with both high spatial and temporal resolution. IR spectroscopy provides an approach that can be used to characterize the environments at specific locations in proteins and their dynamics on very fast timescales, while the protein is in solution at room temperature. However, the extent that 1D IR spectra can be interpreted in a rigorous manner is limited. 2D IR spectroscopy can provide richer information, including the spectral dynamics.
What made you choose the path of two distinct chemistry disciplines, these being protein and enzyme biochemistry and biophysics, and ultrafast laser spectroscopy?
I've always been amazed by the complexity of living systems and how it could evolve. My undergraduate major was biochemistry, and all my work since has focused on proteins. I also find the quantitative nature of physical chemistry satisfying. Together these formed my interests in biophysics and spectroscopy. Multidimensional spectroscopy is more powerful than linear, and its implementation requires ultrafast lasers. I also really enjoy working with optical systems, building and optimizing the instrument, like a kid with a really expensive set of Legos.
How do you go about building a research team for this specialized work?
All of our group thinks about proteins and spectroscopy, but some focus more on the ultrafast 2D IR spectroscopy, while others focus on the biophysical questions, the biochemical aspects of labeling proteins with IR probe groups, and the challenge of detecting weak absorptions by 1D IR spectroscopy. I try to excite prospective members by interest in the questions we address and the fun and challenge of building the ultrafast spectroscopic system.
Where would you like to see this research expand into the future?
We are always working to improve and extend the experimental approach of 1D and 2D IR spectroscopy on biomolecules. We are just beginning to investigate the human cytochrome P450s that are important to human health. Our studies of proline-rich motifs are tied to a general interest in intrinsically disordered parts of proteins. They are very common, but less well understood than structured proteins, and their dynamics are inherently key to function. Another continued interest is biological electron transfer and photosynthesis. We have plans to implement non-equilibrium 2D IR experiments to investigate how proteins mediate electron transfer.
What is your most optimistic view of the fundamental understanding of proteins or enzymatic action that you would hope to achieve over your lifetime?
I hope to understand the mechanisms by which dynamics are tuned through evolution for function, and through our understanding, design protein dynamics for selective or promiscuous activity. We can rationalize how to introduce enthalpic contributions to binding through display of chemical functionality, but controlling entropic contributions is more challenging. Regarding method development, my hope is that biomolecular 2D IR spectroscopy could be used to directly follow all the interactions and their dynamics among all possible carbon deuterium labeled parts and the rest of the functional groups in a protein, providing a spatially complete picture nearing the level currently provided by NMR spectroscopy, but with faster temporal resolution.