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Overall Graduate Prize Winner 2018

I completed my Bachelor’s degree in Biochemistry at the University of Wisconsin-Madison (USA), during which I conducted research at the National Magnetic Resonance Facility at Madison (NMRFAM). At NMRFAM, I was introduced to molecular chaperones, a class of proteins that prevent the misfolding and aggregation of other proteins, and NMR spectroscopy, which can provide atomic-level insight into the structures and dynamics of proteins. I wanted to learn more about molecular chaperones and NMR, and so applied to various PhD programs in the USA, and to one USA-UK program named the National Institutes of Health (NIH)-Oxford/Cambridge (NIH-OxCam) program. I was awarded a fellowship from the NIH and enrolled in the NIH-OxCam program, joining the laboratories of Prof. Andrew Baldwin (Pembroke) and Prof. Justin Benesch (University) at Oxford and Dr. Ad Bax at the NIH.

My research across these three laboratories focused on (1) understanding the molecular mechanisms of small heat shock proteins, a class of molecular chaperones that prevent protein misfolding and aggregation, and (2) studying various aspects of protein folding. While my research utilizes a variety of biophysical tools and approaches, the primary method is NMR spectroscopy. A review that I wrote highlighted the utility of NMR in studying molecular chaperones at the atomic level (Alderson TR, et al. 2016 Structure) and a commentary on a Nature publication (Alderson TR and Bax A, 2016 Nature) discussed the importance of in-cell NMR for the analysis of the conformation and dynamics of the intrinsically disordered protein alpha-synuclein inside neuronal cells.

Small heat shock proteins (sHSPs) typically assemble into large, dynamic oligomers with average molecular masses in excess of 500 kDa. Their oligomeric states are plastic, however, and depend strongly on environmental conditions (pH, temperature, post-translational modifications, etc.), which provides the cell with mechanisms to control sHSP assembly. In field of sHSP biochemistry, the oligomeric plasticity of these chaperones and their complex concentration-dependent equilibria have caused challenges when trying to understand which form is the “active” species: monomers, dimers, small oligomers, or large oligomers? The field overwhelmingly supports the dimer as the active species. My research instead demonstrated that the monomeric state of HSP27 is the most active form of this sHSP. Moreover, using NMR spectroscopy, I showed that monomerization of HSP27 leads to the unfolding of the region responsible for dimerization. Therefore, the HSP27 monomer exists as a partially disordered protein, yet highly potent molecular chaperone (Alderson TR, et al. 2018 Nature Communications).

The second theme of my DPhil research related to the application of NMR to investigate aspects of protein folding. Two such studies involved the study of cis-proline formation in unfolded proteins (Alderson TR, Benesch JLP, Baldwin AJ, 2017 Cell Stress and Chaperones) (Alderson TR, et al. 2018 ChemBioChem). As many biochemists know, the peptide bond can exist in two states: the cis or trans conformation, with >99.9% of peptide bonds not involving proline adopting the trans conformer. When the peptide bond contains a proline residue, however, the geometrical restrictions imposed by its side-chain render the energy difference between trans and cis conformers close to zero. As such, the population of the cis conformation is significantly increased in peptide bonds containing proline. Inside the cell, the slow isomerization of cis- to trans-proline comprises an essential step in the folding of many proteins, and the ribosome interacts with a cis-trans proline isomerase to specifically catalyze this reaction. Using NMR spectroscopy, we were able to quantify exactly how much cis-proline exists in a given disordered or unfolded protein. The precise values of cis-proline that we measured (3–10%, mean of 6% across 15 X-Pro bonds) are significantly lower than values commonly reported in textbooks (e.g. 20% cis-proline in the

textbook by Voet and Voet), as those data are derived from studies performed on small peptides. We further showed that small peptides contain elevated values of cis-proline due to favorable electrostatic interactions between the oppositely charged N- and C-termini, which are proximal in small peptides but far apart in disordered/unfolded proteins. A caveat to our study is that the 15 X-Pro bonds that were investigated contained a limited set of amino acids in the “X” position; the nature of this amino acid influences the amount of cis-proline, with aromatics capable of producing 20% or more cis. However, in general, non-aromatic residues involved in a peptide bond with proline will contain 10% or less cis-proline. For example, all five X-Pro bonds in alpha-synuclein range from 3-5% cis-proline.

Finally, at the NIH, I was fortunate to participate in the development of new NMR methodology to investigate protein folding at the atomic level with millisecond resolution. Ad Bax and his colleague Dr. Philip Anfinfrud at the NIH constructed novel hardware to rapidly and repeatedly switch the pressure inside the NMR tube within a few milliseconds (ms). For proteins that unfold at high pressures (3000 bar), this enables multidimensional NMR studies of protein folding with ms resolution, whereas previous NMR studies of protein folding were restricted to slowly folding proteins that took seconds or longer to reach their final state. Numerous single domain proteins – including the model proteins used for many folding studies – fold on the millisecond timescale or faster. Hydrogen-deuterium exchange (HDX) coupled to mass spectrometry has been employed to study proteins that fold on this timescale, albeit the approach provides limited resolution, as it requires proteolytic digestion to yield a set of peptides. Using the novel hardware developed by Ad and Philip, we developed an NMR method that enables measurement of amide-resolved solvent exchange (HX) rates during protein folding, enabling dissection of the times required to form hydrogen bonds as the protein is folding (Alderson TR, et al. 2017 Journal of the American Chemical Society). Our results revealed that a variant of ubiquitin, which folds in ca. 150 ms, first forms hydrogen bonds around 70–80 ms, but remains in a molten globule state; the overall folding is limited by the slow formation of a reverse type-II turn near the C-terminus of the protein. This paper was recommended by F1000 Prime. Two subsequent papers further demonstrated the utility of the rapid pressure-switch approach to studying the folding of the ubiquitin variant (Charlier C, Alderson TR, et al. 2018 Proceedings of the National Academy of Sciences) (Charlier C, et al. 2018 Journal of the American Chemical Society), with the former publication highlighted by the magazine Chemical & Engineering News.

I submitted my DPhil in October 2018 and am presently learning the Croatian language in Zagreb. Afterward, I will begin a postdoctoral research appointment in the laboratory of Prof. Lewis Kay (FRS) from the University of Toronto, furthering my training in the field of protein NMR.

 Publications during my DPhil:

1. Alderson TR and Bax A* (2016). Parkinson’s disease: Disorder in the court. Nature 530, 38-39. DOI: 10.1038/nature16871

2. Alderson TR*, Kim JH, Markley JL (2016). Dynamical structures of Hsp70 and Hsp70–Hsp40 complexes. Structure 24, 1014-30. DOI: 10.1016/j.str.2016.05.011

3. Alderson TR, Benesch JLP*, Baldwin AJ* (2017) Proline isomerization in the C-terminal region of HSP27. Cell Stress and Chaperones 22, 639-51. DOI: 10.1007/s12192-017-0791-z

4. Pritišanac I, Degiacomi MT, Alderson TR, Carneiro M, Ab E, Siegal G, Baldwin AJ* (2017) Automatic assignment of methyl-NMR spectra of supramolecular machines using graph theory. Journal of the American Chemical Society 139, 9523-33. DOI: 10.1021/jacs.6b11358

5. Alderson TR§, Charlier C§, Anfinrud P*, Bax A* (2017) Monitoring hydrogen exchange during protein folding by fast pressure jump NMR spectroscopy. Journal of the American Chemical Society 139, 11036-39. DOI: 10.1021/jacs.7b06676 Recommended by F1000 Prime

6. Alderson TR, Lee JH, Charlier C, Ying J, Bax A*. (2018) Propensity for cis-proline formation in unfolded proteins. ChemBioChem 19: 37-42. DOI: 10.1002/cbic.201700548

7. Charlier C, Alderson TR, Courtney JM, Ying J, Anfinrud P*, Bax A*. (2018) Study of protein folding under native conditions by rapid switching of the hydrostatic pressure inside an NMR sample cell. Proc. Natl. Acad. Sci. USA 115: E4169-78. DOI: 10.1073/pnas.1803642115 Covered by Chemical and Engineering News magazine

8. Charlier C, Courtney JM, Alderson TR, Ying J, Bax A*. (2018) Monitoring 15N chemical shifts during protein folding by pressure-jump NMR. J. Am. Chem. Soc., in press.

9. Alderson TR, Roche J, Gastall HY, Dias DM, Pritišanac I, Bax A, Benesch JLP*, Baldwin AJ*. Local unfolding of the HSP27 monomer regulates chaperone activity. Nat. Comms., in press.