NSRRC Activity Report 2022

Life Science 053 References 1. S. Shen, Y. Tong, Y. Luo, L. Huang, W. Gao, Nat. Prod. Rep. 39 , 1856 (2022). 2. L. Niu, Y. Wang, C. Wang, Y. Wang, X. Jiang, L. Ma, C. Wu, Y. Yu, Q. Chen, Biochem. Biophys. Res. Commun. 493 , 718 (2017). 3. C.-C. Wu, T.-K. Li, L. Farh, L.-Y. Lin, T.-S. Lin, Y.-J. Yu, T.-J. Yen, C.-W. Chiang, N.-L. Chan, Science 333 , 459 (2011). 4. W. Lau, E. S. Sattely, Science 349 , 1224 (2015). 5. H. Tang, M.-H. Wu, H.-Y. Lin, M.-R. Han, Y.-H. Tu, Z.-J. Yang, T.-C. Chien, N.-L. Chan, W.-C. Chang, PNAS 119 , e2113770119 (2022). 6. M. Lazzarotto, L. Hammerer, M. Hetmann, A. Borg, L. Schmermund, L. Steiner, P. Hartmann, F. Belaj, W. Kroutil, K. Gruber, M. Fuchs, Angew. Chem. Int. Ed. Engl. 58 , 8226 (2019). Applications of Small-Angle X-ray Scattering in Protein Folding Studies Small-angle X-ray scattering is a powerful tool for investigating protein structures and dynamics under diverse experimental conditions. It is particularly useful in investigating conformational changes pertinent to protein folding and misfolding. U nderstanding the mechanism by which polypeptide chains fold into specific three-dimensional (3D) structures to confer biological functions has been a decade- long challenge. The protein folding problem is not only fundamental to physical chemistry but also medically relevant because the failure of a protein to fold correctly or the tendency to misfold can cause debilitating diseases, such as cancer and neurodegenerative diseases. Protein folding can be considered a conformational sampling process of a funnel-like free-energy landscape, which involves all possible conformation combinations according to the degrees of freedom accessible to the polypeptide chain. Thus, the most energetically favorable state is assumed to correspond to the native state. The free-energy funnel often exhibits different degrees of roughness, or frustrations, that can trap folding intermediates in local minima. In addition to globular proteins that exhibit robust folding characteristics, a significant proportion of the proteome encodes for intrinsically disordered regions covering segments or the entirety of given proteins is becoming increasingly evident. The free-energy landscapes of these intrinsically disordered proteins (IDPs) exhibit many energetically equivalent local minima to populate an ensemble of conformations that can differ vastly. Many IDPs exhibit high aggregation propensities and self-associate into amorphous oligomers before forming highly ordered filamentous assemblies, amyloid fibrils. Environmental stress conditions, posttranslational modifications, or genetic mutations can also considerably perturb the native states of proteins such that their hydrophobic cores become more exposed, increasing their aggregation propensities. Therefore, understanding the structure- function relationships of disease-associated proteins requires in-depth understanding of the atomic structures and dynamics involved in protein folding. Although protein crystallography has been instrumental in providing atomic insights into protein structures, it primarily addresses how the target protein folds into the defined native structure in its crystalline state. Likewise, the emergence of cryo-electron microscopy (cryo-EM) has pushed the frontier of structural biology, but conformational heterogeneity remains a limiting factor for effective single-particle reconstruction. Solution-state nuclear magnetic resonance (NMR) spectroscopy can be used to determine protein structures at atomic resolution under either native or nonnative (denaturing) conditions. NMR spectroscopy can be used to quantitatively describe protein dynamics at atomic resolution across a broad range of timescales. However, a major disadvantage of solution- state NMR spectroscopy is the size limitation of the system of interest, which is typically less than 30 kDa. Advanced NMR experiments aided by elaborately stable isotope labeling can increase size limits to the order of MDa, but such applications are exceptional. In addition to the big three of structural biology—X-ray crystallography, NMR spectroscopy, and cryo-EM—small- angle X-ray scattering (SAXS) is exceptionally versatile in providing structural information of proteins across a broad range of spatial and temporal resolutions. 1 SAXS data can essentially be collected under any experimental conditions. Furthermore, SAXS does not have fundamental size limitations, although it is intrinsically more sensitive to larger particles. SAXS is particularly suited for protein folding studies that require the use of concentrated