NSRRC Activity Report 2022

054 NSRRC ACTIVITY REPORT 2022 chemical denaturants, extreme pH values, high temperatures, or high pressures. The one-dimensional (1D) SAXS profile as a function of the momentum transfer, q, which is proportional to the scattering angle, is encoded by the 3D structure (or ensemble of structures) of the system of interest. Robust modeling procedures have been developed to generate ab initio models that fit experimental SAXS profiles, with the caveat that degenerate solutions exist because the 3D solutions are derived from 1D information. A more practical use of SAXS data is to back-calculate the theoretical SAXS profile of a 3D atomic model to evaluate whether the model agrees with experimental SAXS data ( Fig. 1(a) ). When structural polymorphism and molecular dynamics are considered, an ensemble average of SAXS profiles can be generated to quantitatively describe the conformational features of the protein of interest. In protein folding studies, model-free SAXS analysis is particularly useful in characterizing solution structures as a function of different physicochemical variables. First, the radius of gyration R g of the particle of interest can be approximated using a Guinier plot—ln I(q) vs. q 2 —that focuses on the low-q region (q•R g < 1.3; Figs. 1(b) and 1(c) ). Second, the Kratky plot—I(q)q 2 vs. q—indicates the degree of folding of the protein; a bell-shaped profile indicates a well-folded structure ( Fig. 1(d) ). By contrast, the up-tilting profile in the high-q region is indicative of conformational disorder. Third, the pair-wise distance distribution, P(r), of the SAXS profile is used as the input for ab initio modeling. However, the raw SAXS profile already indicates the overall dimension, D max , and the likely number of globular domains of the system ( Fig. 1(e) ). The laboratory of Shang-Te Danny Hsu (Academia Sinica) has systematically used SAXS to verify whether the analyzed protein crystal structures resemble their solution- state counterparts. 2,3 Importantly, the team members have extensively used SAXS for protein folding studies. For instance, they demonstrated that an engineered, truly 3 1 -knotted YibK without open ends followed the same dimension scaling law as predicated using polymer theory. 4 They also reported an unusual case of a chemically induced protein folding intermediate of 5 2 -knotted UCH-L1 that can form a specific dimer despite the loss of most secondary structures. 5 Furthermore, the integration of SAXS, far UV circular dichroism, and intrinsic fluorescence spectroscopy enabled them to comprehensively describe the folding pathway of the most complex 6 1 -knotted DehI 6 and the smallest 3 1 -knotted MJ0366. 7 More recently, they used SAXS to investigate how several cancer-associated mutations affect the structure-function relationship of the 5 2 -knotted ubiquitin C-terminal hydrolase (UCH) domain of BRCA1-associated protein 1 (BAP1). Some mutations significantly destabilized the UCH domain of BAP1, leading to a substantial increase in their R g values, which correlated strongly with reduction in melting temperature and enthalpy of unfolding ( Figs. 1(f) and 1(g) ). 8 In some cases, the mutations substantially enhanced aggregation propensity, and the BAP1 variants readily formed intracellular aggregates that limited their ability to enter Fig. 1 : (a) Structural mapping of cancer-associated high-frequency mutations in BAP1-UCH. The homology model of BAP1-UCH is shown in a ribbon representation, whereas ubiquitin is shown in a surface representation. The positions of the Cα atoms of the mutation sites are shown as colored spheres. Inset: Comparison of the experimental SAXS profiles (open circles) and back-calculated SAXS profile based on a homology model of BAP1-UCH in a complex with ubiquitin (gray). (b) Overlay of experimental SAXS profiles with wild type (WT) shown in black and those of the other mutants shown in colors corresponding with those in (a). (c) Overlay of Guinier plots of BAP1-UCH variants with estimated R g values indicated. (d) Overlay of Kratky plots of BAP1-UCH variants. (e) Overlay of P(r) profiles of BAP1-UCH variants. (f) Differential scanning calorimetry (DSC) isotherm of WT BAP1-UCH. The profile was deconvoluted into two Gaussian functions, the areas under the curve of which indicate enthalpies of unfolding, ∆H 1 and ∆H 2 . (g) Correlation plot of SAXS-derived R g values corresponding to ∆H 1 and ∆ H2 derived from DSC. [Reproduced from Ref. 8] (a) (b) (c) (f) (d) (e) (g)