Analysis of RNA structure using small-angle X-ray scattering

SAXS Ensemble Refinement of ESCRT-III CHMP3 Conformational Transitions

Nuclear magnetic resonance (NMR) spectroscopy and small angle X-ray scattering (SAXS) are solution-based methods that can be used to analyze and determine molecular structure. Here, we review the use of NMR and SAXS for the structural analysis of RNA molecules, with a focus on RNAs involved in translational control. Practical considerations, experimental requirements, and data analysis are discussed. We also review a recently developed hybrid approach that combines NMR and SAXS. The hybrid NMR-SAXS approach shows great promise for expanding the scope of biophysical studies on biological molecules in solution. Finally, we review examples of these methods applied to RNA systems such as programmed ribosomal frameshift sites, internal ribosome entry sites, ribosome-binding structural elements, RNA thermometers, and riboswitches.

Biopolymers are versatile molecules that perform myriad cellular functions such as gene regulation and high-energy molecule synthesis. Alterations in biomolecular structure such as those arising from mutations can disrupt important cellular processes and lead to disease. Therefore, accurate biopolymer structure characterization plays an outsized role in improving human health. X-ray crystallography, cryogenic electron microscopy (Cryo-EM), and nuclear magnetic resonance (NMR) spectroscopy are the most powerful techniques for obtaining high-resolution, 3D structures.Valuable structural insight is also obtained using optical rotation, small angle X-ray scattering (SAXS), circular dichroism (CD) spectroscopy, and Forster resonance energy transfer (FRET) spectroscopy. Over the past few years, remarkable advances in computational power and algorithm development have revolutionized the field of biopolymer structure prediction. For example, leveraging machine learning (ML) and artificial intelligence (AI), AlphaFold and Open fold have demonstrated a remarkable ability to predict biopolymer structure from sequence. Although powerful, these techniques are limited in their ability to provide information about challenging systems where the structure is either unknown or highly flexible under physiological conditions. In this work, studies are presented in which structural information is gleaned from challenging biopolymer systems using novel computational and experimental methods. In Chapter 2, a new structure characterization methodology based on hydrogen-deuterium exchange-mass spectrometry (HDX-MS) in combination with molecular dynamics (MD) simulations is introduced. The approach is used to describe the structural motifs of the unstructured peptide, Nt17, a conserved sequence in Huntingtin protein and associated with Huntington’s Disease progression. The model is used to show that, although highly flexible, Nt17 is intrinsically primed for facile conversion to α-helical conformation upon binding with molecular partners. Having shown that HDX reactivity is predictive of the degree of structural flexibility and bias (propensity to form 2° structural elements such as α-helix, β-sheet, and β-turn) for intrinsically disordered regions (IDRs), the work in Chapter 3 explores an extension of structure characterization to protein-ligand systems. In this study human FK506 binding protein (FKBP12) structure is examined in the absence and presence of two, well-recognized ligand molecules (i.e., FK506 and rapamycin). The work provides a better understanding of binding/unbinding events, competitive binding, and folded/unfolded conformer heterogeneity. Native MS and HDX-MS were utilized in conjunction with MD simulations to understand this select intrinsically disordered region (IDR) and IDR-drug conformational landscape. Chapter 4 explores the functional property prediction of Proteorhodopsin, by integrating experimental studies with MD simulations validation. Here, a novel linear regression model accurately predicts the spectral

Methods for using new conceptual tools and parameters to assess RNA structure by small-angle X-ray scattering.

Cytoplasmic dynein complexes play crucial roles in intracellular transport of cellular organelles. While the motor domain of dynein is well characterized by techniques such as X-ray crystallography and cryo-electron microscopy (Cryo-EM), structural representations of dynein usually include only the more packed and easily resolved regions and omit the long flexible and poorly structured regions. One such flexible region is the N-terminal half of the intermediate chain (IC), which contains almost 300 amino acids that are predicted to be disordered. This level of disorder makes IC impossible to study by X-ray crystallography and Cryo-EM, but amenable to study by solution nuclear magnetic resonance (NMR), a powerful technique that can elucidate residue-specific information in adynamic ensemble of structures, and transient binding interactions of associated proteins. Here, we describe the methods we use to characterize flexible and disordered proteins including protein expression, purification, sample preparation, and NMR data acquisition and analysis.

Histotoxic clostridia produce collagenases responsible for extensive tissue destruction in gas gangrene. The C-terminal collagen-binding domain (CBD) of these enzymes is the minimal segment required to bind to collagen fibril. Collagen binding efficiency of CBD is more pronounced in the presence of Ca(2+). We have shown that CBD can be functional to anchor growth factors in local tissue. A (1)H-(15)N HSQC NMR titration study with three different tropocollagen analogues ((POG)(10))(3), ((GPOG)(7)PRG)(3), and (GPRG(POG)(7)C-carbamidomethyl)(3), mapped a saddle-like binding cleft on CBD. NMR titrations with three nitroxide spin-labeled analogues of collagenous peptide, (PROXYL-G(POG)(7)PRG)(3), (PROXYL-G(POG)(7))(3), and (GPRG(POG)(7)C-PROXYL)(3) (where PROXYL represents 2,2,5,5-tetramethyl-l-pyrrolidinyloxy), unambiguously demonstrated unidirectional binding of CBD to the tropocollagen analogues. Small angle x-ray scattering data revealed that CBD binds closer to a terminus for each of the five different tropocollagen analogues, which in conjunction with NMR titration studies, implies a binding mode where CBD binds to the C terminus of the triple helix.

Common features of the extracellular carbohydrate-active virulence factors involved in host-pathogen interactions are their large sizes and modular complexities. This has made them recalcitrant to structural analysis, and therefore our understanding of the significance of modularity in these important proteins is lagging. Clostridium perfringens is a prevalent human pathogen that harbors a wide array of large, extracellular carbohydrate-active enzymes and is an excellent and relevant model system to approach this problem. Here we describe the complete structure of C. perfringens GH84C (NagJ), a 1001-amino acid multimodular homolog of the C. perfringens micro-toxin, which was determined using a combination of small angle x-ray scattering and x-ray crystallography. The resulting structure reveals unprecedented insight into how catalysis, carbohydrate-specific adherence, and the formation of molecular complexes with other enzymes via an ultra-tight protein-protein interaction are spatially coordinated in an enzyme involved in a host-pathogen interaction.

Plasmodium falciparum is a unicellular protozoan parasite that is commonly known to cause malaria in humans. The symptoms of malaria associated with repeated rounds of parasite replication, egress, and invasion into the red blood cells. During the red blood cell stage of malaria infection, PfBDP1 is shown to bind at the transcriptional start sites of invasion-related genes and regulates their expression. A recent spike in malaria infections may be attributed to drug-resistance in P. falciparum, thus we need a better understanding of the parasite's life cycle to produce more effective antimalarial drugs. The P. falciparum genome encodes for ten bromodomain-containing proteins. Previously, the bromodomain of PfBDP1 was shown to preferentially bind acetylated lysine marks on histones, effectively tethering the PfBDP1 transcriptional activator complex to modified nucleosomes within specific chromatin regions. However, the molecular mechanisms driving chromatin binding and recognition by PfBDP1 are not well understood. PfBDP1 contains a unique combination of six ankyrin repeats (ANK) domain followed by a bromodomain (BRD). Bromodomains are evolutionary conserved protein-protein interaction modules (110 amino acids long) that recognize acetylated lysine (Kac) on histones and other proteins. We hypothesized that the bromodomain would modulate the interaction of PfBDP1 with a subset of acetylated histone modifications. We used a structure-function approach including X-ray crystallography, Nuclear Magnetic Resonance (NMR), Small Angle X-ray Scattering (SAXS), analytical ultracentrifugation, and Isothermal Titration Calorimetry (ITC) to characterize the interaction of PfBDP1 with chromatin ligands. The crystal structure of PfBDP1-BRD at 2.0 Å shows that it has a conserved bromodomain fold, and an acetylated lysine binding pocket comprised of four alpha helices. As previously reported, PfBDP1 has been shown to interact with acetylated histone H3, but our in vitro binding experiments revealed that PfBDP1-BRD preferentially binds to tetra-acetylated histone H4. Our data indicate that PfBDP1 has a unique histone ligand binding mechanism that might be leveraged for the design novel therapeutic treatments, and suggest that PfBDP1 may have additional, yet unidentified roles in the P. falciparum life cycle. Plasmodium falciparum is a unicellular protozoan parasite that is commonly known to cause malaria in humans. The symptoms of malaria associated with repeated rounds of parasite replication, egress, and invasion into the red blood cells. During the red blood cell stage of malaria infection, PfBDP1 is shown to bind at the transcriptional start sites of invasion-related genes and regulates their expression. A recent spike in malaria infections may be attributed to drug-resistance in P. falciparum, thus we need a better understanding of the parasite's life cycle to produce more effective antimalarial drugs. The P. falciparum genome encodes for ten bromodomain-containing proteins. Previously, the bromodomain of PfBDP1 was shown to preferentially bind acetylated lysine marks on histones, effectively tethering the PfBDP1 transcriptional activator complex to modified nucleosomes within specific chromatin regions. However, the molecular mechanisms driving chromatin binding and recognition by PfBDP1 are not well understood. PfBDP1 contains a unique combination of six ankyrin repeats (ANK) domain followed by a bromodomain (BRD). Bromodomains are evolutionary conserved protein-protein interaction modules (110 amino acids long) that recognize acetylated lysine (Kac) on histones and other proteins. We hypothesized that the bromodomain would modulate the interaction of PfBDP1 with a subset of acetylated histone modifications. We used a structure-function approach including X-ray crystallography, Nuclear Magnetic Resonance (NMR), Small Angle X-ray Scattering (SAXS), analytical ultracentrifugation, and Isothermal Titration Calorimetry (ITC) to characterize the interaction of PfBDP1 with chromatin ligands. The crystal structure of PfBDP1-BRD at 2.0 Å shows that it has a conserved bromodomain fold, and an acetylated lysine binding pocket comprised of four alpha helices. As previously reported, PfBDP1 has been shown to interact with acetylated histone H3, but our in vitro binding experiments revealed that PfBDP1-BRD preferentially binds to tetra-acetylated histone H4. Our data indicate that PfBDP1 has a unique histone ligand binding mechanism that might be leveraged for the design novel therapeutic treatments, and suggest that PfBDP1 may have additional, yet unidentified roles in the P. falciparum life cycle.

Interferon-stimulated gene-15 (ISG15) is an interferon-induced protein with two ubiquitin-like (Ubl) domains linked by a short peptide chain and is a conjugated protein of the ISGylation system. Similar to ubiquitin and other Ubls, ISG15 is ligated to its target proteins through a series of E1, E2, and E3 enzymes known as Uba7, Ube2L6/UbcH8, and HERC5, respectively. Ube2L6/UbcH8 plays a central role in ISGylation, underscoring it as an important drug target for boosting innate antiviral immunity. Depending on the type of conjugated protein and the ultimate target protein, E2 enzymes have been shown to function as monomers, dimers, or both. UbcH8 has been crystallized in both monomeric and dimeric forms, but its functional state remains unclear. Here, we used a combined approach of small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) spectroscopy to characterize UbcH8's oligomeric state in solution. SAXS revealed a dimeric UbcH8 structure that could be dissociated when fused N-terminally to glutathione S-transferase. NMR spectroscopy validated the presence of a concentration-dependent monomer-dimer equilibrium and suggested a back-side dimerization interface. Chemical shift perturbation and peak intensity analysis further suggest dimer-induced conformational dynamics at the E1 and E3 interfaces, providing hypotheses for the protein's functional mechanisms. Our study highlights the power of combining NMR and SAXS techniques to provide structural information about proteins in solution.

The structural basis for the functional comparability of factor VIII and the long‐acting variant recombinant factor VIII Fc fusion protein

The composition and morphology of bones implanted with stainless steel (316L-SS) and a metal alloy made of zinc, aluminum, and copper (Zinalco) are compared. Small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) results show that with time Zinalco is corroded and zinc, aluminum, and copper diffuse into the osseous tissue, promoting nonhomogeneous bone. Instead, 316L-SS does not incorporate into bone, and the bone recovers homogeneously at a lower speed.

The double-gyroid phase of nanoporous silica films formed by evaporation-induced self-assembly (EISA) has been shown to possess facile mass-transport properties and may be used as a robust template for the nanofabrication of metal and semiconductor nanostructures. Recently, we developed a new synthesis of double-gyroid nanoporous silica films where the aging time of the coating solution prior to EISA was the key parameter required to control the interfacial curvature that results upon self-assembly of the film. Here, we use 29Si nuclear magnetic resonance (NMR) and small-angle X-ray scattering (SAXS) to investigate the nanoscale structure of the coating solutions used to obtain double-gyroid nanoporous silica films. NMR and SAXS were carried out on the water, ethanol, silica, and poly(ethylene oxide)-b-poly(propylene oxide)-b-alkyl (EO17-PO12-C14) surfactant coating solutions as well as similar solutions that excluded either the silica or the surfactant. NMR data reveal that the silica monomers in the coating solution condense very rapidly to form rings and connected ring species. After 1 day of aging, all monomers and dimers have disappeared, and the distribution is dominated by Q2 and Q3 species, where the superscript in Qn describes the number of silicon atoms in the second coordination shell of the central silicon. Over the course of the next 9 days, the Q3 population slowly rises at the expense of the Q2 and Q3t populations. Absolute intensity SAXS measurements reveal that the size of the silica clusters increases steadily during this aging period, reaching an average radius of gyration of 9.0 A after 9 days of aging. Longer aging results in the continued growth of clusters with a mass fractal dimension of 1.8. Absolute intensity SAXS data also reveals that micelles are not present in the coating solution. At 9% volume fraction of surfactant, the coating solution is far above the aqueous critical micellar concentration. However, even a small amount of ethanol inhibits micellization. SAXS data also shows that when surfactant is present the radius of gyration is larger but increases more slowly. This indicates that there are weak associative interactions between the silica clusters and surfactant in solution that reduce the cluster-cluster growth rate. In part II of this work, we use the results discovered here to interpret the effects of aging on interfacial curvature in the nanostructured films that self-assemble from these solutions.

Multi-state modeling of antibody-antigen complexes with SAXS profiles and deep-learning models.

Small-angle X-ray scattering (SAXS) and Nuclear Magnetic Resonance (NMR) are established methods to analyze the structure and structural transitions of biological macromolecules in solution. Both methods are directly applicable to near-native macromolecular solutions and allow one to study structural responses to physical and chemical changes or ligand additions. Whereas SAXS is applied to elucidate overall structure, interactions and flexibility over a wide range of particle sizes, NMR yields atomic resolution detail for moderately sized macromolecules. NMR is arguably the most powerful technique for the experimental analysis of dynamics. The joint application of these two highly complementary techniques provides an extremely useful approach that facilitates comprehensive characterization of biomacromolecular solutions.

Chapter 4 - NMR-based approaches to the study of GPCRs and GPCR-ligand interactions