Conformational Changes Research Articles

Machine learning-augmented molecular dynamics simulations (MD) reveal insights into the disconnect between affinity and activation of ZTP riboswitch ligands.

The challenge of targeting RNA with small molecules necessitates a better understanding of RNA-ligand interaction mechanisms. However, the dynamic nature of nucleic acids, their ligand-induced stabilization, and how conformational changes influence gene expression pose significant difficulties for experimental investigation. This work employs a combination of computational and experimental methods to address these challenges. By integrating structure-informed design, crystallography, and machine learning-augmented all-atom molecular dynamics simulations (MD) we synthesized, biophysically and biochemically characterized, and studied the dissociation of a library of small molecule activators of the ZTP riboswitch, a ligand-binding RNA motif that regulates bacterial gene expression. We uncovered key interaction mechanisms, revealing valuable insights into the role of ligand binding kinetics on riboswitch activation. Further, we established that ligand on-rates determine activation potency as opposed to binding affinity and elucidated RNA structural differences, which provide mechanistic insights into the interplay of RNA structure on riboswitch activation.

Molecular simulation reveals that pathogenic mutations in BTB/ANK domains of Arabidopsis thaliana NPR1 circumscribe the EDS1-mediated immune regulation

The NPR1 (nonexpressor of pathogenesis-related genes 1) is a key regulator of the salicylic-acid-mediated immune response caused by pathogens in Arabidopsis thaliana. Mutations C150Y and H334Y in the BTB/ANK domains of NPR1 inhibit the defense response, and transcriptional co-activity with enhanced disease susceptibility 1 (EDS1) has been revealed experimentally. This study examined the conformational changes and reduced NPR1–EDS1 interaction upon mutation using a molecular dynamics simulation. Initially, BTBC150YNPR1 and ANKH334YNPR1 were categorized as pathological mutations rather than others based on sequence conservation. A distant ortholog was used to map the common residues shared among the wild-type because the mutations were highly conserved. Overall, 179 of 373 residues were determining the secondary structures and fold versatility of conformations. In addition, the mutational hotspots Cys150, Asp152, Glu153, Cys155, His157, Cys160, His334, Arg339 and Lys370 were crucial for oligomer-to-monomer exchange. Subsequently, the atomistic simulations with free energy (MM/PB(GB)SA) calculations predicted structural displacements engaging in the N-termini α5133-178α7 linker connecting the central ANK regions (α13260–290α14 and α18320–390α22), where prominent long helices (α516) and short helices (α310) replaced with β-turns and loops disrupting hydrogen bonds and salt bridges in both mutants implicating functional regulation and activation. Furthermore, the mutation repositions the intact stability of multiple regions (L13C149-N356α20BTB/ANK-α17W301-E357α21N−ter/coiled-coil) compromising a dynamic interaction of NPR1–EDS1. By unveiling the transitions between the distinct functions of mutational perception, this study paves the way for future investigation to orchestrate additive host-adapted transcriptional reprogramming that controls defense-related regulatory mechanisms of NPR1s in plants.

CRISPR RNA binding drives structural ordering that primes Cas7-11 for target cleavage.

Type III-E CRISPR-Cas effectors, of which Cas7-11 is the first, are single proteins that cleave target RNAs without nonspecific collateral cleavage, opening new possibilities for RNA editing. Biochemical experiments combined with amide hydrogen-deuterium exchange (HDX-MS) experiments provide a first glimpse of the conformational dynamics of apo Cas7-11. HDX-MS revealed the backbone comprised of the four Cas7 zinc-binding RRM folds are well-folded but insertion sequences are highly dynamic and fold upon binding crRNA. The crRNA causes folding of disordered catalytic loops and β-hairpins, stronger interactions at domain-domain interfaces, and folding of the Cas7.1 processing site. Target RNA binding causes only minor ordering around the catalytic loops of Cas7.2 and Cas7.3. We show that Cas7-11 cannot fully process the CRISPR array and that binding of partially processed crRNA induces multiple states in Cas7-11 and reduces target RNA cleavage. The insertion domain shows the most ordering upon binding of mature crRNA. Finally, we show a crRNA-induced conformational change in one of the TPR-CHAT binding sites providing an explanation for why crRNA binding facilitates TPR-CHAT binding. The results provide the first glimpse of the apo state of Cas7-11 and reveal how its structure and function are regulated by crRNA binding.

Supramolecular scaffold-directed two-dimensional assembly of pentacene into a configuration to facilitate singlet fission.

Molecular assemblies featuring two-dimensionality have attracted increasing attention, whereas such structures are difficult to construct simply relying on spontaneous molecular assembly. Here, we present two-dimensional assemblies of acene chromophores achieved using a tripodal triptycene supramolecular scaffold, which have been shown to exhibit a strong ability to assemble molecular and polymer motifs two-dimensionally. We designed pentacene and anthracene derivatives sandwiched by two triptycene units. These compounds assemble into expected two-dimensional structures, with the pentacene chromophores having both sufficient overlap to cause singlet fission and space for conformational change to facilitate the dissociation of a triplet pair into free triplets, which is not the case for the anthracene analog. Detailed spectroscopic analysis revealed that the pentacene chromophore in the assembly undergoes singlet fission with a quantum yield of 88 ± 5%, giving rise to triplet pairs, from which free triplets are efficiently generated (ΦT = 130 ± 8.8%). This demonstrates the utility of the triptycene-based scaffold to design functional π-electronic molecular assemblies.

A Deep Learning Approach to Uncover Voltage-Gated Ion Channels' Intermediate States.

Owing to recent advancements in cryo-electron microscopy, voltage-gated ion channels have gained a greater comprehension of their structural characteristics. However, a significant enigma remains unsolved for a large majority of these channels: their gating mechanism. This mechanism, which encompasses the conformational changes between open and closed states, is pivotal to their proper functioning. Beyond the binary states of open and closed, an ensemble of intermediate states defines the transition path in-between. Due to the lack of experimental data, one might resort to molecular dynamics simulations as an alternative to decipher these states and the transitions between them. However, the high-energy barriers and the colossal time scales involved hinder access to the latter. We present here an application of deep learning as a reliable pipeline for a comprehensive exploration of voltage-gated ion channel conformational rearrangements during gating. We showcase the pipeline performance specifically on the Kv1.2 voltage sensor domain and confront the results with existing data. We demonstrate how our physics-based deep learning approach contributes to the theoretical understanding of these channels and how it might provide further insights into the exploration of channelopathies.

Interactions of human serum albumin with phosphate and Tris buffers: impact on paclitaxel binding and nanoparticles self-assembly

Aim To investigate the conformational changes in human serum albumin (HSA) caused by chemical (CD) and thermal denaturation (TD) at pH 7.4 and 9.9, crucial for designing controlled drug delivery systems with paclitaxel (PTX). Methods Experimental and computational methods, including differential scanning calorimetry (DSC), UV-Vis and intrinsic fluorescence spectroscopy, mean diameter, polydispersity index (PDI), ζ-potential, encapsulation efficiency (EE), in vitro release and protein docking studies were conducted to study the HSA denaturation and nanoparticles (NPs) preparation. Results TD at pH 7.4 produced smaller NPs (287.1 ± 12.9 nm) than CD at pH 7.4 with NPs (584.2 ± 47.7 nm). TD at pH 9.9 exhibited high EE (97.3 ± 0.2%w/w) with rapid PTX release (50% within 1h), whereas at pH 7.4 (96.4 ± 2.1%w/w), release only 40%. ζ-potentials were around −30 mV. Conclusion Buffer type and pH significantly influence NP properties. TD in PBS at pH 7.4, provided optimal conditions for a stable and efficient drug delivery system.

RAFT polymerization assisted P(NIPAm-co-AAc)-AEMR integrated PVA hydrogels: Dual responsive features, texture analysis, and cytotoxicity studies

Modified P(NIPAm-co-AAc) copolymers are prepared using Radical Addition Fragmentation Chain Transfer (RAFT) copolymerization by varying the concentration of castor oil sourced acrylated epoxy methyl ricinoleate (AEMR). Subsequently, hydrogels are prepared by integrating copolymers with polyvinyl alcohol (PVA) via freeze-thaw process. The employment of RAFT polymerization yielded copolymers with dispersity (D) value of 1.2–1.3 revealing the formation of structurally well controlled polymer chains. The lower critical solution temperature (LCST) of the copolymers (∼30–35 °C) are tuned to the range of physiological temperature (∼37 °C) in PVA hydrogel system. Temperature dependent viscoelastic properties indicated that the characteristics of copolymeric solutions and hydrogels are composition dependent while undergoing noncovalent interactions and the conformational changes and the samples showed extremely elastic behaviour beyond LCST. Swelling ratio of hydrogels are also found to be pH dependent, which displayed higher swelling ratio in alkaline and reduced swelling ratio in acidic medium. Cytotoxicity studies with L929 cells showed that the copolymers and hydrogels exhibited desirable biocompatibility, which gets improved with AEMR concentrations. Thus, these dual responsive PA-AEMR-PVA smart hydrogels can be used as a viable functional material for possible bio-medical applications.

The study on characterization of interaction between casticin and model proteins using spectroscopic and computational methodologies

The present studies were conducted to investigate the interaction of casticin to model proteins (BSA, HSA, OVA and HEL) using fluorescence spectra, Fourier transform infrared spectra, molecular docking and molecular dynamics simulation methodologies. Our work displayed that the endogenous fluorescence intensities of BSA, HSA, OVA and HEL were quenched by various concentrations of casticin were the typical static quenching process. The binding constants (Ka) of casticin for these model proteins were in the range of 104–105 mol·L-1, with the highest affinity for HSA. On the basis of thermodynamic parameters and computational simulation studies, it was suggested that the binding force of casticin to BSA, HSA, OVA and HEL were mainly hydrogen bonds, van der Waals forces and hydrophobic interactions. The spectral results illustrated that the binding of casticin to BSA, HSA, OVA and HEL induced conformational and microenvironmental changes. Casticin could decrease the α-helix percentages of BSA, HSA and OVA, but increase that of HEL. Binding substitution studies confirmed that casticin bound to the Sudlow site I of BSA and HSA. Molecular docking predicted the binding models of casticin to BSA, HSA, OVA and HEL, followed closely by molecular dynamic simulations to assess the binding stability.

Insights into Ligand-Mediated Activation of an Oligomeric Ring-Shaped Gene-Regulatory Protein from Solution- and Solid-State NMR

The 91 kDa oligomeric ring-shaped ligand binding protein TRAP (trp RNA binding attenuation protein) regulates the expression of a series of genes involved in tryptophan (Trp) biosynthesis in bacilli. When cellular Trp levels rise, the free amino acid binds to sites buried in the interfaces between each of the 11 (or 12, depending on the species) protomers in the ring. Crystal structures of Trp-bound TRAP show the Trp ligands are sequestered from solvent by a pair of loops from adjacent protomers that bury the bound ligand via polar contacts to several threonine residues. Binding of the Trp ligands occurs cooperatively, such that successive binding events occur with higher apparent affinity but the structural basis for this cooperativity is poorly understood. We used solution methyl-TROSY NMR relaxation experiments focused on threonine and isoleucine sidechains, as well as magic angle spinning solid-state NMR 13C–13C and 15N-13C chemical shift correlation spectra on uniformly labeled samples recorded at 800 and 1200 MHz, to characterize the structure and dynamics of the protein. Methyl 13C relaxation dispersion experiments on ligand-free apo TRAP revealed concerted exchange dynamics on the µs-ms time scale, consistent with transient sampling of conformations that could allow ligand binding. Cross-correlated relaxation experiments revealed widespread disorder on fast timescales. Chemical shifts for methyl-bearing side chains in apo- and Trp-bound TRAP revealed subtle changes in the distribution of sampled sidechain rotameric states. These observations reveal a pathway and mechanism for induced conformational changes to generate homotropic Trp-Trp binding cooperativity.

Phe265 of the D1 protein is required to stabilize plastoquinone binding in the QB-binding site of photosystem II in Synechocystis sp. PCC 6803

In Photosystem II electrons from water splitting pass through a primary quinone electron acceptor (QA) to the secondary plastoquinone (QB). The D2 protein forms the QA-binding site and the D1 protein forms the QB-binding site. A non-heme iron sits between QA and QB resulting in a quinone-Fe-acceptor complex that must be activated before assembly of the oxygen-evolving complex can occur. An extended loop (residues 223–266) between the fourth (helix D) and fifth (helix E) helices of the D1 protein activates forward electron transfer via a conformational change that stabilizes a bidentate bicarbonate ligand to the non-heme iron while simultaneously stabilizing the binding of QB. We show that positioning of D1:Phe265 to provide a hydrogen bond to the distal oxygen of QB is required for forward electron transfer. In addition, mutations targeting D1:Phe265, resulted in a 50 mV decrease in the QB/QB– midpoint potential.

In(III) pyridinecarboxylate complexes: Composition, solution equilibria estimation, bioevaluation and interactions with HSA

Two In(III) – pyridinecarboxylates ([In(Pic)2(NO3)(H2O)] (InPic; HPic = picolinic acid), [In(HDpic)(Dpic)(H2O)2]·5H2O (InDpic; H2Dpic = dipicolinic acid), have been synthesized by one-step procedure. The complexes composition was confirmed by physicochemical analyses and X-ray diffraction confirmed molecular structure of both complexes. Moreover, complex species speciation was described in both systems by potentiometry and 1H NMR spectroscopy and mononuclear complex species were determined; [In(Pic)]2+ (logβ011 = 6.94(4)), [In(Pic)2]+ (logβ021 = 11.98(9)), [In(Dpic)]+ (logβ011 = 10.42(6)), [In(Dpic)2]− (logβ021 = 17.58(7)) and [In(Dpic)2(OH)]2− (logβ−121 = 10.18(6)). To confirm the complexes stability in 1 % DMSO, 1H NMR spectra were measured (immediately after dissolution up to 96 h). Antimicrobial and anticancer assays indicate a more significant sensitivity of S. aureus bacteria and MDA-MB-231 cancer cells to the InPic complex (IC50 = 25 and 340.7 μM) than to the InDpic (IC50 = 50 and 975.4 μM). The interaction and binding mechanism of picolinic/dipicolinic acid and their indium(III) complexes with HSA (human serum albumin) were studied using fluorescence and CD spectroscopy. The results confirmed that the studied compounds had bound successfully to HSA, and the binding parameters and constants (KSV, Kq, Kb) were calculated together with the number of binding sites. The binding forces were identified based on calculated thermodynamic parameters (ΔG, ΔH, ΔS). Synchronous spectra were used to study the microenvironment of Tyr and Trp residues and displacement assays revealed that site I was the preferred binding site. After binding, conformational changes were found to have occurred in the HSA molecule and the % α-helical content had decreased.

Revealing the pH-dependent conformational changes in sol g 2.1 protein and potential ligands binding

Sol g 2, a major protein found in the venom of the tropical fire ant (Solenopsis geminata), is well-known for its ability to bind various hydrophobic molecules. In this study, we investigate the binding activity of recombinant Sol g 2.1 protein (rSol g 2.1) with potential molecules, including (E)-β-Farnesene, α-Caryophyllene, and 1-Octen-3-ol at different pH levels (pH 7.4 and 5.5) using fluorescence competitive binding assays (FCBA). Our results revealed that Sol g 2.1 protein has higher affinity binding with these ligands at neutral pH. Relevance to molecular docking and molecular dynamics simulations were utilized to provide insights into the stability and conformational dynamics of Sol g 2.1 and its ligand complexes. After simulation, we found that Sol g 2.1 protein has higher affinity binding with these ligands as well as high structural stability at pH 7.4 than at an acidic pH level, indicating by RMSD, RMSF, Rg, SASA, and principal component analysis (PCA). Additionally, the Sol g 2.1 protein complexes at pH 7.4 showed significantly lower binding free energy (∆Gbind) and higher total residue contributions, particularly from key non-polar amino acids such as Trp36, Met40, Cys62, and Ile104, compared to the lower pH environment. These explain why they exhibited higher binding affinity than the lower pH. Therefore, we suggested that Sol g 2.1 protein is a pH-responsive carrier protein. These findings also expand our understanding of protein–ligand interactions and offer potential avenues for the development of innovative drug delivery strategies targeting Sol g 2.1 protein.

Alpha-1 antitrypsin inhibits Clostridium botulinum C2 toxin, Corynebacterium diphtheriae diphtheria toxin and B. anthracis fusion toxin

The bacterium Clostridium botulinum, well-known for producing botulinum neurotoxins, which cause the severe paralytic illness known as botulism, produces C2 toxin, a binary AB-toxin with ADP-ribosyltranferase activity. C2 toxin possesses two separate protein components, an enzymatically active A-component C2I and the binding and translocation B-component C2II. After proteolytic activation of C2II to C2IIa, the heptameric structure binds C2I and is taken up via receptor-mediated endocytosis into the target cells. Due to acidification of endosomes, the C2IIa/C2I complex undergoes conformational changes and consequently C2IIa forms a pore into the endosomal membrane and C2I can translocate into the cytoplasm, where it ADP-ribosylates G-actin, a key component of the cytoskeleton. This modification disrupts the actin cytoskeleton, resulting in the collapse of cytoskeleton and ultimately cell death. Here, we show that the serine-protease inhibitor α1-antitrypsin (α1AT) which we identified previously from a hemofiltrate library screen for PT from Bordetella pertussis is a multitoxin inhibitor. α1AT inhibits intoxication of cells with C2 toxin via inhibition of binding to cells and inhibition of enzyme activity of C2I. Moreover, diphtheria toxin and an anthrax fusion toxin are inhibited by α1AT. Since α1AT is commercially available as a drug for treatment of the α1AT deficiency, it could be repurposed for treatment of toxin-mediated diseases.

Exploring the Origins of Association of Poly(acrylic acid) Polyelectrolyte with Lysozyme in Aqueous Environment through Molecular Simulations and Experiments.

This study provides a detailed picture of how a protein (lysozyme) complexes with a poly(acrylic acid) polyelectrolyte (PAA) in water at the atomic level using a combination of all-atom molecular dynamics simulations and experiments. The effect of PAA and temperature on the protein's structure is explored. The simulations reveal that a lysozyme's structure is relatively stable except from local conformational changes induced by the presence of PAA and temperature increase. The effect of a specific thermal treatment on the complexation process is investigated, revealing both structural and energetic changes. Certain types of secondary structures (i.e., α-helix) are found to undergo a partially irreversible shift upon thermal treatment, which aligns qualitatively with experimental observations. This uncovers the origins of thermally induced aggregation of lysozyme with PAA and points to new PAA/lysozyme bonds that are formed and potentially enhance the stability in the complexes. As the temperature changes, distinct amino acids are found to exhibit the closest proximity to PAA, resulting into different PAA/lysozyme interactions; consequently, a different complexation pathway is followed. Energy calculations reveal the dominant role of electrostatic interactions. This detailed information can be useful for designing new biopolymer/protein materials and understanding protein function under immobilization of polyelectrolytes and upon mild denaturation processes.

Film-forming modifications and mechanistic studies of soybean protein isolate by glycerol plasticization and thermal denaturation: A molecular interaction perspective

Plasticizer and thermal denaturation are indeed important factors for soybean protein film formation. The objective of the study was to investigate the effects of glycerol and thermal denaturation on the film-forming performances of soybean protein isolate (SPI) and elucidate the underlying mechanisms. From the results, glycerol had almost no effect on the protein’s secondary and tertiary structures. Indeed, the dispersion of glycerol diminished the intra- or intermolecular hydrogen bonding of SPI and interacted with the amino acids of subunits through hydrogen bonding and van der Waals forces. By interfering with protein network interactions, the glycerol molecule achieved a plasticizing effect on SPI films. The effects of heat treatment on SPI film properties were mainly realized through the changes in molecular conformation caused by protein denaturation, which manifested in the enhancement of light barrier and mechanical capabilities, and markedly altered the distribution of water states within the film network. This study provided valuable insights to clarify the mechanism of protein film formation.

Mechanistic Insights into Substrate Recognition of Human Nucleoside Diphosphate Kinase C Based on Nucleotide-Induced Structural Changes.

Nucleoside diphosphate kinases (NDPKs) are encoded by nme genes and exist in various isoforms. Based on interactions with other proteins, they are involved in signal transduction, development and pathological processes such as tumorigenesis, metastasis and heart failure. In this study, we report a 1.25 Å resolution structure of human homohexameric NDPK-C bound to ADP and describe the yet unknown complexes formed with GDP, UDP and cAMP, all obtained at a high resolution via X-ray crystallography. Each nucleotide represents a distinct group of mono- or diphosphate purine or pyrimidine bases. We analyzed different NDPK-C nucleotide complexes in the presence and absence of Mg2+ and explain how this ion plays an essential role in NDPKs' phosphotransferase activity. By analyzing a nucleotide-depleted NDPK-C structure, we detected conformational changes upon substrate binding and identify flexible regions in the substrate binding site. A comparison of NDPK-C with other human isoforms revealed a strong similarity in the overall composition with regard to the 3D structure, but significant differences in the charge and hydrophobicity of the isoforms' surfaces. This may play a role in isoform-specific NDPK interactions with ligands and/or important complex partners like other NDPK isoforms, as well as monomeric and heterotrimeric G proteins. Considering the recently discovered role of NDPK-C in different pathologies, these high-resolution structures thus might provide a basis for interaction studies with other proteins or small ligands, like activators or inhibitors.

Luminol tautomers and their interaction with zinc cation – A DFT treatment

Luminol is a chemiluminescent material having variety of applications. In the present study, its 1,3-proton tautomers have been considered within the restrictions of density functional theory at the level of B3LYP/6-31++G(d,p) and B3LYP/6-311++G(d,p) levels. Also, interaction of luminol tautomers with zinc dication at the level of B3LYP/6-31++G(d,p) is considered. All the structures considered presently are thermally favored and electronically stable at the standard states. The effect of zinc dication on the tautomers of luminol is not drastic but causes some conformational changes and enhancing the hydrogen bond formation in some cases. Some electron population has been transferred from the organic partner of the composite to the zinc cation, thereby lowering the initial formal charge of the cation. Various structural and quantum chemical data have been collected and discussed, including IR and UV-VIS spectra. Also the NICS (0) data have been obtained for the tautomers.

Calcium Role in Gap Junction Channel Gating: Direct Electrostatic or Calmodulin-Mediated?

The chemical gating of gap junction channels is mediated by cytosolic calcium (Ca2+i) at concentrations ([Ca2+]i) ranging from high nanomolar (nM) to low micromolar (µM) range. Since the proteins of gap junctions, connexins/innexins, lack high-affinity Ca2+-binding sites, most likely gating is mediated by a Ca2+-binding protein, calmodulin (CaM) being the best candidate. Indeed, the role of Ca2+-CaM in gating is well supported by studies that have tested CaM blockers, CaM expression inhibition, testing of CaM mutants, co-localization of CaM and connexins, existence of CaM-binding sites in connexins/innexins, and expression of connexins (Cx) mutants, among others. Based on these data, since 2000, we have published a Ca2+-CaM-cork gating model. Despite convincing evidence for the Ca2+-CaM role in gating, a recent study has proposed an alternative gating model that would involve a direct electrostatic Ca2+-connexin interaction. However, this study, which tested the effect of unphysiologically high [Ca2+]i on the structure of isolated junctions, reported that neither changes in the channel's pore diameter nor connexin conformational changes are present, in spite of exposure of isolated gap junctions to [Ca2+]i as high at the 20 mM. In conclusion, data generated in the past four decades by multiple experimental approaches have clearly demonstrated the direct role of Ca2+-CaM in gap junction channel gating.

Controlled mechanochemical coupling of anti-junctions in DNA origami arrays

Allostery is a hallmark of cellular function and important in every biological system. Still, we are only starting to mimic it in the laboratory. Here, we introduce an approach to study aspects of allostery in artificial systems. We use a DNA origami domino array structure which–upon binding of trigger DNA strands–undergoes a stepwise allosteric conformational change. Using two FRET probes placed at specific positions in the DNA origami, we zoom in into single steps of this reaction cascade. Most of the steps are strongly coupled temporally and occur simultaneously. Introduction of activation energy barriers between different intermediate states alters this coupling and induces a time delay. We then apply these approaches to release a cargo DNA strand at a predefined step in the reaction cascade to demonstrate the applicability of this concept in tunable cascades of mechanochemical coupling with both spatial and temporal control.

Selective deuteration of an RNA:RNA complex for structural analysis using small-angle scattering.

The structures of RNA:RNA complexes regulate many biological processes. Despite their importance, protein-free RNA:RNA complexes represent a tiny fraction of experimentally-determined structures. Here, we describe a joint small-angle X-ray and neutron scattering (SAXS/SANS) approach to structurally interrogate conformational changes in a model RNA:RNA complex. Using SAXS, we measured the solution structures of the individual RNAs in their free state and of the overall RNA:RNA complex. With SANS, we demonstrate, as a proof-of-principle, that isotope labeling and contrast matching (CM) can be combined to probe the bound state structure of an RNA within a selectively deuterated RNA:RNA complex. Furthermore, we show that experimental scattering data can validate and improve predicted AlphaFold 3 RNA:RNA complex structures to reflect its solution structure. Our work demonstrates that in silico modeling, SAXS, and CM-SANS can be used in concert to directly analyze conformational changes within RNAs when in complex, enhancing our understanding of RNA structure in functional assemblies.