Results for “molecular biophysics” — Page 11

DOE OSTI2020

Identification of preferred multimodal ligand‐binding regions on IgG1 F C using nuclear magnetic resonance and molecular dynamics simulations

Abstract In this study, the binding of multimodal chromatographic ligands to the IgG1 F C domain were studied using nuclear magnetic resonance and molecular dynamics simulations. Nuclear magnetic resonance experiments carried out with chromatographic ligands and a perdeuterated 15 N‐labeled F C domain indicated that while single‐mode ion exchange ligands interacted very weakly throughout the F C surface, multimodal ligands containing negatively charged and aromatic moieties interacted with specific clusters of residues with relatively high affinity, forming distinct binding regions on the F C . The multimodal ligand‐binding sites on the F C were concentrated in the hinge region and near the interface of the C H 2 and C H 3 domains. Furthermore, the multimodal binding sites were primarily composed of positively charged, polar, and aliphatic residues in these regions, with histidine residues exhibiting some of the strongest binding affinities with the multimodal ligand. Interestingly, comparison of protein surface property data with ligand interaction sites indicated that the patch analysis on F C corroborated molecular‐level binding information obtained from the nuclear magnetic resonance experiments. Finally, molecular dynamics simulation results were shown to be qualitatively consistent with the nuclear magnetic resonance results and to provide further insights into the binding mechanisms. An important contribution to multimodal ligand‐F C binding in these preferred regions was shown to be electrostatic interactions and π–π stacking of surface‐exposed histidines with the ligands. This combined biophysical and simulation approach has provided a deeper molecular‐level understanding of multimodal ligand–F C interactions and sets the stage for future analyses of even more complex biotherapeutics.

Gudhka, Ronak B.↗

DOE OSTI2024

RrA, an enzyme from Rhodospirillum rubrum , is a prototype of a new family of short‐chain L‐asparaginases

Abstract L‐Asparaginases (ASNases) catalyze the hydrolysis of L‐Asn to L‐Asp and ammonia. Members of the ASNase family are used as drugs in the treatment of leukemia, as well as in the food industry. The protomers of bacterial ASNases typically contain 300–400 amino acids (typical class 1 ASNases). In contrast, the chain of ASNase from Rhodospirillum rubrum , reported here and referred to as RrA, consists of only 172 amino acid residues. RrA is homologous to the N‐terminal domain of typical bacterial class 1 ASNases and exhibits millimolar affinity for L‐Asn. In this study, we demonstrate that RrA belongs to a unique family of cytoplasmic, short‐chain ASNases (scASNases). These proteins occupy a distinct region in the sequence space, separate from the regions typically assigned to class 1 ASNases. The scASNases are present in approximately 7% of eubacterial species, spanning diverse bacterial lineages. They seem to be significantly enriched in species that encode for more than one class 1 ASNase. Here, we report biochemical, biophysical, and structural properties of RrA, a member of scASNases family. Crystal structures of the wild‐type RrA, both with and without bound L‐Asp, as well as structures of several RrA mutants, reveal topologically unique tetramers. Moreover, the active site of one protomer is complemented by two residues (Tyr21 and Asn26) from another protomer. Upon closer inspection, these findings clearly outline scASNases as a stand‐alone subfamily of ASNases that can catalyze the hydrolysis of L‐Asn to L‐Asp despite the lack of the C‐terminal domain that is present in all ASNases described structurally to date.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Complex water networks visualized by cryogenic electron microscopy of RNA

The stability and function of biomolecules are directly influenced by their myriad interactions with water. Here we investigated water through cryogenic electron microscopy (cryo-EM) on a highly solvated molecule: the Tetrahymena ribozyme. By using segmentation-guided water and ion modelling (SWIM), an approach combining resolvability and chemical parameters, we automatically modelled and cross-validated water molecules and Mg 2+ ions in the ribozyme core, revealing the extensive involvement of water in mediating RNA non-canonical interactions. Unexpectedly, in regions where SWIM does not model ordered water, we observed highly similar densities in both cryo-EM maps. In many of these regions, the cryo-EM densities superimpose with complex water networks predicted by molecular dynamics, supporting their assignment as water and suggesting a biophysical explanation for their elusiveness to conventional atomic coordinate modelling. Our study demonstrates an approach to unveil both rigid and flexible waters that surround biomolecules through cryo-EM map densities, statistical and chemical metrics, and molecular dynamics simulations.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

National User Resource for Biological Accelerator Mass Spectrometry

The National User Resource for Biological Accelerator Mass Spectrometry (User Resource) will provide isotopic analysis (primarily radiocarbon or 14C) by accelerator mass spectrometry (AMS) for NIH- funded researchers across the United States and will be the only User Resource of its type in the United States. The User Resource will provide measurement capability and expertise to a research community that requires highly sensitive, quantitative isotope analyses. Since commissioning a new accelerator mass spectrometer in June 2014, we have measured over 4000 samples a year for collaborators and service users. The User Resource will enable us to continue to meet these research needs, as well as provide for new users whose research programs would benefit from AMS as a measurement tool. The User Resource’s forte will be ultra-high sensitivity quantitation of radiocarbon and selected other radioisotopes for research studies where isotopes are required. Radioisotope labeling studies have been and will continue to be an important tool for addressing many complex biomedical science problems. AMS is a specialized and unique type of mass spectrometry that provides absolute quantitation of radiocarbon and other relevant radioisotopes with extreme sensitivity, having limits of detection in real samples on the order of a few attomol/mg of sample at measurement precisions of ~3%. It is the only instrumental method capable of quantifying radioisotope-labeled agents routinely in real-world samples with such precision and sensitivity. The sensitivity of AMS allows for the quantification of radiolabeled metabolites in extremely complex matrices of cells and organisms at very low concentrations and in small samples. AMS allows studies to be conducted without perturbing metabolism leading to more relevant quantification of metabolic rates and pathways. In addition, it enables quantification of pharmacokinetic and metabolic properties of toxicants at environmentally relevant concentrations in model systems as well as the ability to quantify pharmacokinetics and other molecular endpoints directly in humans. Such quantitative assessments can 1) improve risk assessment for toxicants, 2) address safety and efficacy considerations for therapeutic entities, 3) deepen understanding of xenobiotic and intermediary metabolism, 4) help understand the interactions between critical molecular pathways, and 5) improve efforts to model and predict various metabolic and biological states. These capabilities have been applied in a number of areas including research in carcinogenesis, toxicology, nutrition, pharmacology/drug development and basic biological science. As a NIGMS National Resource the National User Resource for Biological Accelerator Mass Spectrometry will help NIH funded scientists achieve a deeper understanding of the etiology of human health concerns by (1) enabling the quantification of pharmacokinetics and other molecular endpoints directly in humans; (2) offering the ability to conduct quantitative studies using biologics such as proteins or lipids, and thereby reducing the amount of radioisotope usage in biomedical labs; and (3) enabling more relevant studies of metabolic pathways in health and disease through the use of much lower, more biologically-relevant, concentrations of metabolic substrates in cells and intact organisms. Such studies support NIGMS’s basic biomedical research areas that contribute to the understanding of fundamental cellular and physiological principles and enable research supported by the Biophysics, Biomedical Technology, and Computational Biosciences (BBCB); Genetics and Molecular, Cellular, and Developmental Biology (GMCDB); Pharmacology, Physiology, Biological Chemistry (PPBC) and Training, Workforce Development, and Diversity (TWD) Divisions.

46 INSTRUMENTATION RELATED TO NUCLEAR SCIENCE AND ↗

DOE OSTI2025

National User Resource for Biological Accelerator Mass Spectrometry (Final Report)

The National User Resource for Biological Accelerator Mass Spectrometry (User Resource) will provide isotopic analysis (primarily radiocarbon or 14C) by accelerator mass spectrometry (AMS) for NIH- funded researchers across the United States and will be the only User Resource of its type in the United States. The User Resource will provide measurement capability and expertise to a research community that requires highly sensitive, quantitative isotope analyses. Since commissioning a new accelerator mass spectrometer in June 2014, we have measured over 4000 samples a year for collaborators and service users. The User Resource will enable us to continue to meet these research needs, as well as provide for new users whose research programs would benefit from AMS as a measurement tool. The User Resource’s forte will be ultra-high sensitivity quantitation of radiocarbon and selected other radioisotopes for research studies where isotopes are required. Radioisotope labeling studies have been and will continue to be an important tool for addressing many complex biomedical science problems. AMS is a specialized and unique type of mass spectrometry that provides absolute quantitation of radiocarbon and other relevant radioisotopes with extreme sensitivity, having limits of detection in real samples on the order of a few attomol/mg of sample at measurement precisions of ~3%. It is the only instrumental method capable of quantifying radioisotope-labeled agents routinely in real-world samples with such precision and sensitivity. The sensitivity of AMS allows for the quantification of radiolabeled metabolites in extremely complex matrices of cells and organisms at very low concentrations and in small samples. AMS allows studies to be conducted without perturbing metabolism leading to more relevant quantification of metabolic rates and pathways. In addition, it enables quantification of pharmacokinetic and metabolic properties of toxicants at environmentally relevant concentrations in model systems as well as the ability to quantify pharmacokinetics and other molecular endpoints directly in humans. Such quantitative assessments can 1) improve risk assessment for toxicants, 2) address safety and efficacy considerations for therapeutic entities, 3) deepen understanding of xenobiotic and intermediary metabolism, 4) help understand the interactions between critical molecular pathways, and 5) improve efforts to model and predict various metabolic and biological states. These capabilities have been applied in a number of areas including research in carcinogenesis, toxicology, nutrition, pharmacology/drug development and basic biological science. As a NIGMS National Resource the National User Resource for Biological Accelerator Mass Spectrometry will help NIH funded scientists achieve a deeper understanding of the etiology of human health concerns by (1) enabling the quantification of pharmacokinetics and other molecular endpoints directly in humans; (2) offering the ability to conduct quantitative studies using biologics such as proteins or lipids, and thereby reducing the amount of radioisotope usage in biomedical labs; and (3) enabling more relevant studies of metabolic pathways in health and disease through the use of much lower, more biologically-relevant, concentrations of metabolic substrates in cells and intact organisms. Such studies support NIGMS’s basic biomedical research areas that contribute to the understanding of fundamental cellular and physiological principles and enable research supported by the Biophysics, Biomedical Technology, and Computational Biosciences (BBCB); Genetics and Molecular, Cellular, and Developmental Biology (GMCDB); Pharmacology, Physiology, Biological Chemistry (PPBC) and Training, Workforce Development, and Diversity (TWD) Divisions. Over the next five years, our goals are to: 1. Improve the efficiency of operation for AMS measurements through installation of new interfaces to our AMS systems, technical modifications to improve gas accepting ion source efficiency and upgrading our data analysis software for improved ease of use and data reporting. 2. Increase the accessibility and visibility of ultra-sensitive 14C measurements for the biomedical research community by training of new investigators and expanding our national user base. 3. Provide high throughput, ultra-sensitive 14C analysis for the NIGMS and NIH user community.

47 OTHER INSTRUMENTATION↗

DOE OSTI2022

Modelling Hyperglycaemia in an Epithelial Membrane Model: Biophysical Characterisation

Biomimetic models are valuable platforms to improve our knowledge on the molecular mechanisms governing membrane-driven processes in (patho)physiological conditions, including membrane permeability, transport, and fusion. However, current membrane models are over simplistic and do not include the membrane’s lipid remodelling in response to extracellular stimuli. Our study describes the synthesis of glycated dimyristoyl-phosphatidylethanolamine (DMPE-glyc), which was structurally characterised by mass spectrometry (ESI-MS) and quantified by NMR spectroscopy to be further incorporated in a complex phospholipid (PL) membrane model enriched in cholesterol (Chol) and (glyco)sphingolipids (GSL) designed to mimic epithelial membranes (PL/Chol/GSL) under hyperglycaemia conditions. Characterisation of synthesised DMPE-glyc adducts by tandem mass spectrometry (ESI-MS/MS) show that synthetic DMPE-glyc adducts correspond to Amadori products and quantification by 1H NMR spectroscopy show that the yield of glycation reaction was 8%. The biophysical characterisation of the epithelial membrane model shows that excess glucose alters the thermotropic behaviour and fluidity of epithelial membrane models likely to impact permeability of solutes. The epithelial membrane models developed to mimic normo- and hyperglycaemic scenarios are the basis to investigate (poly)phenol-lipid and drug–membrane interactions crucial in nutrition, pharmaceutics, structural biochemistry, and medicinal chemistry.

Reis, Ana (ORCID:0000000245379902)↗

DOE OSTI2026

Classifying biophysical subpopulations of insulin secretory granules using quantitative whole-cell structure analysis

Pancreatic beta cells contain insulin secretory granules (ISGs), organelles where proinsulin is converted into insulin. As ISGs mature, they undergo extensive biophysical remodeling, producing a spectrum of subpopulations with heterogeneous molecular and spatial characteristics. However, systematic methods to define ISG subpopulations remain underdeveloped. To address this gap in knowledge, we employed soft X-ray tomography (SXT), which can quantitatively measure the biochemical density of ISGs within whole beta cells. Using unsupervised clustering, we classified subpopulations based on molecular density, size, and spatial positioning. Across different insulin secretory stimuli, we observed shifts toward mature and releasable subtypes, demonstrating that exogenous signals can dynamically remodel ISG subpopulation distributions. We extended this methodology to primary beta cells characterized using volume electron microscopy (vEM). Integrating subpopulations from SXT and vEM uncovered insights inaccessible by a single method in isolation. This strategy establishes a framework for defining therapeutic approaches aimed at enriching physiologically beneficial ISG subpopulations.

dense-core granules↗

DOE OSTI2023

Plants use molecular mechanisms mediated by biomolecular condensates to integrate environmental cues with development

This review highlights recent literature on biomolecular condensates in plant development and discusses challenges for fully dissecting their functional roles. Plant developmental biology has been inundated with descriptive examples of biomolecular condensate formation, but it is only recently that mechanistic understanding has been forthcoming. Here, we discuss recent examples of potential roles biomolecular condensates play at different stages of the plant life cycle. We group these examples based on putative molecular functions, including sequestering interacting components, enhancing dwell time, and interacting with cytoplasmic biophysical properties in response to environmental change. We explore how these mechanisms could modulate plant development in response to environmental inputs and discuss challenges and opportunities for further research into deciphering molecular mechanisms to better understand the diverse roles that biomolecular condensates exert on life.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Crystal structures of OrfX1, OrfX2 and the OrfX1–OrfX3 complex from the orfX gene cluster of botulinum neurotoxin E1

Botulinum neurotoxins (BoNTs) are among the most lethal toxins known to humans, comprising seven established serotypes termed BoNT/A–G encoded in two types of gene clusters (ha and orfX) in BoNT-producing clostridia. The ha cluster encodes four non-toxic neurotoxin-associated proteins (NAPs) that assemble with BoNTs to protect and enhance their oral toxicity. However, the structure and function of the orfX-type NAPs remain largely unknown. Here, we report the crystal structures for OrfX1, OrfX2, and an OrfX1–OrfX3 complex, which are encoded in the orfX cluster of a BoNT/E1-producing Clostridium botulinum strain associated with human foodborne botulism. These structures lay the foundation for future studies on the potential roles of OrfX proteins in oral intoxication and pathogenesis of BoNTs.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2023

Data-driven prediction of α IIb β 3 integrin activation paths using manifold learning and deep generative modeling

The integrin heterodimer is a transmembrane protein critical for driving cellular process and is a therapeutic target in the treatment of multiple diseases linked to its malfunction. Activation of integrin involves conformational transitions between bent and extended states. Some of the conformations that are intermediate between bent and extended states of the heterodimer have been experimentally characterized, but the full activation pathways remain unresolved both experimentally due to their transient nature and computationally due to the challenges in simulating rare barrier crossing events in these large molecular systems. An understanding of the activation pathways can provide new fundamental understanding of the biophysical processes associated with the dynamic interconversions between bent and extended states and unveil new putative therapeutic targets. In this work, we apply nonlinear manifold learning to coarse-grained molecular dynamics simulations of bent, extended, and two intermediate states of αI I b β3 integrin to learn a low-dimensional embedding of the configurational phase space. We then train deep generative models to learn an inverse mapping between the low-dimensional embedding and high-dimensional molecular space and use these models to interpolate the molecular configurations constituting the activation pathways between the experimentally characterized states. Furthermore, this work furnishes plausible predictions of integrin activation pathways and reports a generic and transferable multi-scale technique to predict transition pathways for biomolecular systems.

97 MATHEMATICS AND COMPUTING↗

DOE OSTI2025

Integrated multi-omic characterizations of the synapse reveal RNA processing factors and ubiquitin ligases associated with neurodevelopmental disorders

The molecular composition of the excitatory synapse is incompletely defined due to its dynamic nature across developmental stages and neuronal populations. To address this gap, we apply proteomic mass spectrometry to characterize the synapse in multiple biological models including the fetal human brain and hiPSC-derived neurons. To prioritize the identified proteins, we develop an orthogonal multi-omic screen of genomic, transcriptomic, interactomic, and structural data. This data-driven framework identifies proteins with key molecular features intrinsic to the synapse, including characteristic patterns of biophysical interactions and cross-tissue expression. The multi-omic analysis captures synaptic proteins across developmental stages and experimental systems, including 493 synaptic candidates supported by proteomics. We further investigate three such proteins that are associated with neurodevelopmental disorders – the CUL3 E3 ubiquitin ligase, the DDX3X and YBX1 nucleic-acid binding proteins – by mapping their networks of physically interacting synapse proteins or transcripts. Our study demonstrates the potential of an integrated multi-omic approach to systematically and more comprehensively resolve the synaptic architecture.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2026

Ultrafast Carrier Self-Trapping Driven by Strong Exciton–Phonon Coupling in 2D MA 3 Bi 2 I 6 Cl 3 Perovskite

Two-dimensional (2D) hybrid bismuth halide perovskites have emerged as promising lead-free materials for optoelectronic applications due to their solution processability and tunable structures. Here, in this study, we investigate 2D layered hybrid perovskite MA 3 Bi 2 I 6 Cl 3 using temperature-dependent photoluminescence (PL) and femtosecond transient absorption spectroscopy. Our results reveal strong coupling between excitons and phonons, evidenced by giant Huang–Rhys factors, coherent phonon oscillations, and ultrafast carrier self-trapping into small-polaron and self-trapped exciton (STE) states. These processes appear as time-dependent ground-state bleach and photoinduced absorption features, highlighting the influence of the lattice in carrier dynamics. Wavelength- and time-resolved measurements reveal that PL emission is dominated by STEs, while free exciton emission is weak and short-lived. By comparing 2D MA 3 Bi 2 I 6 Cl 3 with 0D MA 3 Bi 2 I 9 , which contains molecularly isolated [BiI 6 ] 3− octahedra and 2D MA 3 Bi 2 Br 9 perovskites, we demonstrate how halide composition and structural dimensionality influence the balance between free exciton populations and carrier localization. These insights uncover the intrinsic kinetic landscape of photoexcited states in MA 3 Bi 2 I 6 Cl 3 . Overall, our study contributes to a mechanistic understanding of exciton–phonon interactions in lead-free 2D perovskites.

Pradeep, Kodimana Ramakrishnan [Northwestern Univ.↗

DOE OSTI2020

Mathematical Model of ATM Activation and Chromatin Relaxation by Ionizing Radiation

We propose a comprehensive mathematical model to study the dynamics of ionizing radiation induced Ataxia-telangiectasia mutated (ATM) activation that consists of ATM activation through dual mechanisms: the initiative activation pathway triggered by the DNA damage-induced local chromatin relaxation and the primary activation pathway consisting of a self-activation loop by interplay with chromatin relaxation. The model is expressed as a series of biochemical reactions, governed by a system of differential equations and analyzed by dynamical systems techniques. Radiation induced double strand breaks (DSBs) cause rapid local chromatin relaxation, which is independent of ATM but initiates ATM activation at damage sites. Key to the model description is how chromatin relaxation follows when active ATM phosphorylates KAP-1, which subsequently spreads throughout the chromatin and induces global chromatin relaxation. Additionally, the model describes how oxidative stress activation of ATM triggers a self-activation loop in which PP2A and ATF2 are released so that ATM can undergo autophosphorylation and acetylation for full activation in relaxed chromatin. In contrast, oxidative stress alone can partially activate ATM because phosphorylated ATM remains as a dimer. The model leads to predictions on ATM mediated responses to DSBs, oxidative stress, or both that can be tested by experiments.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2023

Monatomic ions influence substrate permeation across bacterial microcompartment shells

Abstract Bacterial microcompartments (BMCs) are protein organelles consisting of an inner enzymatic core encased within a selectively permeable shell. BMC shells are modular, tractable architectures that can be repurposed with new interior enzymes for biomanufacturing purposes. The permeability of BMC shells is function-specific and regulated by biophysical properties of the shell subunits, especially its pores. We hypothesized that ions may interact with pore residues in a manner that influences the substrate permeation process. In vitro activity comparisons between native and broken BMCs demonstrated that increasing NaCl negatively affects permeation rates. Molecular dynamics simulations of the dominant shell protein (BMC-H) revealed that chloride ions preferentially occupy the positive pore, hindering substrate permeation, while sodium cations remain excluded. Overall, these results demonstrate that shell properties influence ion permeability and leverages the integration of experimental and computational techniques to improve our understanding of BMC shells towards their repurposing for biotechnological applications.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Structures of a synthetic antibody selected against and bound to the C‐terminal domain of Clostridium perfringens enterotoxin

Abstract Clostridium perfringensenterotoxin (CpE) causes cytotoxic gastrointestinal disease in mammalian epithelium by binding membrane protein receptors called claudins. Claudins direct the formation of cell/cell tight junctions through oligomerization and govern the transport of molecules between individual cells. CpE binds claudins through its C‐terminal domain (cCpE) and induces cytotoxicity through its N‐terminal domain. The non‐toxic cCpE is a useful tool to study claudins, tight junctions, and for translational applications, such as increasing the permeability of restrictive tissues like the blood–brain barrier or selective targeting of claudin overexpressing cancers. Conversely, there are no specialized molecular tools to study CpE or cCpE, or to modulate or inhibit their functions. We previously reported the development of synthetic antigen‐binding fragments (sFabs) that bind cCpE, and low‐resolution structures of them bound to claudin/cCpE complexes. Here, we determine high‐resolution structures of sFab COP‐2 bound to cCpE using X‐ray crystallography and cryogenic electron microscopy. The structures and biophysical findings provide the mechanism of COP‐2 binding to cCpE and the molecular determinants driving their interactions. These insights can advance the design of new antibody‐based tools from our COP‐2 scaffold to study or alter cCpE function and give rise to a “Trojan horse” strategy that exploits cCpE's tight junction barrier disrupting function to selectively deliver conjugated therapeutics through normally impermeable tissues.

Biochemistry & Molecular Biology↗

DOE OSTI2022

The allosteric mechanism leading to an open-groove lipid conductive state of the TMEM16F scramblase

TMEM16F is a Ca 2+ -activated phospholipid scramblase in the TMEM16 family of membrane proteins. Unlike other TMEM16s exhibiting a membrane-exposed hydrophilic groove that serves as a translocation pathway for lipids, the experimentally determined structures of TMEM16F shows the groove in a closed conformation even under conditions of maximal scramblase activity. It is currently unknown if/how TMEM16F groove can open for lipid scrambling. Here we describe the analysis of ~400 µs all-atom molecular dynamics (MD) simulations of the TMEM16F revealing an allosteric mechanism leading to an open-groove, lipid scrambling competent state of the protein. The groove opens into a continuous hydrophilic conduit that is highly similar in structure to that seen in other activated scramblases. The allosteric pathway connects this opening to an observed destabilization of the Ca 2+ ion bound at the distal site near the dimer interface, to the dynamics of specific protein regions that produces the open-groove state to scramble phospholipids.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Multivalent interactions between CsoS2 and Rubisco mediate α-carboxysome formation

Carboxysomes are bacterial microcompartments that function as the centerpiece of the bacterial CO 2 -concentrating mechanism by facilitating high CO 2 concentrations near the carboxylase Rubisco. The carboxysome self-assembles from thousands of individual proteins into icosahedral-like particles with a dense enzyme cargo encapsulated within a proteinaceous shell. In the case of the α-carboxysome, there is little molecular insight into protein–protein interactions that drive the assembly process. In this work, studies on the α-carboxysome from Halothiobacillus neapolitanus demonstrate that Rubisco interacts with the N terminus of CsoS2, a multivalent, intrinsically disordered protein. X-ray structural analysis of the CsoS2 interaction motif bound to Rubisco reveals a series of conserved electrostatic interactions that are only made with properly assembled hexadecameric Rubisco. Although biophysical measurements indicate that this single interaction is weak, its implicit multivalency induces high-affinity binding through avidity. Taken together, our results indicate that CsoS2 acts as an interaction hub to condense Rubisco and enable efficient α-carboxysome formation.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Unveiling the nanoscale architectures and dynamics of protein assembly with in situ atomic force microscopy

Proteins play a vital role in different biological processes by forming complexes through precise folding with exclusive inter- and intra-molecular interactions. Understanding the structural and regulatory mechanisms underlying protein complex formation provides insights into biophysical processes. Furthermore, the principle of protein assembly gives guidelines for new biomimetic materials with potential applications in medicine, energy, and nanotechnology. Atomic force microscopy (AFM) is a powerful tool for investigating protein assembly and interactions across spatial scales (single molecules to cells) and temporal scales (milliseconds to days). It has significantly contributed to understanding nanoscale architectures, inter- and intra-molecular interactions, and regulatory elements that determine protein structures, assemblies, and functions. This review describes recent advancements in elucidating protein assemblies with in situ AFM. We discuss the structures, diffusions, interactions, and assembly dynamics of proteins captured by conventional and high-speed AFM in near-native environments and recent AFM developments in the multimodal high-resolution imaging, bimodal imaging, live cell imaging, and machine-learning-enhanced data analysis. These approaches show the significance of broadening the horizons of AFM and enable unprecedented explorations of protein assembly for biomaterial design and biomedical research.

36 MATERIALS SCIENCE↗