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At least 37 records · Page 2
All sourcesNASA NTRSDOE OSTI
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↗
DOE OSTI2025

Experimental and Computational Insights into the Structural Dynamics of the Fc Fragment of IgG1 Subtype from Biosimilar VEGF‐Trap

The constant fragment (Fc) of the immunoglobulin G1 (IgG1) subtype is increasingly recognized as a crucial scaffold in the development of advanced therapeutics due to its enhanced specificity, efficacy, and extended half‐life. A prime example is VEGF‐Trap (Aflibercept), a recombinant fusion protein that merges the Fc region of the IgG1 subtype with the binding domains of vascular endothelial growth factor receptors (VEGFR)‐1 and VEGFR‐2. The Fc region's role in N‐glycosylation is particularly important, as it significantly influences protein stability. Herein, the first near‐physiological temperature structures of the N‐glycan‐bound Fc fragment of IgG1 subtype from a biosimilar VEGF‐Trap are presented, determined using the SPring‐8 Angstrom Compact free electron LAser (SACLA) and the Turkish Light Source (Turkish DeLight). Comparative analysis with cryogenic structures, including existing data, reveals alternate conformations within the glycan‐binding pocket. Furthermore, molecular dynamics simulations indicate the presence of a high degree of structural plasticity, explaining how the protein adapts its structure through conformational changes. The observed structural fluctuations/conformational changes demonstrate the effect of N‐glycans on protein stability. These findings offer new insights into the molecular basis of Fc‐mediated functions and provide valuable information for the design of next‐generation therapeutics.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

A Highly Ordered Nitroxide Side Chain for Distance Mapping and Monitoring Slow Structural Fluctuations in Proteins

Abstract Site-directed spin labeling electron paramagnetic resonance (SDSL-EPR) is an established tool for exploring protein structure and dynamics. Although nitroxide side chains attached to a single cysteine via a disulfide linkage are commonly employed in SDSL-EPR, their internal flexibility complicates applications to monitor slow internal motions in proteins and to structure determination by distance mapping. Moreover, the labile disulfide linkage prohibits the use of reducing agents often needed for protein stability. To enable the application of SDSL-EPR to the measurement of slow internal dynamics, new spin labels with hindered internal motion are desired. Here, we introduce a highly ordered nitroxide side chain, designated R9, attached at a single cysteine residue via a non-reducible thioether linkage. The reaction to introduce R9 is highly selective for solvent-exposed cysteine residues. Structures of R9 at two helical sites in T4 Lysozyme were determined by X-ray crystallography and the mobility in helical sequences was characterized by EPR spectral lineshape analysis, Saturation Transfer EPR, and Saturation Recovery EPR. In addition, interspin distance measurements between pairs of R9 residues are reported. Collectively, all data indicate that R9 will be useful for monitoring slow internal structural fluctuations, and applications to distance mapping via dipolar spectroscopy and relaxation enhancement methods are anticipated.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Lessons from combined experimental and theoretical examination of the FTIR and 2D-IR spectroelectrochemistry of the amide I region of cytochrome c</em

Amide I difference spectroscopy is widely used to investigate protein function and structure changes. In this article, we show that the common approach of assigning features in amide I difference signals to distinct secondary structure elements in many cases may not be justified. Evidence comes from Fourier transform infrared (FTIR) and 2D-IR spectroelectrochemistry of the protein cytochrome c in the amide I range, in combination with computational spectroscopy based on molecular dynamics (MD) simulations. This combination reveals that each secondary structure unit, such as an alpha-helix or a beta-sheet, exhibits broad overlapping contributions, usually spanning a large part of the amide I region, which in the case of difference absorption experiments (such as in FTIR spectroelectrochemistry) may lead to intensity-compensating and even sign-changing contributions. We use cytochrome c as the test case, as this small electron-transferring redox-active protein contains different kinds of secondary structure units. Upon switching its redox-state, the protein exhibits a different charge distribution while largely retaining its structural scaffold. Our theoretical analysis suggests that the change in charge distribution contributes to the spectral changes and that structural changes are small. However, in order to confidently interpret FTIR amide I difference signals in cytochrome c and proteins in general, MD simulations in combination with additional experimental approaches such as isotope labeling, the insertion of infrared labels to selectively probe local structural elements will be required. In case these data are not available, a critical assessment of previous interpretations of protein amide I 1D- and 2D-IR difference spectroscopy data is warranted.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

All-Atom Simulations Uncover Structural and Dynamical Properties of STING Proteins in the Membrane System

Recent studies have shown that the stimulator of interferon gene (STING) protein plays a central role in the immune system by facilitating the production of type I interferons in cells. The STING signaling pathway is also a prominent activator of cancer-killing T cells that initiate a powerful adaptive immune response. Since biomolecular signaling pathways are complicated and not easily identified through traditional experiments, molecular dynamics (MD) has often been used to study structural and dynamical responses of biological pathways. In this work, we carried out MD simulations for full-length chicken and human STING (chSTING and hSTING) proteins. Specifically, we investigated ligand-bound closed (holo) and ligand-unbound open (apo) forms of STING in the membrane system by comparing their conformational and dynamical differences. Our research provides clues for understanding the mechanism of the STING signaling pathway by uncovering detailed insights for the examined systems: the residues from each chain in the binding pocket are strongly correlated to one another in the open STING structure compared with those in the closed STING structure. Ligand-bound closed STING displays ~174° rotation of the ligand-binding domain (LBD) relative to the open STING structure. The dynamical analysis of residue Cys148 located in the linker region of hSTING does not support the earlier hypothesis that Cys148 can form disulfide bonds between adjacent STING dimers. We also demonstrate that using the full-length proteins is critical, since the MD simulations of the LBD portion alone cannot properly describe the global conformational properties of STING.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Dynamical Signatures of Thermotoga maritima Maltose-Binding Proteins Affected by Ligand Binding

Functional segregation among protein isoforms depends on the interplay of their overall structures and the molecular dynamics of these structures. Thermotoga maritima maltose-binding protein (tmMBP) isoforms show size-dependent differential binding of maltose and malto-oligomers while maintaining remarkable fold conservation. This differential behavior needs detailed characterization in native-like aqueous conditions to understand the effects of protein dynamics on ligand binding and recognition. Small-angle neutron scattering (SANS), neutron spin echo (NSE) spectroscopy, and dynamic light scattering (DLS) were used in conjunction with previously published computational molecular dynamics (MD) simulations to understand the dynamic behavior of tmMBPs experimentally. SANS provided information on the overall structure of the molecules, while NSE was used to determine the dynamics in the nanosecond time scale. Both tmMBP2 and tmMBP3 have a bidomain architecture linked with a flexible hinge, with the binding pocket sitting in the cleft between the two domains. tmMBP2 and tmMBP3 showed different solution dynamics, with the translational and rotational components dominating the dynamics of both systems, resulting in a clear differentiation of their diffusion pattern. A faster dynamics component was also observed and was attributed to segmental dynamics. Differences observed between the ligand-free (apo) and ligand-bound (holo) states of the two proteins are attributed to conformational entropy. Our results highlight the intricacies of how structure and dynamics can together shape binding to a repertoire of substrates in structurally similar proteins.

Diffusion↗
DOE OSTI2021

Reproducibility of protein x-ray diffuse scattering and potential utility for modeling atomic displacement parameters

Protein structure and dynamics can be probed using x-ray crystallography. Whereas the Bragg peaks are only sensitive to the average unit-cell electron density, the signal between the Bragg peaks—diffuse scattering—is sensitive to spatial correlations in electron-density variations. Although diffuse scattering contains valuable information about protein dynamics, the diffuse signal is more difficult to isolate from the background compared to the Bragg signal, and the reproducibility of diffuse signal is not yet well understood. We present a systematic study of the reproducibility of diffuse scattering from isocyanide hydratase in three different protein forms. Both replicate diffuse datasets and datasets obtained from different mutants were similar in pairwise comparisons (Pearson correlation coefficient ≥0.8). The data were processed in a manner inspired by previously published methods using custom software with modular design, enabling us to perform an analysis of various data processing choices to determine how to obtain the highest quality data as assessed using unbiased measures of symmetry and reproducibility. The diffuse data were then used to characterize atomic mobility using a liquid-like motions (LLM) model. This characterization was able to discriminate between distinct anisotropic atomic displacement parameter (ADP) models arising from different anisotropic scaling choices that agreed comparably with the Bragg data. Our results emphasize the importance of data reproducibility as a model-free measure of diffuse data quality, illustrate the ability of LLM analysis of diffuse scattering to select among alternative ADP models, and offer insights into the design of successful diffuse scattering experiments.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Interplays of electron and nuclear motions along CO dissociation trajectory in myoglobin revealed by ultrafast X-rays and quantum dynamics calculations

Significance In this multidisciplinary study, we investigated local and global structural responses upon carbon monoxide (CO) dissociation from the heme iron(II) in myoglobin in solution using ultrafast X-ray absorption and scattering. CO dissociation was optically triggered and pulsed X-rays probed structural and electronic changes as a function of time, revealing an iron(II) spin state transition and nuclear motions propagating from the active site throughout the entire protein. Quantum mechanical computational results were used to assign the iron K-edge absorption features, and to map out electronic and nuclear trajectories during Fe–CO bond elongation and heme macrocycle doming. The study has important implications for heme–protein function and the interaction of small molecule substrates with the metal active centers in enzymes.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Photosynthesis Protein Collective Vibrations Role in Energy Transfer and Dissipation (Final Technical Report)

This project investigates how low‑frequency collective vibrations of photosynthetic proteins control energy transfer and dissipation, using terahertz (THz) spectroscopy and advanced theoretical analysis. The central hypothesis is that specific long‑range protein scaffold motions, rather than just local chromophore dynamics, steer exciton transport and photoprotection by modulating energy gaps and coupling between pigments.

14 SOLAR ENERGY↗
DOE OSTI2025

Acetylcholinesterase: Structure, dynamics, and interactions with organophosphorus compounds

Acetylcholinesterase (AChE) is an enzyme that hydrolyzes the neurotransmitter acetylcholine (ACh), removing it from the synaptic cleft after the transmission of an electrical signal, making it an essential component of chemical neurotransmission. AChE is a serine hydrolase, containing a catalytic triad of Ser/His/Glu. AChE is a prime target for pharmaceuticals treating a variety of neurological disorders. It is also the target of synthetic organophosphorus (OP) compounds that have been used as pesticides and chemical warfare agents. OP compounds contain a potent leaving group, such as fluorine, and act by forming a covalent adduct with the catalytic serine of the AChE active site. A wealth of structural information is available for AChE, including over 300 structures, including a subset of structures in complex with drugs as well as OP compounds. This review will highlight the interactions between OP compounds and AChE from a structural and computational perspective, with a discussion of access to the active site, as well as side reactions that lead to dealkylation of the OP-catalytic serine adduct, a process known as aging. We conclude that while the majority of the conformational changes needed to accommodate the OP compounds are localized to the acyl loop in the crystal structures, molecular dynamics simulations highlight the potential for a far more dynamic enzyme.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Study of Protein Dynamics via Neutron Spin Echo Spectroscopy

Most human body proteins' activity and functionality are related to configurational changes of entire subdomains within the protein crystal structure. The crystal structures build the basis for any calculation that describes the structure or dynamics of a protein, most of the time with strong geometrical restrictions. However, these restrictions from the crystal structure are not present in the solution. The structure of the proteins in the solution may differ from the crystal due to rearrangements of loops or subdomains on the pico to nanosecond time scale (i.e., the internal protein dynamics time regime). This study describes how slow motions on timescales of several tens of nanoseconds can be accessed using neutron scattering. In particular, the dynamical characterization of two major human proteins, an intrinsically disordered protein that lacks a well-defined secondary structure and a classical antibody protein, is addressed by neutron spin echo spectroscopy (NSE) combined with a wide range of laboratory characterization methods. Further insights into protein domain dynamics were achieved using mathematical modeling to describe the experimental neutron data and determine the crossover between combined diffusive and internal protein motions. The extraction of the internal dynamic contribution to the intermediate scattering function obtained from NSE, including the timescale of the various movements, allows further vision into the mechanical properties of single proteins and the softness of proteins in their nearly natural environment in the crowded protein solution.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Comparative Pore Structure and Dynamics for Bacterial Microcompartment Shell Protein Assemblies in Sheets or Shells

Bacterial microcompartments (BMCs) are protein-bound organelles found in some bacteria that encapsulate enzymes for enhanced catalytic activity. These compartments spatially sequester enzymes within semipermeable shell proteins, analogous to many membrane-bound organelles. The shell proteins assemble into multimeric tiles; hexamers, trimers, and pentamers, and these tiles self-assemble into larger assemblies with icosahedral symmetry. While icosahedral shells are the predominant form in vivo , the tiles can also form nanoscale cylinders or sheets. The individual multimeric tiles feature central pores that are key to regulating transport across the protein shell. Our primary interest is to quantify pore shape changes in response to alternative component morphologies at the nanoscale. We used molecular modeling tools to develop atomically detailed models for both planar sheets of tiles and curved structures representative of the complete shells found in vivo . Subsequently, these models were animated using classical molecular dynamics simulations. From the resulting trajectories, we analyzed the overall structural stability, water accessibility to individual residues, water residence time, and pore geometry for the hexameric and trimeric protein tiles from the Haliangium ochraceu m model BMC shell. These exhaustive analyses suggest no substantial variation in pore structure or solvent accessibility between the flat and curved shell geometries. We additionally compare our analysis to hydroxyl radical footprinting data to serve as a check against our simulation results, highlighting specific residues where water molecules are bound for a long time. Although with little variation in morphology or water interaction, we propose that the planar and capsular morphology can be used interchangeably when studying permeability through BMC pores.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Repetitive proteins that undergo large conformational changes evade structural prediction algorithms

Protein structure prediction algorithms, such as AlphaFold, have accelerated protein design and advanced the understanding of the relationship between amino acid sequence and protein structure. However, these algorithms are limited in their ability to predict the structures of conformationally dynamic, intrinsically disordered, and stimuli-responsive proteins. To evaluate sequence-to-structure predictions of such challenging proteins, we explored a class of conformationally dynamic, repeats-in-toxin (RTX) proteins. RTX proteins adopt intrinsically disordered conformations in the absence of calcium and undergo reversible folding into β-roll structures upon binding to calcium. RTX proteins are characterized by tandem repeats of the sequence GGXGXDXUX, in which X can be any amino acid and U is an aliphatic amino acid. We designed RTX sequence variants with global substitutions of nonconserved amino acids, tandem repeats of consensus sequences GGAGXDTLY, and tandem repeats of scrambled sequences GGAGXDTYL. AlphaFold2 and AlphaFold3 predicted that all of these RTX variants adopt β-roll structures, characteristic of wild-type RTX bound to calcium. However, modeling the predicted structures with molecular dynamics simulations and characterizing the protein variants with circular dichroism spectroscopy, small-angle x-ray scattering, and x-ray crystallography revealed that variants adopt diverse, sequence-dependent structures in the absence and presence of calcium. To better design proteins for applications in biotechnology and sustainability, it is critical to build predictive tools that consider intrinsically disordered protein states and validate these tools with multi-mode, multi-scale experimental data.

Chang, Marina P. [Stanford Univ., CA (United State↗
DOE OSTI2022

In vitro investigation of protein assembly by combined microscopy and infrared spectroscopy at the nanometer scale

The nanoscale structure and dynamics of proteins on surfaces has been extensively studied using various imaging techniques, such as transmission electron microscopy and atomic force microscopy (AFM) in liquid environments. These powerful imaging techniques, however, can potentially damage or perturb delicate biological material and do not provide chemical information, which prevents a fundamental understanding of the dynamic processes underlying their evolution under physiological conditions. Here, we use a platform developed in our laboratory that enables acquisition of infrared (IR) spectroscopy and AFM images of biological material in physiological liquids with nanometer resolution in a cell closed by atomically thin graphene membranes transparent to IR photons. In this work, we studied the self-assembly process of S-layer proteins at the graphene-aqueous solution interface. The graphene acts also as the membrane separating the solution containing the proteins and Ca 2+ ions from the AFM tip, thus eliminating sample damage and contamination effects. The formation of S-layer protein lattices and their structural evolution was monitored by AFM and by recording the amide I and II IR absorption bands, which reveal the noncovalent interaction between proteins and their response to the environment, including ionic strength and solvation. Our measurement platform opens unique opportunities to study biological material and soft materials in general.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

All-Atom Modeling and Simulation of Biopolymer Interface: Dual Role of Antifouling Polymer Brushes

Antifouling polymer brushes are well-known for their exceptional resistance to unwanted protein adsorption. While experimental studies have extensively characterized protein–polymer brush interactions, computational investigations remain limited, largely due to the challenges in accurately modeling and integrating these complex, multicomponent systems without resorting to oversimplification. To address this challenge, this study presents one of the most comprehensive and realistic model systems to date, comprising the substrate, grafted polymers, and proteins. Our work interprets the interactions between polymer brush and protein based on realistic modeling without simplification. In particular, this study utilizes molecular modeling and simulation of polycationic and polyzwitterionic brushes─poly(dimethylaminoethyl methacrylate) (PDMAEMA), poly(2-(N-oxide-N,N-dimethylamino)ethyl methacrylate) (PNOMA), and poly(2-(N-3-sulfopropyl-N,N-dimethylammonium)ethyl methacrylate) (PSBMA)─grafted onto α-quartz substrates via polymerization initiator linkers. The brush models were developed to closely replicate experimentally synthesized samples and to provide detailed insights into structural and dynamical changes at the molecular level during protein adsorption. Using steered molecular dynamics simulations, we show that the PSBMA brush, due to its high local density, exhibits the greatest resistance to protein insertion. Cα root-mean-square deviation and interaction pattern analyses further reveal that the PSBMA brush also induces the most significant destabilization of lysozyme, while the PDMAEMA brush enhances protein stability through ion-mediated interactions. The PNOMA brush, while requiring the lowest force for protein adsorption, induces greater protein destabilization than the PDMAEMA brush, primarily due to electrostatic repulsion caused by a short carbon spacer length. Hydration analysis reveals that both the PSBMA brush and the lysozyme interacting with it exhibit the most rapid dehydration, attributed to the brush’s high local chain density, which results in the greatest lysozyme destabilization and the highest adsorption force. These findings highlight the dual role of antifouling polymer brushes: resisting protein adsorption and modulating protein structural dynamics. In conclusion, this study provides valuable insights for the rational design of next-generation antifouling materials and offers a framework for realistic model development in complex multicomponent systems.

Adsorption↗