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At least 109 records · Page 6
All sourcesNASA NTRSDOE OSTI
NASA NTRS2012

The Origins of Transmembrane Ion Channels

Even though membrane proteins that mediate transport of ions and small molecules across cell walls are among the largest and least understood biopolymers in contemporary cells, it is still possible to shed light on their origins and early evolution. The central observation is that transmembrane portions of most ion channels are simply bundles of -helices. By combining results of experimental and computer simulation studies on synthetic models and natural channels, mostly of non-genomic origin, we show that the emergence of -helical channels was protobiologically plausible, and did not require highly specific amino acid sequences. Despite their simple structure, such channels could possess properties that, at the first sight, appear to require markedly larger complexity. Specifically, we explain how the antiamoebin channels, which are made of identical helices, 16 amino acids in length, achieve efficiency comparable to that of highly evolved channels. We further show that antiamoebin channels are extremely flexible, compared to modern, genetically coded channels. On the basis of our results, we propose that channels evolved further towards high structural complexity because they needed to acquire stable rigid structures and mechanisms for precise regulation rather than improve efficiency. In general, even though architectures of membrane proteins are not nearly as diverse as those of water-soluble proteins, they are sufficiently flexible to adapt readily to the functional demands arising during evolution.

Pohorille, Andrew↗
DOE OSTI2022

Transient local secondary structure in the intrinsically disordered C‐term of the Albino3 insertase

Albino3 (Alb3) is an integral membrane protein fundamental to the targeting and insertion of light‐harvesting complex (LHC) proteins into the thylakoid membrane. Alb3 contains a stroma‐exposed C‐terminus (Alb3‐Cterm) that is responsible for binding the LHC‐loaded transit complex before LHC membrane insertion. Alb3‐Cterm has been reported to be intrinsically disordered, but precise mechanistic details underlying how it recognizes and binds to the transit complex are lacking, and the functional roles of its four different motifs have been debated. Using a novel combination of experimental and computational techniques such as single‐molecule fluorescence resonance energy transfer, circular dichroism with deconvolution analysis, site‐directed mutagenesis, trypsin digestion assays, and all‐atom molecular dynamics simulations in conjunction with enhanced sampling techniques, we show that Alb3‐Cterm contains transient secondary structure in motifs I and II. The excellent agreement between the experimental and computational data provides a quantitatively consistent picture and allows us to identify a heterogeneous structural ensemble that highlights the local and transient nature of the secondary structure. This structural ensemble was used to predict both the inter‐residue distance distributions of single molecules and the apparent unfolding free energy of the transient secondary structure, which were both in excellent agreement with those determined experimentally. We hypothesize that this transient local secondary structure may play an important role in the recognition of Alb3‐Cterm for the LHC‐loaded transit complex, and these results should provide a framework to better understand protein targeting by the Alb3‐Oxa1‐YidC family of insertases.

Okoto, Patience S.↗
DOE OSTI2021

Assembly and Analysis of Cell-Scale Membrane Envelopes

The march toward exascale computing will enable routine molecular simulation of larger and more complex systems, for example, simulation of entire viral particles, on the scale of approximately billions of atoms-a simulation size commensurate with a small bacterial cell. Anticipating the future hardware capabilities that will enable this type of research and paralleling advances in experimental structural biology, efforts are currently underway to develop software tools, procedures, and workflows for constructing cell-scale structures. Herein, we describe our efforts in developing and implementing an efficient and robust workflow for construction of cell-scale membrane envelopes and embedding membrane proteins into them. A new approach for construction of massive membrane structures that are stable during the simulations is built on implementing a subtractive assembly technique coupled with the development of a structure concatenation tool (fastmerge), which eliminates overlapping elements based on volumetric criteria rather than adding successive molecules to the simulation system. Using this approach, we have constructed two "protocells" consisting of MARTINI coarse-grained beads to represent cellular membranes, one the size of a cellular organelle and another the size of a small bacterial cell. The membrane envelopes constructed here remain whole during the molecular dynamics simulations performed and exhibit water flux only through specific proteins, demonstrating the success of our methodology in creating tight cell-like membrane compartments. Extended simulations of these cell-scale structures highlight the propensity for nonspecific interactions between adjacent membrane proteins leading to the formation of protein microclusters on the cell surface, an insight uniquely enabled by the scale of the simulations. We anticipate that the experiences and best practices presented here will form the basis for the next generation of cell-scale models, which will begin to address the addition of soluble proteins, nucleic acids, and small molecules essential to the function of a cell.

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

Lifetime of actin-dependent protein nanoclusters

Protein nanoclusters (PNCs) are dynamic collections of a few proteins that spatially organize in nanometer-length clusters. PNCs are one of the principal forms of spatial organization of membrane proteins, and they have been shown or hypothesized to be important in various cellular processes, including cell signaling. PNCs show remarkable diversity in size, shape, and lifetime. In particular, the lifetime of PNCs can vary over a wide range of timescales. The diversity in size and shape can be explained by the interaction of the clustering proteins with the actin cytoskeleton or the lipid membrane, but very little is known about the processes that determine the lifetime of the nanoclusters. In this paper, using mathematical modeling of the cluster dynamics, we model the biophysical processes that determine the lifetime of actin-dependent PNCs. In particular, we investigated the role of actin aster fragmentation, which had been suggested to be a key determinant of the PNC lifetime, and we found that it is important only for a small class of PNCs. A simple extension of our model allowed us to investigate the kinetics of protein-ligand interaction near PNCs. We found an anomalous increase in the lifetime of ligands near PNCs, which agrees remarkably well with experimental data on RAS-RAF kinetics. In particular, analysis of the RAS-RAF data through our model provides falsifiable predictions and novel hypotheses that will not only shed light on the role of RAS-RAF kinetics in various cancers, but also will be useful in studying membrane protein clustering in general.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Tau and Membranes: Interactions That Promote Folding and Condensation

Tau misfolding and assembly is linked to a number of neurodegenerative diseases collectively described as tauopathies, including Alzheimer’s disease (AD) and Parkinson’s disease. Anionic cellular membranes, such as the cytosolic leaflet of the plasma membrane, are sites that concentrate and neutralize tau, primarily due to electrostatic interactions with tau’s microtubule binding repeat domain (RD). In addition to electrostatic interactions with lipids, tau also has interactions with membrane proteins, which are important for tau’s cellular functions. Tau also interacts with lipid tails to facilitate direct translocation across the membrane and can form stable protein-lipid complexes involved in cell-to-cell transport. Concentrated tau monomers at the membrane surface can form reversible condensates, change secondary structures, and induce oligomers, which may eventually undergo irreversible crosslinking and fibril formation. These β-sheet rich tau structures are capable of disrupting membrane organization and are toxic in cell-based assays. Given the evidence for relevant membrane-based tau assembly, we review the emerging hypothesis that polyanionic membranes may serve as a site for phase-separated tau condensation. Membrane-mediated phase separation may have important implications for regulating tau folding/misfolding, and may be a powerful mechanism to spatially direct tau for native membrane-mediated functions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Revealing functional insights into ER proteostasis through proteomics and interactomics

The endoplasmic reticulum (ER), responsible for processing approximately one-third of the human proteome including most secreted and membrane proteins, plays a pivotal role in protein homeostasis (proteostasis). Dysregulation of ER proteostasis has been implicated in a number of disease states. As such, continued efforts are directed at elucidating mechanisms of ER protein quality control which are mediated by transient and dynamic protein-protein interactions with molecular chaperones, co-chaperones, protein folding and trafficking factors that take place in and around the ER. Technological advances in mass spectrometry have played a pivotal role in characterizing and understanding these protein-protein interactions that dictate protein quality control mechanisms. Here, we highlight the recent progress from mass spectrometry-based investigation of ER protein quality control in revealing the topological arrangement of the proteostasis network, stress response mechanisms that adjust the ER proteostasis capacity, and disease specific changes in proteostasis network engagement. We close by providing a brief outlook on underexplored areas of ER proteostasis where mass spectrometry is a tool uniquely primed to further expand our understanding of the regulation and coordination of protein quality control processes in diverse diseases.

60 APPLIED LIFE SCIENCES↗
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 OSTI2023

Tandem repeats in giant archaeal Borg elements undergo rapid evolution and create new intrinsically disordered regions in proteins

Borgs are huge, linear extrachromosomal elements associated with anaerobic methane-oxidizing archaea. Striking features of Borg genomes are pervasive tandem direct repeat (TR) regions. Here, we present six new Borg genomes and investigate the characteristics of TRs in all ten complete Borg genomes. We find that TR regions are rapidly evolving, recently formed, arise independently, and are virtually absent in host Methanoperedens genomes. Flanking partial repeats and A-enriched character constrain the TR formation mechanism. TRs can be in intergenic regions, where they might serve as regulatory RNAs, or in open reading frames (ORFs). TRs in ORFs are under very strong selective pressure, leading to perfect amino acid TRs (aaTRs) that are commonly intrinsically disordered regions. Proteins with aaTRs are often extracellular or membrane proteins, and functionally similar or homologous proteins often have aaTRs composed of the same amino acids. We propose that Borg aaTR-proteins functionally diversify Methanoperedens and all TRs are crucial for specific Borg–host associations and possibly cospeciation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Ligand-induced transmembrane conformational coupling in monomeric $\mathrm{EGFR}$

Single pass cell surface receptors regulate cellular processes by transmitting ligand-encoded signals across the plasma membrane via changes to their extracellular and intracellular conformations. This transmembrane signaling is generally initiated by ligand binding to the receptors in their monomeric form. While subsequent receptor-receptor interactions are established as key aspects of transmembrane signaling, the contribution of monomeric receptors has been challenging to isolate due to the complexity and ligand-dependence of these interactions. By combining membrane nanodiscs produced with cell-free expression, single-molecule Förster Resonance Energy Transfer measurements, and molecular dynamics simulations, we report that ligand binding induces intracellular conformational changes within monomeric, full-length epidermal growth factor receptor (EGFR). Our observations establish the existence of extracellular/intracellular conformational coupling within a single receptor molecule. We implicate a series of electrostatic interactions in the conformational coupling and find the coupling is inhibited by targeted therapeutics and mutations that also inhibit phosphorylation in cells. Collectively, these results introduce a facile mechanism to link the extracellular and intracellular regions through the single transmembrane helix of monomeric EGFR, and raise the possibility that intramolecular transmembrane conformational changes upon ligand binding are common to single-pass membrane proteins.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Dissecting the structural heterogeneity of proteins by native mass spectrometry

Abstract A single gene yields many forms of proteins via combinations of posttranscriptional/posttranslational modifications. Proteins also fold into higher‐order structures and interact with other molecules. The combined molecular diversity leads to the heterogeneity of proteins that manifests as distinct phenotypes. Structural biology has generated vast amounts of data, effectively enabling accurate structural prediction by computational methods. However, structures are often obtained heterologously under homogeneous states in vitro. The lack of native heterogeneity under cellular context creates challenges in precisely connecting the structural data to phenotypes. Mass spectrometry (MS) based proteomics methods can profile proteome composition of complex biological samples. Most MS methods follow the “bottom‐up” approach, which denatures and digests proteins into short peptide fragments for ease of detection. Coupled with chemical biology approaches, higher‐order structures can be probed via incorporation of covalent labels on native proteins that are maintained at the peptide level. Alternatively, native MS follows the “top‐down” approach and directly analyzes intact proteins under nondenaturing conditions. Various tandem MS activation methods can dissect the intact proteins for in‐depth structural elucidation. Herein, we review recent native MS applications for characterizing heterogeneous samples, including proteins binding to mixtures of ligands, homo/hetero‐complexes with varying stoichiometry, intrinsically disordered proteins with dynamic conformations, glycoprotein complexes with mixed modification states, and active membrane protein complexes in near‐native membrane environments. We summarize the benefits, challenges, and ongoing developments in native MS, with the hope to demonstrate an emerging technology that complements other tools by filling the knowledge gaps in understanding the molecular heterogeneity of proteins.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Cinematic reflectometry using QIKR, the quite intense kinetics reflectometer

The Quite Intense Kinetics Reflectometer (QIKR) will be a general-purpose, horizontal-sample-surface neutron reflectometer. Reflectometers measure the proportion of an incident probe beam reflected from a surface as a function of wavevector (momentum) transfer to infer the distribution and composition of matter near an interface. The unique scattering properties of neutrons make this technique especially useful in the study of soft matter, biomaterials, and materials used in energy storage. Exploiting the increased brilliance of the Spallation Neutron Source Second Target Station, QIKR will collect specular and off-specular reflectivity data faster than the best existing such machines. It will often be possible to collect complete specular reflectivity curves using a single instrument setting, enabling “cinematic” operation, wherein the user turns on the instrument and “films” the sample. Samples in time-dependent environments (e.g., temperature, electrochemical, or undergoing chemical alteration) will be observed in real time, in favorable cases with frame rates as fast as 1 Hz. Cinematic data acquisition promises to make time-dependent measurements routine, with time resolution specified during post-experiment data analysis. This capability will be deployed to observe such processes as in situ polymer diffusion, battery electrode charge–discharge cycles, hysteresis loops, and membrane protein insertion into lipid layers.

47 OTHER INSTRUMENTATION↗
DOE OSTI2021

Effect of docetaxel on mechanical properties of ovarian cancer cells

Docetaxel could inhibit the proliferation of tumor cells by targeting microtubules. The extension of cellular microtubules plays an important role in the invasion and metastasis of tumor cells. This paper aims to study the distribution and mechanical properties of cytoskeletal proteins with low concentration of docetaxel. MTT assay was used to detect the minimum drug activity concentration of docetaxel on SKOV-3 cells, fluorescence staining was used to analyze the distribution of cytoskeleton proteins, scanning electron microscopy(SEM) was used to observe the morphology of single cells, and atomic force microscopy(AFM) was used to determine the microstructure and mechanical properties of cells. The results showed that the IC{sub 10} of docetaxel was 1 ng/ml. Docetaxel can effectively inhibit the formation of cell pseudopodia, hinder the indirectness between cells, reduce the cell extension area, and make the cells malformed. In addition, when AFM analyzes the effects of drugs on cell microstructure and mechanical properties, the average cell surface roughness and cell height are positively correlated with the concentration of docetaxel. Especially when the concentration was 100 ng/ml, the adhesion decreased by 37.04% and Young's modulus increased by 1.57 times compared with the control group. This may be because docetaxel leads to microtubule remodeling and membrane protein aggregation, which affects cell microstructure and increases cell strength, leading to significant changes in the mechanical properties of ovarian cells. This is of great significance to the study of the formation mechanism of tumor cell invasion and migration activities mediated by actin.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

High Mass Analysis with a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer: From Inorganic Salt Clusters to Antibody Conjugates and Beyond

Analysis of proteins and complexes under native mass spectrometric (MS) and solution conditions was typically performed using time-of-flight (ToF) analyzers, due to their routine high m/z transmission and detection capabilities. However, over recent years, the ability of Orbitrap-based mass spectrometers to transmit and detect a range of high molecular weight species is well documented. Herein, we describe how a 15 Tesla Fourier transform ion cyclotron resonance mass spectrometer (15 T FT-ICR MS) is more than capable of analyzing a wide range of ions in the high m/z scale (>5000), in both positive and negative instrument polarities, ranging from the inorganic cesium iodide salt clusters; a humanized IgG1k monoclonal antibody (mAb; 148.2 kDa); an IgG1-mertansine drug conjugate (148.5 kDa, drug-to-antibody ratio; DAR 2.26); an IgG1-siRNA conjugate (159.1 kDa; ribonucleic acid to antibody ratio; RAR 1); the membrane protein aquaporin-Z (97.2 kDa) liberated from a C8E4 detergent micelle; the empty MSP1D1-nanodisc (142.5 kDa) and the tetradecameric chaperone protein complex GroEL (806.2 kDa; GroEL dimer at 1.6 MDa). We also investigate different regions of the FT-ICR MS that impact ion transmission and desolvation. Finally, we demonstrate how the transmission of these species and resultant spectra are highly consistent with those previously generated on both quadrupole-ToF (Q-ToF) and Orbitrap instrumentation. This article serves as an impactful example of how FT-ICR mass analyzers are competitive to Q-ToFs and Orbitraps for high mass detection at high m/z.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Saccharomyces cerevisiae CellWall Remodeling in the Absence of Knr4 and Kre6 Revealed by Nano-FourierTransform Infrared Spectroscopy

The cell wall integrity (CWI) signaling pathway regulates yeast cell wall biosynthesis, cell division, and responses to external stress. The cell wall, comprised of a dense network of chitin, β-1,3- and β-1,6- glucans, and mannoproteins, is very thin, <100 nm. Alterations in cell wall composition may activate the CWI pathway. Saccharomyces cerevisiae, a model yeast, was used to study the role of individual wall components in altering the structure and biophysical properties of the yeast cell wall. Near-field Fourier transform infrared spectroscopy (nano-FT-IR) was used for the first direct, spectrochemical identification of cell wall composition in a background (wild-type) strain and two deletion mutants from the yeast knock-out collection: kre6Δ and knr4Δ. Killer toxin resistant 6 (Kre6) is an integral membrane protein required for biosynthesis of β-1,6-glucan, while Knr4 is a cell signaling protein involved in the control of cell wall biosynthesis, in particular, biosynthesis and deposition of chitin. Complementary spectral data were obtained with far-field (FF)-FT-IR, in transmission, and with attenuated total reflectance (ATR) spectromicroscopy with 3 –10 μm wavelength-dependent spatial resolution. The FF-FT-IR spectra of cells and spectra of isolated cell wall components showed that components of the cell body dominated transmission spectra and were still evident in ATR spectra. In contrast, the nano-FT-IR at ~25 nm spatial resolution could be used to characterize the yeast wall chemical structure. Our results show that the β-1,6-glucan content is decreased in kre6Δ, while all glucan content is decreased in the knr4Δ cell wall. The latter may be thinner than in wild type, since not only are mannan and chitin detectable by nano-FT-IR, but also lipid membranes and protein, indicative of cell interior.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Structural Basis of Clostridium perfringens Enterotoxin Activation and Oligomerization by Trypsin

Clostridium perfringens enterotoxin (CpE) is a β-pore forming toxin that disrupts gastrointestinal homeostasis in mammals by binding membrane protein receptors called claudins. Although structures of CpE fragments bound to claudins have been determined, the mechanisms that trigger CpE activation and oligomerization that lead to the formation of cytotoxic β-pores remain undetermined. Proteolysis of CpE in the gut by trypsin has been shown to play a role in this and subsequent cytotoxicity processes. Here, we report solution structures of full-length and trypsinized CpE using small-angle X-ray scattering (SAXS) and crystal structures of trypsinized CpE and its C-terminal claudin-binding domain (cCpE) using X-ray crystallography. Mass spectrometry and SAXS uncover that removal of the CpE N-terminus by trypsin alters the CpE structure to expose areas that are normally unexposed. Crystal structures of trypsinized CpE and cCpE reveal unique dimer interfaces that could serve as oligomerization sites. Moreover, comparisons of these structures to existing ones predict the functional implications of oligomerization in the contexts of cell receptor binding and β-pore formation. This study sheds light on trypsin’s role in altering CpE structure to activate its function via inducing oligomerization on its path toward cytotoxic β-pore formation. Its findings can incite new approaches to inhibit CpE-based cytotoxicity with oligomer-disrupting therapeutics.

59 BASIC BIOLOGICAL SCIENCES↗