17 results on '"Myungwoon Lee"'
Search Results
2. Exploring the complexity of amyloid-beta fibrils: structural polymorphisms and molecular interactions.
- Author
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Yoongyeong Baek and Myungwoon Lee
- Subjects
- *
X-ray crystallography technique , *MOLECULAR structure , *IONIC interactions , *MOLECULAR interactions , *HYDROPHOBIC interactions , *AMYLOID beta-protein - Abstract
The aggregation of amyloid-beta (Aβ) peptides into cross-β structures forms a variety of distinct fibril conformations, potentially correlating with variations in neurodegenerative disease progression. Recent advances in techniques such as X-ray crystallography, solid-state NMR, and cryo-electron microscopy have enabled the development of high-resolution molecular structures of these polymorphic amyloid fibrils, which are either grown in vitro or isolated from human and transgenic mouse brain tissues. This article reviews our current understanding of the structural polymorphisms in amyloid fibrils formed by Aβ40 and Aβ42, as well as disease-associated mutants of Aβ peptides. The aim is to enhance our understanding of various molecular interactions, including hydrophobic and ionic interactions, within and among cross-β structures. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
3. Quantitative Correlation between Bound Water and Mechanical Stress Relaxation in Dehydrated Metal-Coordinate Polymer Networks
- Author
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Sungjin Kim, Myungwoon Lee, Mei Hong, and Niels Holten-Andersen
- Subjects
General Chemical Engineering ,Materials Chemistry ,General Chemistry - Published
- 2022
4. Structures of brain-derived 42-residue amyloid-β fibril polymorphs with unusual molecular conformations and intermolecular interactions
- Author
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Myungwoon Lee, Wai-Ming Yau, John M. Louis, and Robert Tycko
- Subjects
Multidisciplinary - Abstract
Fibrils formed by the 42-residue amyloid-β peptide (Aβ42), a main component of amyloid deposits in Alzheimer's disease (AD), are known to be polymorphic, i.e., to contain multiple possible molecular structures. Previous studies of Aβ42 fibrils, including fibrils prepared entirely in vitro or extracted from brain tissue and using solid-state NMR (ssNMR) or cryogenic electron microscopy (cryo-EM) methods, have found polymorphs with differences in amino acid sidechain orientations, lengths of structurally ordered segments, and contacts between cross-β subunit pairs within a single filament. Despite these differences, Aβ42 molecules adopt a common S-shaped conformation in all previously described high-resolution Aβ42 fibril structures. Here we report two cryo-EM-based structures of Aβ42 fibrils that are qualitatively different, in samples derived from AD brain tissue by seeded growth. In type A fibrils, residues 12 to 42 adopt a ν-shaped conformation, with both intra-subunit and intersubunit hydrophobic contacts to form a compact core. In type B fibrils, residues 2 to 42 adopt an υ-shaped conformation, with only intersubunit contacts and internal pores. Type A and type B fibrils have opposite helical handedness. Cryo-EM density maps and molecular dynamics simulations indicate intersubunit K16-A42 salt bridges in type B fibrils and partially occupied K28-A42 salt bridges in type A fibrils. The coexistence of two predominant polymorphs, with differences in N-terminal dynamics, is supported by ssNMR data, as is faithful propagation of structures from first-generation to second-generation brain-seeded Aβ42 fibril samples. These results demonstrate that Aβ42 fibrils can exhibit a greater range of structural variations than seen in previous studies.
- Published
- 2023
5. Early stages and final results of amyloid-beta self-assembly
- Author
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Robert Tycko, Jaekyun Jeon, Myungwoon Lee, Kent Thurber, and Wai-Ming Yau
- Subjects
Biophysics - Published
- 2023
6. Double-stranded RNA drives SARS-CoV-2 nucleocapsid protein to undergo phase separation at specific temperatures
- Author
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Christine A Roden, Yifan Dai, Catherine A Giannetti, Ian Seim, Myungwoon Lee, Rachel Sealfon, Grace A McLaughlin, Mark A Boerneke, Christiane Iserman, Samuel A Wey, Joanne L Ekena, Olga G Troyanskaya, Kevin M Weeks, Lingchong You, Ashutosh Chilkoti, and Amy S Gladfelter
- Subjects
Binding Sites ,biology ,Coronavirus disease 2019 (COVID-19) ,SARS-CoV-2 ,Chemistry ,viruses ,Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ,fungi ,Temperature ,RNA-Binding Proteins ,RNA ,Translation (biology) ,Phosphoproteins ,biology.organism_classification ,Article ,In vitro ,Cell biology ,RNA silencing ,Genetics ,Coronavirus Nucleocapsid Proteins ,RNA, Viral ,Nucleic acid structure ,Betacoronavirus ,RNA, Double-Stranded - Abstract
Nucleocapsid protein (N-protein) is required for multiple steps in betacoronaviruses replication. SARS-CoV-2-N-protein condenses with specific viral RNAs at particular temperatures making it a powerful model for deciphering RNA sequence specificity in condensates. We identify two separate and distinct double-stranded, RNA motifs (dsRNA stickers) that promote N-protein condensation. These dsRNA stickers are separately recognized by N-protein's two RNA binding domains (RBDs). RBD1 prefers structured RNA with sequences like the transcription-regulatory sequence (TRS). RBD2 prefers long stretches of dsRNA, independent of sequence. Thus, the two N-protein RBDs interact with distinct dsRNA stickers, and these interactions impart specific droplet physical properties that could support varied viral functions. Specifically, we find that addition of dsRNA lowers the condensation temperature dependent on RBD2 interactions and tunes translational repression. In contrast RBD1 sites are sequences critical for sub-genomic (sg) RNA generation and promote gRNA compression. The density of RBD1 binding motifs in proximity to TRS-L/B sequences is associated with levels of sub-genomic RNA generation. The switch to packaging is likely mediated by RBD1 interactions which generate particles that recapitulate the packaging unit of the virion. Thus, SARS-CoV-2 can achieve biochemical complexity, performing multiple functions in the same cytoplasm, with minimal protein components based on utilizing multiple distinct RNA motifs that control N-protein interactions.
- Published
- 2021
7. Fully hydrophobic HIV gp41 adopts a hemifusion-like conformation in phospholipid bilayers
- Author
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Myungwoon Lee, Mei Hong, and Chloe A. Morgan
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Gene Expression Regulation, Viral ,Models, Molecular ,Protein Conformation, alpha-Helical ,0301 basic medicine ,Protein Conformation ,Lipid Bilayers ,Phospholipid ,HIV Infections ,Gp41 ,Biochemistry ,Cell membrane ,03 medical and health sciences ,chemistry.chemical_compound ,Protein Domains ,medicine ,Humans ,Lipid bilayer ,Molecular Biology ,Phospholipids ,030102 biochemistry & molecular biology ,Chemistry ,Cell Membrane ,Water ,Lipid bilayer fusion ,Cell Biology ,Virus Internalization ,HIV Envelope Protein gp41 ,Transmembrane domain ,030104 developmental biology ,medicine.anatomical_structure ,Ectodomain ,Structural biology ,Protein Structure and Folding ,HIV-1 ,Biophysics ,Protein Conformation, beta-Strand ,Hydrophobic and Hydrophilic Interactions - Abstract
The HIV envelope glycoprotein mediates virus entry into target cells by fusing the virus lipid envelope with the cell membrane. This process requires large-scale conformational changes of the fusion protein gp41. Current understanding of the mechanisms with which gp41 induces membrane merger is limited by the fact that the hydrophobic N-terminal fusion peptide (FP) and C-terminal transmembrane domain (TMD) of the protein are challenging to characterize structurally in the lipid bilayer. Here we have expressed a gp41 construct that contains both termini, including the FP, the fusion peptide–proximal region (FPPR), the membrane-proximal external region (MPER), and the TMD. These hydrophobic domains are linked together by a shortened water-soluble ectodomain. We reconstituted this “short NC” gp41 into a virus-mimetic lipid membrane and conducted solid-state NMR experiments to probe the membrane-bound conformation and topology of the protein. (13)C chemical shifts indicate that the C-terminal MPER-TMD is predominantly α-helical, whereas the N-terminal FP-FPPR exhibits β-sheet character. Water and lipid (1)H polarization transfer to the protein revealed that the TMD is well-inserted into the lipid bilayer, whereas the FPPR and MPER are exposed to the membrane surface. Importantly, correlation signals between the FP-FPPR and the MPER are observed, providing evidence that the ectodomain is sufficiently collapsed to bring the N- and C-terminal hydrophobic domains into close proximity. These results support a hemifusion-like model of the short NC gp41 in which the ectodomain forms a partially folded hairpin that places the FPPR and MPER on the opposing surfaces of two lipid membranes.
- Published
- 2019
8. Solid-State Nuclear Magnetic Resonance Investigation of the Structural Topology and Lipid Interactions of a Viral Fusion Protein Chimera Containing the Fusion Peptide and Transmembrane Domain
- Author
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Hongwei Yao, Mei Hong, Shu-Yu Liao, and Myungwoon Lee
- Subjects
0301 basic medicine ,Fusion ,Chemistry ,Membrane Proteins ,Lipid bilayer fusion ,010402 general chemistry ,Lipids ,01 natural sciences ,Biochemistry ,Fusion protein ,0104 chemical sciences ,stomatognathic diseases ,03 medical and health sciences ,Transmembrane domain ,030104 developmental biology ,Membrane ,Ectodomain ,Solid-state nuclear magnetic resonance ,Membrane curvature ,Biophysics ,Nuclear Magnetic Resonance, Biomolecular ,Viral Fusion Proteins ,human activities - Abstract
The fusion peptide (FP) and transmembrane domain (TMD) of viral fusion proteins play important roles during virus-cell membrane fusion, by inducing membrane curvature and transient dehydration. The structure of the water-soluble ectodomain of viral fusion proteins has been extensively studied crystallographically, but the structures of the FP and TMD bound to phospholipid membranes are not well understood. We recently investigated the conformations and lipid interactions of the separate FP and TMD peptides of parainfluenza virus 5 (PIV5) fusion protein F using solid-state nuclear magnetic resonance. These studies provide structural information about the two domains when they are spatially well separated in the fusion process. To investigate how these two domains are structured relative to each other in the postfusion state, when the ectodomain forms a six-helix bundle that is thought to force the FP and TMD together in the membrane, we have now expressed and purified a chimera of the FP and TMD, connected by a Gly-Lys linker, and measured the chemical shifts and interdomain contacts of the protein in several lipid membranes. The FP-TMD chimera exhibits α-helical chemical shifts in all the membranes examined and does not cause strong curvature of lamellar membranes or membranes with negative spontaneous curvature. These properties differ qualitatively from those of the separate peptides, indicating that the FP and TMD interact with each other in the lipid membrane. However, no
- Published
- 2016
9. Solid-State NMR Investigation of the Conformation, Proton Conduction, and Hydration of the Influenza B Virus M2 Transmembrane Proton Channel
- Author
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Jonathan K. Williams, Jun Wang, Daniel Tietze, Mei Hong, and Myungwoon Lee
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Models, Molecular ,0301 basic medicine ,Protein Conformation ,010402 general chemistry ,medicine.disease_cause ,01 natural sciences ,Biochemistry ,Article ,Catalysis ,Virus ,Viral Matrix Proteins ,03 medical and health sciences ,Colloid and Surface Chemistry ,Influenza A virus ,medicine ,Histidine ,Lipid bilayer ,Nuclear Magnetic Resonance, Biomolecular ,Chemistry ,Chemical shift ,Cell Membrane ,Water ,General Chemistry ,Nuclear magnetic resonance spectroscopy ,Hydrogen-Ion Concentration ,Transmembrane protein ,0104 chemical sciences ,Cold Temperature ,Influenza B virus ,Crystallography ,030104 developmental biology ,Membrane ,Solid-state nuclear magnetic resonance ,Protons - Abstract
Together with the influenza A virus, influenza B virus causes seasonal flu epidemics. The M2 protein of influenza B (BM2) forms a tetrameric proton-conducting channel that is important for the virus lifecycle. BM2 shares little sequence homology with AM2, except for a conserved HxxxW motif in the transmembrane (TM) domain. Unlike AM2, no antiviral drugs have been developed to block the BM2 channel. To elucidate the proton-conduction mechanism of BM2 and to facilitate the development of BM2 inhibitors, we have employed solid-state NMR spectroscopy to investigate the conformation, dynamics, and hydration of the BM2 TM domain in lipid bilayers. BM2 adopts an α-helical conformation in lipid membranes. At physiological temperature and low pH, the proton-selective residue, His19, shows relatively narrow (15)N chemical exchange peaks for the imidazole nitrogens, indicating fast proton shuttling that interconverts cationic and neutral histidines. Importantly, pH-dependent (15)N chemical shifts indicate that His19 retains the neutral population to much lower pH than His37 in AM2, indicating larger acid-dissociation constants or lower pKa's. We attribute these dynamical and equilibrium differences to the presence of a second titratable histidine, His27, which may increase the proton-dissociation rate of His19. Two-dimensional (1)H-(13)C correlation spectra probing water (1)H polarization transfer to the peptide indicates that the BM2 channel becomes much more hydrated at low pH than at high pH, particularly at Ser12, indicating that the pore-facing serine residues in BM2 mediate proton relay to the proton-selective histidine.
- Published
- 2016
10. Interplay Between Membrane Curvature and Protein Conformational Equilibrium Investigated by Solid-State NMR
- Author
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Shu Y. Liao, Myungwoon Lee, Mei Hong, and Massachusetts Institute of Technology. Department of Chemistry
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Models, Molecular ,Protein Conformation ,Antiviral Agents ,Article ,Cell membrane ,Viral Matrix Proteins ,03 medical and health sciences ,chemistry.chemical_compound ,Protein structure ,Protein Domains ,Structural Biology ,medicine ,Amantadine ,Nuclear Magnetic Resonance, Biomolecular ,030304 developmental biology ,Phosphatidylethanolamine ,0303 health sciences ,Carbon Isotopes ,biology ,Nitrogen Isotopes ,Chemistry ,030302 biochemistry & molecular biology ,Transmembrane protein ,Kinetics ,Membrane ,medicine.anatomical_structure ,M2 proton channel ,Membrane protein ,Membrane curvature ,Influenza A virus ,biology.protein ,Biophysics ,Protein Binding - Abstract
Many membrane proteins sense and induce membrane curvature for function, but structural information about how proteins modulate their structures to cause membrane curvature is sparse. We review our recent solid-state NMR studies of two virus membrane proteins whose conformational equilibrium is tightly coupled to membrane curvature. The influenza M2 proton channel has a drug-binding site in the transmembrane (TM) pore. Previous chemical shift data indicated that this pore-binding site is lost in an M2 construct that contains the TM domain and a curvature-inducing amphipathic helix. We have now obtained chemical shift perturbation, protein-drug proximity, and drug orientation data that indicate that the pore-binding site is restored when the full cytoplasmic domain is present. This finding indicates that the curvature-inducing amphipathic helix distorts the TM structure to interfere with drug binding, while the cytoplasmic tail attenuates this effect. In the second example, we review our studies of a parainfluenza virus fusion protein that merges the cell membrane and the virus envelope during virus entry. Chemical shifts of two hydrophobic domains of the protein indicate that both domains have membrane-dependent backbone conformations, with the β-strand structure dominating in negative-curvature phosphatidylethanolamine (PE) membranes. 31 P NMR spectra and 1 H- 31 P correlation spectra indicate that the β-strand-rich conformation induces saddle-splay curvature to PE membranes and dehydrates them, thus stabilizing the hemifusion state. These results highlight the indispensable role of solid-state NMR to simultaneously determine membrane protein structures and characterize the membrane curvature in which these protein structures exist. ©2019, NIH (grant no.GM088204), NIH (grant no. GM066976)
- Published
- 2018
11. Conformation and Trimer Association of the Transmembrane Domain of the Parainfluenza Virus Fusion Protein in Lipid Bilayers from Solid-State NMR: Insights into the Sequence Determinants of Trimer Structure and Fusion Activity
- Author
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Hongwei Yao, Byungsu Kwon, Myungwoon Lee, Chandan Singh, Alan J. Waring, Peter Ruchala, Mei Hong, Massachusetts Institute of Technology. Department of Chemistry, Hong, Mei, Lee, Myungwoon, Yao, Hongwei, Kwon, Byungsu, and Singh, Chandan
- Subjects
Models, Molecular ,Protein Conformation, alpha-Helical ,0301 basic medicine ,Protein Conformation ,Lipid Bilayers ,Trimer ,010402 general chemistry ,01 natural sciences ,Article ,Cell membrane ,03 medical and health sciences ,chemistry.chemical_compound ,Protein structure ,Protein Domains ,Structural Biology ,Scattering, Small Angle ,medicine ,Computer Simulation ,Amino Acid Sequence ,Lipid bilayer ,Nuclear Magnetic Resonance, Biomolecular ,Molecular Biology ,POPC ,Cell Membrane ,Fusion protein ,0104 chemical sciences ,Transmembrane domain ,Cholesterol ,030104 developmental biology ,medicine.anatomical_structure ,chemistry ,Membrane curvature ,Parainfluenza Virus 5 ,Phosphatidylcholines ,Biophysics ,Peptides ,Viral Fusion Proteins - Abstract
Enveloped viruses enter cells by using their fusion proteins to merge the virus lipid envelope and the cell membrane. While crystal structures of the water-soluble ectodomains of many viral fusion proteins have been determined, the structure and assembly of the C-terminal transmembrane domain (TMD) remains poorly understood. Here we use solid-state NMR to determine the backbone conformation and oligomeric structure of the TMD of the parainfluenza virus 5 fusion protein. 13C chemical shifts indicate that the central leucine-rich segment of the TMD is α-helical in POPC/cholesterol membranes and POPE membranes, while the Ile- and Val-rich termini shift to the β-strand conformation in the POPE membrane. Importantly, lipid mixing assays indicate that the TMD is more fusogenic in the POPE membrane than in the POPC/cholesterol membrane, indicating that the β-strand conformation is important for fusion by inducing membrane curvature. Incorporation of para-fluorinated Phe at three positions of the α-helical core allowed us to measure interhelical distances using 19F spin diffusion NMR. The data indicate that, at peptide:lipid molar ratios of ~ 1:15, the TMD forms a trimeric helical bundle with inter-helical distances of 8.2–8.4 Å for L493F and L504F and 10.5 Å for L500F. These data provide high-resolution evidence of trimer formation of a viral fusion protein TMD in phospholipid bilayers, and indicate that the parainfluenza virus 5 fusion protein TMD harbors two functions: the central α-helical core is the trimerization unit of the protein, while the two termini are responsible for inducing membrane curvature by transitioning to a β-sheet conformation. Keywords: magic-angle-spinning NMR; trimer formation; conformational plasticity; spin diffusion, National Institutes of Health (U.S.) (Grant GM066976)
- Published
- 2017
12. Zinc-binding structure of a catalytic amyloid from solid-state NMR
- Author
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William F. DeGrado, Haifan Wu, Tuo Wang, Olga V. Makhlynets, Jan Stöhr, Ivan V. Korendovych, Yibing Wu, Nicholas F. Polizzi, Pallavi M. Gosavi, Myungwoon Lee, and Mei Hong
- Subjects
0301 basic medicine ,Models, Molecular ,Amyloid ,Magnetic Resonance Spectroscopy ,1.1 Normal biological development and functioning ,010402 general chemistry ,01 natural sciences ,metal–peptide framework ,Turn (biochemistry) ,03 medical and health sciences ,Models ,Underpinning research ,Metalloproteins ,magic angle spinning ,Side chain ,Histidine ,Binding site ,Multidisciplinary ,Binding Sites ,metal-peptide framework ,Chemistry ,Ligand ,Water ,Computational Biology ,Molecular ,metalloprotein ,Nuclear magnetic resonance spectroscopy ,histidine ,0104 chemical sciences ,Crystallography ,Zinc ,030104 developmental biology ,Physical Sciences ,Protein folding ,Generic health relevance ,Protein ligand - Abstract
Throughout biology, amyloids are key structures in both functional proteins and the end product of pathologic protein misfolding. Amyloids might also represent an early precursor in the evolution of life because of their small molecular size and their ability to self-purify and catalyze chemical reactions. They also provide attractive backbones for advanced materials. When β-strands of an amyloid are arranged parallel and in register, side chains from the same position of each chain align, facilitating metal chelation when the residues are good ligands such as histidine. High-resolution structures of metalloamyloids are needed to understand the molecular bases of metal-amyloid interactions. Here we combine solid-state NMR and structural bioinformatics to determine the structure of a zinc-bound metalloamyloid that catalyzes ester hydrolysis. The peptide forms amphiphilic parallel β-sheets that assemble into stacked bilayers with alternating hydrophobic and polar interfaces. The hydrophobic interface is stabilized by apolar side chains from adjacent sheets, whereas the hydrated polar interface houses the Zn2+-binding histidines with binding geometries unusual in proteins. Each Zn2+ has two bis-coordinated histidine ligands, which bridge adjacent strands to form an infinite metal-ligand chain along the fibril axis. A third histidine completes the protein ligand environment, leaving a free site on the Zn2+ for water activation. This structure defines a class of materials, which we call metal-peptide frameworks. The structure reveals a delicate interplay through which metal ions stabilize the amyloid structure, which in turn shapes the ligand geometry and catalytic reactivity of Zn2.
- Published
- 2017
13. Cryoprotection of lipid membranes for high-resolution solid-state NMR studies of membrane peptides and proteins at low temperature
- Author
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Myungwoon Lee and Mei Hong
- Subjects
Lipid Bilayers ,Membrane Proteins ,Membranes, Artificial ,Polyethylene glycol ,Biochemistry ,Trehalose ,Article ,NMR spectra database ,chemistry.chemical_compound ,Membrane ,chemistry ,Solid-state nuclear magnetic resonance ,Membrane protein ,Freezing ,Biophysics ,Organic chemistry ,Dimethylformamide ,Peptides ,Lipid bilayer ,Nuclear Magnetic Resonance, Biomolecular ,Spectroscopy - Abstract
Solid-state NMR spectra of membrane proteins often show significant line broadening at cryogenic temperatures. Here we investigate the effects of several cryoprotectants to preserve the spectral resolution of lipid membranes and membrane peptides at temperatures down to ~200 K. Trehalose, glycerol, dimethylsulfoxide (DMSO), dimethylformamide (DMF), and polyethylene glycol (PEG), were chosen. These compounds are commonly used in protein crystallography and cryobiology. 13C and 1H magic-angle-spinning spectra of several types of lipid membranes show that DMSO provides the best resolution enhancement over unprotected membranes and also best retards ice formation at low temperature. DMF and PEG-400 show slightly weaker cryoprotection, while glycerol and trehalose neither prevent membrane line broadening nor prevent ice formation under the conditions of our study. Neutral saturated-chain phospholipids are the most amenable to cryoprotection, whereas negatively charged and unsaturated lipids attenuate cryoprotection. 13C-1H dipolar couplings and 31P chemical shift anisotropies indicate that high spectral resolution at low temperature is correlated with stronger immobilization of the lipids at high temperature, indicating that line narrowing results from reduction of the conformational space sampled by the lipid molecules at high temperature. DMSO selectively narrowed the linewidths of the most disordered residues in the influenza M2 transmembrane peptide, while residues that exhibit narrow linewidths in the unprotected membrane are less impacted. A relatively rigid β-hairpin antimicrobial peptide, PG-1, showed a linewidth increase of ~0.5 ppm over a ~70 K temperature drop both with and without cryoprotection. Finally, a short-chain saturated lipid, DLPE, exhibits excellent linewidths, suggesting that it may be a good medium for membrane protein structure determination. The three best cryoprotectants found in this work-DMSO, PEG, and DMF-should be useful for low-temperature membrane-protein structural studies by SSNMR without compromising spectral resolution.
- Published
- 2014
14. Efficient DNP NMR of membrane proteins: sample preparation protocols, sensitivity, and radical location
- Author
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Tuo Wang, Shu Y. Liao, Myungwoon Lee, Ivan V. Sergeyev, Mei Hong, Massachusetts Institute of Technology. Department of Chemistry, Liao, Shu-Yu, Lee, Myungwoon, Wang, Tuo, and Hong, Mei
- Subjects
010405 organic chemistry ,Radical ,Membrane lipids ,Temperature ,Analytical chemistry ,Phospholipid ,Membrane Proteins ,010402 general chemistry ,Photochemistry ,01 natural sciences ,Biochemistry ,Article ,0104 chemical sciences ,NMR spectra database ,Membrane Lipids ,chemistry.chemical_compound ,Membrane ,chemistry ,Solid-state nuclear magnetic resonance ,Peptides ,Lipid bilayer ,Nuclear Magnetic Resonance, Biomolecular ,Spectroscopy ,Macromolecule - Abstract
Although dynamic nuclear polarization (DNP) has dramatically enhanced solid-state NMR spectral sensitivities of many synthetic materials and some biological macromolecules, recent studies of membrane-protein DNP using exogenously doped paramagnetic radicals as polarizing agents have reported varied and sometimes surprisingly limited enhancement factors. This motivated us to carry out a systematic evaluation of sample preparation protocols for optimizing the sensitivity of DNP NMR spectra of membrane-bound peptides and proteins at cryogenic temperatures of ~110 K. We show that mixing the radical with the membrane by direct titration instead of centrifugation gives a significant boost to DNP enhancement. We quantify the relative sensitivity enhancement between AMUPol and TOTAPOL, two commonly used radicals, and between deuterated and protonated lipid membranes. AMUPol shows ~fourfold higher sensitivity enhancement than TOTAPOL, while deuterated lipid membrane does not give net higher sensitivity for the membrane peptides than protonated membrane. Overall, a ~100 fold enhancement between the microwave-on and microwave-off spectra can be achieved on lipid-rich membranes containing conformationally disordered peptides, and absolute sensitivity gains of 105–160 can be obtained between low-temperature DNP spectra and high-temperature non-DNP spectra. We also measured the paramagnetic relaxation enhancement of lipid signals by TOTAPOL and AMUPol, to determine the depths of these two radicals in the lipid bilayer. Our data indicate a bimodal distribution of both radicals, a surface-bound fraction and a membrane-bound fraction where the nitroxides lie at ~10 Å from the membrane surface. TOTAPOL appears to have a higher membrane-embedded fraction than AMUPol. These results should be useful for membrane-protein solid-state NMR studies under DNP conditions and provide insights into how biradicals interact with phospholipid membranes., National Institutes of Health (U.S.) (NIH Grant GM088204), National Institutes of Health (U.S.) (NIH Grant GM066976)
- Published
- 2015
15. The gene encoding rat nuclear pore glycoprotein p62 is intronless
- Author
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Mae-Jean Miller, Myungwoon Lee, Christopher M. Starr, John A. Hanover, and Mara D'Onofrio
- Subjects
chemistry.chemical_classification ,Reporter gene ,Cell Biology ,Biology ,Biochemistry ,Molecular biology ,Primer extension ,Open reading frame ,chemistry ,Complementary DNA ,Transcriptional regulation ,Nuclear pore ,Glycoprotein ,Molecular Biology ,Gene - Abstract
Glycoproteins of the nuclear pore complex are thought to play an important role in the transport of regulatory proteins and ribonucleoproteins across the nuclear envelope. However, the genetic elements and signals that control the expression of nuclear pore glycoproteins are poorly understood. To study the transcriptional regulation of mammalian nuclear pore glycoprotein biosynthesis, we have isolated the gene coding for the major rat nuclear pore glycoprotein p62. The p62 gene consists of a 2941-base pair region that is linear with the full length p62 cDNA with no intervening sequences. Quantitative Southern analysis revealed that the gene is present in single copy. The p62 gene encodes a 525-amino acid open reading frame that directs the synthesis of the 62-kDa pore glycoprotein in vitro and in transfected cultured cells. The 5‘-flanking region contains two potential transcription start sites; primer extension analysis revealed that the furthest upstream site is preferentially used in vivo. When linked to a reporter gene, the 5‘-flanking region of the p62 gene serves as an active promoter.
- Published
- 1991
16. Zinc-binding structure of a catalytic amyloid from solid-state NMR.
- Author
-
Myungwoon Lee, Tuo Wang, Makhlynets, Olga V., Yibing Wu, Polizzi, Nicholas F., Haifan Wu, Gosavi, Pallavi M., Stöhr, Jan, Korendovych, Ivan V., Degrado, William F., and Mei Hong
- Subjects
- *
NUCLEAR magnetic resonance , *ZINC , *CHEMICAL reactions , *AMYLOID , *HISTIDINE - Abstract
Throughout biology, amyloids are key structures in both functional proteins and the end product of pathologic protein misfolding. Amyloids might also represent an early precursor in the evolution of life because of their small molecular size and their ability to selfpurify and catalyze chemical reactions. They also provide attractive backbones for advanced materials. When ß-strands of an amyloid are arranged parallel and in register, side chains from the same position of each chain align, facilitating metal chelation when the residues are good ligands such as histidine. High-resolution structures of metalloamyloids are needed to understand the molecular bases of metal-amyloid interactions. Here we combine solid-state NMR and structural bioinformatics to determine the structure of a zinc-bound metalloamyloid that catalyzes ester hydrolysis. The peptide forms amphiphilic parallel ß-sheets that assemble into stacked bilayers with alternating hydrophobic and polar interfaces. The hydrophobic interface is stabilized by apolar side chains from adjacent sheets, whereas the hydrated polar interface houses the Zn2+-binding histidines with binding geometries unusual in proteins. Each Zn2+ has two bis-coordinated histidine ligands, which bridge adjacent strands to form an infinite metal-ligand chain along the fibril axis. A third histidine completes the protein ligand environment, leaving a free site on the Zn2+ for water activation. This structure defines a class of materials, which we call metal-peptide frameworks. The structure reveals a delicate interplay through which metal ions stabilize the amyloid structure, which in turn shapes the ligand geometry and catalytic reactivity of Zn2+. [ABSTRACT FROM AUTHOR]
- Published
- 2017
- Full Text
- View/download PDF
17. Solid-State NMR Investigation of the Conformation, Proton Conduction, and Hydration of the Influenza B Virus M2 Transmembrane Proton Channel.
- Author
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Williams, Jonathan K., Tietze, Daniel, Myungwoon Lee, Jun Wang, and Mei Hong
- Published
- 2016
- Full Text
- View/download PDF
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