scholarly journals A strategy to study intrinsically mixed folded proteins: The structure in solution of ataxin-3

2018 ◽  
Author(s):  
A. Sicorello ◽  
G. Kelly ◽  
A. Oregioni ◽  
J. Nováček ◽  
V. Sklenář ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Nuclear Magnetic Resonance ◽  
Low Complexity ◽  
Structural Features ◽  
Resonance Spectroscopy ◽  
Cryo Electron Microscopy ◽  
Per Se ◽  
Folded Proteins ◽  
First Time ◽  
Structure In Solution

AbstractIt has increasingly become clear over the last two decades that proteins can contain both globular domains and intrinsically unfolded regions which both can contribute to function. While equally interesting, the disordered regions are difficult to study because they usually do not crystallize unless bound to partners and are not easily amenable to cryo-electron microscopy studies. Nuclear magnetic resonance spectroscopy remains the best technique to capture the structural features of intrinsically mixed folded proteins and describe their dynamics. These studies rely on the successful assignment of the spectrum, task not easy per se given the limited spread of the resonances of the disordered residues. Here, we describe assignment of the spectrum of ataxin-3, the protein responsible for the neurodegenerative Machado-Joseph disease. We used a 42 kDa construct containing a globular N-terminal josephin domain and a C-terminal tail which comprises thirteen polyglutamine repeats within a low-complexity region. We developed a strategy which allowed us to achieve 87% assignment of the spectrum. We show that the C-terminal tail is flexible with extended helical regions and interacts only marginally with the rest of the protein. We could also, for the first time, deduce the structure of the polyglutamine repeats within the context of the full-length protein and show that it has a strong helical propensity stabilized by the preceding region.

scholarly journals Cryo–electron microscopy structure of the antidiuretic hormone arginine-vasopressin V2 receptor signaling complex

2021 ◽  
Vol 7 (21) ◽  
pp. eabg5628
Author(s):  
Julien Bous ◽  
Hélène Orcel ◽  
Nicolas Floquet ◽  
Cédric Leyrat ◽  
Joséphine Lai-Kee-Him ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Antidiuretic Hormone ◽  
Arginine Vasopressin ◽  
Molecular Mechanisms ◽  
Particle Analysis ◽  
Resonance Spectroscopy ◽  
Gpcr Signaling ◽  
Signaling Complex ◽  
Cryo Electron Microscopy ◽  
V2 Receptor

The antidiuretic hormone arginine-vasopressin (AVP) forms a signaling complex with the V2 receptor (V2R) and the Gs protein, promoting kidney water reabsorption. Molecular mechanisms underlying activation of this critical G protein–coupled receptor (GPCR) signaling system are still unknown. To fill this gap of knowledge, we report here the cryo–electron microscopy structure of the AVP-V2R-Gs complex. Single-particle analysis revealed the presence of three different states. The two best maps were combined with computational and nuclear magnetic resonance spectroscopy constraints to reconstruct two structures of the ternary complex. These structures differ in AVP and Gs binding modes. They reveal an original receptor-Gs interface in which the Gαs subunit penetrates deep into the active V2R. The structures help to explain how V2R R137H or R137L/C variants can lead to two severe genetic diseases. Our study provides important structural insights into the function of this clinically relevant GPCR signaling complex.

scholarly journals Differential Effects of ReplacingEscherichiacoli Ribosomal Protein L27 with Its Homologue from Aquifexaeolicus

2001 ◽  
Vol 183 (22) ◽  
pp. 6565-6572 ◽  
Author(s):  
Bruce A. Maguire ◽  
Anton V. Manuilov ◽  
Robert A. Zimmermann
Keyword(s):  
Growth Rate ◽  
Nuclear Magnetic Resonance ◽  
Magnetic Resonance ◽  
Ribosomal Subunit ◽  
Proton Nuclear Magnetic Resonance ◽  
Resonance Spectroscopy ◽  
Extreme Thermophile ◽  
Ribosomal Subunit Protein ◽  
And Function ◽  
Structure In Solution

ABSTRACT The rpmA gene, which encodes 50S ribosomal subunit protein L27, was cloned from the extreme thermophileAquifex aeolicus, and the protein was overexpressed and purified. Comparison of the A.aeolicus protein with its homologue fromEscherichia coli by circular dichroism analysis and proton nuclear magnetic resonance spectroscopy showed that it readily adopts some structure in solution that is very stable, whereas the E. coli protein is unstructured under the same conditions. A mutant of E.coli that lacks L27 was found earlier to be impaired in the assembly and function of the 50S subunit; both defects could be corrected by expression of E. coliL27 from an extrachromosomal copy of the rpmA gene. WhenA. aeolicus L27 was expressed in the same mutant, an increase in the growth rate occurred and the “foreign” L27 protein was incorporated into E. coliribosomes. However, the presence of A.aeolicus L27 did not promote 50S subunit assembly. Thus, while the A. aeolicus protein can apparently replace its E. coli homologue functionally in completed ribosomes, it does not assist in the assembly of E. coli ribosomes that otherwise lack L27. Possible explanations for this paradoxical behavior are discussed.

scholarly journals Cryo-electron Microscopy Structures of Novel Viruses from Mud Crab Scylla paramamosain with Multiple Infections

2019 ◽  
Vol 93 (7) ◽  
Author(s):  
Yuanzhu Gao ◽  
Shanshan Liu ◽  
Jiamiao Huang ◽  
Qianqian Wang ◽  
Kunpeng Li ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
3D Structure ◽  
Structural Features ◽  
Economic Losses ◽  
Scylla Paramamosain ◽  
Multiple Infections ◽  
Mud Crab ◽  
Cryo Electron Microscopy ◽  
Novel Virus ◽  
Icosahedral Capsid

ABSTRACT Viruses associated with sleeping disease (SD) in crabs cause great economic losses to aquaculture, and no effective measures are available for their prevention. In this study, to help develop novel antiviral strategies, single-particle cryo-electron microscopy was applied to investigate viruses associated with SD. The results not only revealed the structure of mud crab dicistrovirus (MCDV) but also identified a novel mud crab tombus-like virus (MCTV) not previously detected using molecular biology methods. The structure of MCDV at a 3.5-Å resolution reveals three major capsid proteins (VP1 to VP3) organized into a pseudo-T=3 icosahedral capsid, and affirms the existence of VP4. Unusually, MCDV VP3 contains a long C-terminal region and forms a novel protrusion that has not been observed in other dicistrovirus. Our results also reveal that MCDV can release its genome via conformation changes of the protrusions when viral mixtures are heated. The structure of MCTV at a 3.3-Å resolution reveals a T= 3 icosahedral capsid with common features of both tombusviruses and nodaviruses. Furthermore, MCTV has a novel hydrophobic tunnel beneath the 5-fold vertex and 30 dimeric protrusions composed of the P-domains of the capsid protein at the 2-fold axes that are exposed on the virion surface. The structural features of MCTV are consistent with a novel type of virus. IMPORTANCE Pathogen identification is vital for unknown infectious outbreaks, especially for dual or multiple infections. Sleeping disease (SD) in crabs causes great economic losses to aquaculture worldwide. Here we report the discovery and identification of a novel virus in mud crabs with multiple infections that was not previously detected by molecular, immune, or traditional electron microscopy (EM) methods. High-resolution structures of pathogenic viruses are essential for a molecular understanding and developing new disease prevention methods. The three-dimensional (3D) structure of the mud crab tombus-like virus (MCTV) and mud crab dicistrovirus (MCDV) determined in this study could assist the development of antiviral inhibitors. The identification of a novel virus in multiple infections previously missed using other methods demonstrates the usefulness of this strategy for investigating multiple infectious outbreaks, even in humans and other animals.

scholarly journals Cryo-EM reveals unique structural features of the FhuCDB Escherichia coli ferrichrome importer

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Wenxin Hu ◽  
Hongjin Zheng
Keyword(s):  
Electron Microscopy ◽  
Escherichia Coli ◽  
Molecular Basis ◽  
Iron Uptake ◽  
Structural Features ◽  
Biological Processes ◽  
Type Ii ◽  
Functional Studies ◽  
Cryo Electron Microscopy ◽  
Translocation Pathway

AbstractAs one of the most elegant biological processes developed in bacteria, the siderophore-mediated iron uptake demands the action of specific ATP-binding cassette (ABC) importers. Although extensive studies have been done on various ABC importers, the molecular basis of these iron-chelated-siderophore importers are still not fully understood. Here, we report the structure of a ferrichrome importer FhuCDB from Escherichia coli at 3.4 Å resolution determined by cryo electron microscopy. The structure revealed a monomeric membrane subunit of FhuB with a substrate translocation pathway in the middle. In the pathway, there were unique arrangements of residues, especially layers of methionines. Important residues found in the structure were interrogated by mutagenesis and functional studies. Surprisingly, the importer’s ATPase activity was decreased upon FhuD binding, which deviated from the current understanding about bacterial ABC importers. In summary, to the best of our knowledge, these studies not only reveal a new structural twist in the type II ABC importer subfamily, but also provide biological insights in the transport of iron-chelated siderophores.

scholarly journals High-Light versus Low-Light: Effects on Paired Photosystem II Supercomplex Structural Rearrangement in Pea Plants

2020 ◽  
Vol 21 (22) ◽  
pp. 8643
Author(s):  
Alessandro Grinzato ◽  
Pascal Albanese ◽  
Roberto Marotta ◽  
Paolo Swuec ◽  
Guido Saracco ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Photosystem Ii ◽  
Structural Rearrangement ◽  
Structural Features ◽  
Thylakoid Membranes ◽  
Variable Number ◽  
High Light ◽  
Low Light ◽  
Cryo Electron Microscopy ◽  
Light Effects

In plant grana thylakoid membranes Photosystem II (PSII) associates with a variable number of antenna proteins (LHCII) to form different types of supercomplexes (PSII-LHCII), whose organization is dynamically adjusted in response to light cues, with the C2S2 more abundant in high-light and the C2S2M2 in low-light. Paired PSII-LHCII supercomplexes interacting at their stromal surface from adjacent thylakoid membranes were previously suggested to mediate grana stacking. Here, we present the cryo-electron microscopy maps of paired C2S2 and C2S2M2 supercomplexes isolated from pea plants grown in high-light and low-light, respectively. These maps show a different rotational offset between the two supercomplexes in the pair, responsible for modifying their reciprocal interaction and energetic connectivity. This evidence reveals a different way by which paired PSII-LHCII supercomplexes can mediate grana stacking at diverse irradiances. Electrostatic stromal interactions between LHCII trimers almost completely overlapping in the paired C2S2 can be the main determinant by which PSII-LHCII supercomplexes mediate grana stacking in plants grown in high-light, whereas the mutual interaction of stromal N-terminal loops of two facing Lhcb4 subunits in the paired C2S2M2 can fulfil this task in plants grown in low-light. The high-light induced accumulation of the Lhcb4.3 protein in PSII-LHCII supercomplexes has been previously reported. Our cryo-electron microscopy map at 3.8 Å resolution of the C2S2 supercomplex isolated from plants grown in high-light suggests the presence of the Lhcb4.3 protein revealing peculiar structural features of this high-light-specific antenna important for photoprotection.

Structural features of a peptide corresponding to human κ-casein residues 84–101 by 1H-nuclear magnetic resonance spectroscopy

1999 ◽  
Vol 66 (1) ◽  
pp. 53-63 ◽  
Author(s):  
JEFFREY E. PLOWMAN ◽  
LAWRENCE K. CREAMER ◽  
MICHAEL J. LIDDELL ◽  
JENNIFER J. CROSS
Keyword(s):  
Nuclear Magnetic Resonance ◽  
Magnetic Resonance ◽  
Magnetic Resonance Spectroscopy ◽  
Nuclear Magnetic Resonance Spectroscopy ◽  
Structural Features ◽  
Chain Conformation ◽  
Dimethyl Sulphoxide ◽  
Resonance Spectroscopy ◽  
1H Nuclear Magnetic Resonance ◽  
Nuclear Magnetic

The peptide Val–Arg–Arg–Pro–Asn–Leu–His–Pro–Ser–Phe–Ile–Ala–Ile–Pro–Pro–Lys–Lys–Ile, which corresponds to residues 84–101 of human κ-casein, has been synthesized and its conformation preferences determined by 1H-nuclear magnetic resonance spectroscopy in dimethyl sulphoxide. The peptide adopted a largely extended chain conformation in solution and there was evidence for the presence of a β-turn involving residues Pro87–His90 of human κ-casein. The presence of a turn in this position would make the physiologically significant Arg85 residue of human κ-casein (which is equivalent to Arg97 in bovine κ-casein) unavailable for interaction with Asp249of bovine chymosin, and may partly explain why human κ-casein is hydrolysed more slowly than its bovine counterpart by bovine chymosin.

Characterization of Eucalyptus and Chemically Related Exudates by Nuclear Magnetic Resonance Spectroscopy

2007 ◽  
Vol 60 (11) ◽  
pp. 862 ◽  
Author(s):  
Joseph B. Lambert ◽  
Yuyang Wu ◽  
Michael A. Kozminski ◽  
Jorge A. Santiago-Blay
Keyword(s):  
Nuclear Magnetic Resonance ◽  
Solid State ◽  
1H Nmr Spectroscopy ◽  
Nmr Spectra ◽  
Flowering Plants ◽  
Resonance Spectroscopy ◽  
The Family ◽  
First Time ◽  
Genus Eucalyptus

Exudates from six species of the genus Eucalyptus and one of the genus Corymbia (formerly Eucalyptus), from the family Myrtaceae, have been characterized by solid-state 13C and solution 1H NMR spectroscopy for the first time. Although these eucalypt kinos, as these exudates often are called, resemble resin (terpenoid) and gum (carbohydrate) exudates in physical appearance, their NMR spectra are dramatically different. In addition to lacking the characteristic terpene saturated resonances, they exhibit strong unsaturated resonances, which are weak for resins and absent for gums. We additionally report that exudates from genera of several other families of flowering plants (Amyris, Centrolobium, Guaiacum, Liquidambar, and Prosopis) also exhibit part or all of this kino spectroscopic signature.

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