scholarly journals Deciphering molecular details of the RAC–ribosome interaction by EPR spectroscopy

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sandra J. Fries ◽  
Theresa S. Braun ◽  
Christoph Globisch ◽  
Christine Peter ◽  
Malte Drescher ◽  
...  
Keyword(s):  
Protein Folding ◽  
Epr Spectroscopy ◽  
De Novo ◽  
Contact Point ◽  
Ribosomal Subunit ◽  
Paramagnetic Resonance ◽  
40S Ribosomal Subunit ◽  
Site Directed Spin Labeling ◽  
Α Helix ◽  
Chaperone Complex

AbstractThe eukaryotic ribosome-associated complex (RAC) plays a significant role in de novo protein folding. Its unique interaction with the ribosome, comprising contacts to both ribosomal subunits, suggests a RAC-mediated coordination between translation elongation and co-translational protein folding. Here, we apply electron paramagnetic resonance (EPR) spectroscopy combined with site-directed spin labeling (SDSL) to gain deeper insights into a RAC–ribosome contact affecting translational accuracy. We identified a local contact point of RAC to the ribosome. The data provide the first experimental evidence for the existence of a four-helix bundle as well as a long α-helix in full-length RAC, in solution as well as on the ribosome. Additionally, we complemented the structural picture of the region mediating this functionally important contact on the 40S ribosomal subunit. In sum, this study constitutes the first application of SDSL-EPR spectroscopy to elucidate the molecular details of the interaction between the 3.3 MDa translation machinery and a chaperone complex.

scholarly journals Posttranscriptional site-directed spin labeling of large RNAs with an unnatural base pair system under non-denaturing conditions

2020 ◽  
Vol 11 (35) ◽  
pp. 9655-9664
Author(s):  
Yan Wang ◽  
Venkatesan Kathiresan ◽  
Yaoyi Chen ◽  
Yanping Hu ◽  
Wei Jiang ◽  
...  
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Epr Spectroscopy ◽  
Base Pair ◽  
Spin Labeling ◽  
Paramagnetic Resonance ◽  
Site Directed Spin Labeling ◽  
Electron Paramagnetic

Site-directed spin labeling (SDSL) of large RNAs for electron paramagnetic resonance (EPR) spectroscopy has remained challenging to date.

scholarly journals Structure and Dynamics of Type III Secretion Effector Protein ExoU As determined by SDSL-EPR Spectroscopy in Conjunction with De Novo Protein Folding

ACS Omega ◽  
2017 ◽  
Vol 2 (6) ◽  
pp. 2977-2984 ◽  
Author(s):  
Axel W. Fischer ◽  
David M. Anderson ◽  
Maxx H. Tessmer ◽  
Dara W. Frank ◽  
Jimmy B. Feix ◽  
...  
Keyword(s):  
Protein Folding ◽  
Epr Spectroscopy ◽  
Type Iii Secretion ◽  
De Novo ◽  
Effector Protein ◽  
Type Iii ◽  
Structure And Dynamics ◽  
Type Iii Secretion Effector

scholarly journals Structural Dynamics of the N-Extension of Cardiac Troponin I Complexed with Troponin C by Site-Directed Spin Labeling Electron Paramagnetic Resonance

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Chenchao Zhao ◽  
Takayasu Somiya ◽  
Shinji Takai ◽  
Shoji Ueki ◽  
Toshiaki Arata
Keyword(s):  
Secondary Structure ◽  
Cardiac Troponin ◽  
Troponin I ◽  
Cardiac Troponin I ◽  
Spin Labels ◽  
Phosphorylation Sites ◽  
Paramagnetic Resonance ◽  
Single Spin ◽  
Site Directed Spin Labeling ◽  
Α Helix

Abstract The secondary structure of the N-extension of cardiac troponin I (cTnI) was determined by measuring the distance distribution between spin labels attached to the i and i + 4 residues: 15/19, 23/27, 27/31, 35/39, and 43/47. All of the EPR spectra of these regions in the monomeric state were broadened and had a amplitude that was reduced by two-thirds of that of the single spin-labeled spectra and was fit by two residual distance distributions, with a major distribution one spreading over the range from 1 to 2.5 nm and the other minor peak at 0.9 nm. Only slight or no obvious changes were observed when the extension was bound to cTnC in the cTnI-cTnC complex at 0.2 M KCl. However, at 0.1 M KCl, residues 43/47, located at the PKC phosphorylation sites Ser42/44 on the boundary of the extension, exclusively exhibited a 0.9 nm peak, as expected from α-helix in the crystal structure, in the complex. Furthermore, 23/27, which is located on the PKA phosphorylation sites Ser23/24, showed that the major distribution was markedly narrowed, centered at 1.4 nm and 0.5 nm wide, accompanying the spin label immobilization of residue 27. Residues 35 and 69 at site 1 and 2 of cTnC exhibited partial immobilization of the attached spin labels upon complex formation. The results show that the extension exhibited a primarily partially folded or unfolded structure equilibrated with a transiently formed α-helix-like short structure over the length. We hypothesize that the structure binds at least near sites 1 and 2 of cTnC and that the specific secondary structure of the extension on cTnC becomes uncovered when decreasing the ionic strength demonstrating that only the phosphorylation regions of cTnI interact stereospecifically with cTnC.

Site-directed spin labeling EPR spectroscopy in protein research

2013 ◽  
Vol 394 (10) ◽  
pp. 1281-1300 ◽  
Author(s):  
Johann P. Klare
Keyword(s):  
Nucleic Acids ◽  
Epr Spectroscopy ◽  
Protein Function ◽  
Conformational Dynamics ◽  
Spin Labeling ◽  
Paramagnetic Resonance ◽  
Protein Research ◽  
Site Directed Spin Labeling ◽  
Electron Paramagnetic ◽  
Natronomonas Pharaonis

Abstract Site-directed spin labeling (SDSL) in combination with electron paramagnetic resonance (EPR) spectroscopy has emerged as an efficient tool to elucidate the structure and the conformational dynamics of proteins under conditions close to the native state. This review article summarizes the basics as well as the recent progress in SDSL and EPR methods, especially for investigations on protein structure, protein function, and interaction of proteins with other proteins or nucleic acids. Labeling techniques as well as EPR methods are introduced and exemplified with applications to systems that have been studied in the author’s laboratory in the past 15 years, headmost the sensory rhodopsin-transducer complex mediating the photophobic response of the halophilic archaeum Natronomonas pharaonis. Further examples underline the application of SDSL EPR spectroscopy to answer specific questions about the system under investigation, such as the nature and influence of interactions of proteins with other proteins or nucleic acids. Finally, it is discussed how SDSL EPR can be combined with other biophysical techniques to combine the strengths of the different methodologies.

scholarly journals Structure of the 40S ribosomal subunit of Plasmodium falciparum by homology and de novo modeling

2017 ◽  
Vol 7 (1) ◽  
pp. 97-105 ◽  
Author(s):  
Harrison Ndung'u Mwangi ◽  
Peter Wagacha ◽  
Peterson Mathenge ◽  
Fredrick Sijenyi ◽  
Francis Mulaa
Keyword(s):  
Plasmodium Falciparum ◽  
De Novo ◽  
Ribosomal Subunit ◽  
40S Ribosomal Subunit

scholarly journals Site-Directed Spin Labeling EPR for Studying Membrane Proteins

2018 ◽  
Vol 2018 ◽  
pp. 1-13 ◽  
Author(s):  
Indra D. Sahu ◽  
Gary A. Lorigan
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Membrane Proteins ◽  
Epr Spectroscopy ◽  
Dynamic Properties ◽  
Biological Systems ◽  
Spin Labeling ◽  
Paramagnetic Resonance ◽  
Site Directed Spin Labeling ◽  
Living Organisms ◽  
Electron Paramagnetic

Site-directed spin labeling (SDSL) in combination with electron paramagnetic resonance (EPR) spectroscopy is a rapidly expanding powerful biophysical technique to study the structural and dynamic properties of membrane proteins in a native environment. Membrane proteins are responsible for performing important functions in a wide variety of complicated biological systems that are responsible for the survival of living organisms. In this review, a brief introduction of the most popular SDSL EPR techniques and illustrations of recent applications for studying pertinent structural and dynamic properties on membrane proteins will be discussed.

scholarly journals Ribosome-associated Complex Binds to Ribosomes in Close Proximity of Rpl31 at the Exit of the Polypeptide Tunnel in Yeast

2008 ◽  
Vol 19 (12) ◽  
pp. 5279-5288 ◽  
Author(s):  
Kristin Peisker ◽  
Daniel Braun ◽  
Tina Wölfle ◽  
Jendrik Hentschel ◽  
Ursula Fünfschilling ◽  
...  
Keyword(s):  
Slow Growth ◽  
Contact Point ◽  
Ribosomal Subunit ◽  
Proper Function ◽  
Multicopy Suppressor ◽  
Growth Defects ◽  
Yeast Strains ◽  
Highly Sensitive ◽  
Close Proximity ◽  
Chaperone Complex

Ribosome-associated complex (RAC) consists of the Hsp40 homolog Zuo1 and the Hsp70 homolog Ssz1. The chaperone participates in the biogenesis of newly synthesized polypeptides. Here we have identified yeast Rpl31, a component of the large ribosomal subunit, as a contact point of RAC at the polypeptide tunnel exit. Rpl31 is encoded by RPL31a and RPL31b, two closely related genes. Δrpl31aΔrpl31b displayed slow growth and sensitivity to low as well as high temperatures. In addition, Δrpl31aΔrpl31b was highly sensitive toward aminoglycoside antibiotics and suffered from defects in translational fidelity. With the exception of sensitivity at elevated temperature, the phenotype resembled yeast strains lacking one of the RAC subunits or Rpl39, another protein localized at the tunnel exit. Defects of Δrpl31aΔrpl31bΔzuo1 did not exceed that of Δrpl31aΔrpl31b or Δzuo1. However, the combined deletion of RPL31a, RPL31b, and RPL39 was lethal. Moreover, RPL39 was a multicopy suppressor, whereas overexpression of RAC failed to rescue growth defects of Δrpl31aΔrpl31b. The findings are consistent with a model in that Rpl31 and Rpl39 independently affect a common ribosome function, whereas Rpl31 and RAC are functionally interdependent. Rpl31, while not essential for binding of RAC to the ribosome, might be involved in proper function of the chaperone complex.

scholarly journals Molecular Architecture of Full-Length KcsA

2001 ◽  
Vol 117 (2) ◽  
pp. 165-180 ◽  
Author(s):  
D. Marien Cortes ◽  
Luis G. Cuello ◽  
Eduardo Perozo
Keyword(s):  
Three Dimensional ◽  
Full Length ◽  
Spin Interaction ◽  
Structural Constraints ◽  
Molecular Architecture ◽  
Paramagnetic Resonance ◽  
Gating Mechanism ◽  
Potassium Channel Kcsa ◽  
Site Directed Spin Labeling ◽  
Α Helix

The molecular architecture of the NH2 and COOH termini of the prokaryotic potassium channel KcsA has been determined using site-directed spin-labeling methods and paramagnetic resonance EPR spectroscopy. Cysteine mutants were generated (residues 5–24 and 121–160) and spin labeled, and the X-band CW EPR spectra were obtained from liposome-reconstituted channels at room temperature. Data on probe mobility (ΔHo−1), accessibility parameters (ΠO2 and ΠNiEdda), and inter-subunit spin-spin interaction (Ω) were used as structural constraints to build a three-dimensional folding model of these cytoplasmic domains from a set of simulated annealing and restrained molecular dynamics runs. 32 backbone structures were generated and averaged using fourfold symmetry, and a final mean structure was obtained from the eight lowest energy runs. Based on the present data, together with information from the KcsA crystal structure, a model for the three-dimensional fold of full-length KcsA was constructed. In this model, the NH2 terminus of KcsA forms an α-helix anchored at the membrane–water interface, while the COOH terminus forms a right-handed four-helix bundle that extend some 40–50 Å towards the cytoplasm. Functional analysis of COOH-terminal deletion constructs suggest that, while the COOH terminus does not play a substantial role in determining ion permeation properties, it exerts a modulatory role in the pH-dependent gating mechanism.

Localization of metal ions in biomolecules by means of pulsed dipolar EPR spectroscopy

2021 ◽  
Author(s):  
Dinar Abdullin ◽  
Olav Schiemann
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Metal Ions ◽  
Epr Spectroscopy ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Spin Labeling ◽  
Resonance Spectroscopy ◽  
Paramagnetic Resonance ◽  
Paramagnetic Metal ◽  
Site Directed Spin Labeling ◽  
Electron Paramagnetic

A method is introduced in which paramagnetic metal ions are localized by means of trilateration using a combination of site-directed spin labeling and pulsed dipolar electron paramagnetic resonance spectroscopy.

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