Investigating the Dynamics of Destabilized Nucleosomes Using Methyl-TROSY NMR

2018 ◽  
Vol 140 (14) ◽  
pp. 4774-4777 ◽  
Author(s):  
Julianne L. Kitevski-LeBlanc ◽  
Tairan Yuwen ◽  
Pamela N. Dyer ◽  
Johannes Rudolph ◽  
Karolin Luger ◽  
...  
Keyword(s):  
Methyl Trosy ◽  
Methyl Trosy Nmr

Application of Methyl-TROSY NMR to Test Allosteric Models Describing Effects of Nucleotide Binding to Aspartate Transcarbamoylase

2009 ◽  
Vol 387 (3) ◽  
pp. 540-547 ◽  
Author(s):  
Algirdas Velyvis ◽  
Howard K. Schachman ◽  
Lewis E. Kay
Keyword(s):  
Nucleotide Binding ◽  
Aspartate Transcarbamoylase ◽  
Methyl Trosy ◽  
Methyl Trosy Nmr

scholarly journals High Quality Methyl-TROSY NMR Studies of the Interactions Between the Small GTPase ARF1 and Its ArfGAP ASAP1 at the Membrane Surface

2020 ◽  
Vol 118 (3) ◽  
pp. 17a
Author(s):  
Yue Zhang ◽  
Olivier Soubias ◽  
Andrew Byrd
Keyword(s):  
Membrane Surface ◽  
Small Gtpase ◽  
Nmr Studies ◽  
High Quality ◽  
Methyl Trosy ◽  
Methyl Trosy Nmr

Probing Slow Dynamics in High Molecular Weight Proteins by Methyl-TROSY NMR Spectroscopy:  Application to a 723-Residue Enzyme

2004 ◽  
Vol 126 (12) ◽  
pp. 3964-3973 ◽  
Author(s):  
Dmitry M. Korzhnev ◽  
Karin Kloiber ◽  
Voula Kanelis ◽  
Vitali Tugarinov ◽  
Lewis E. Kay
Keyword(s):  
Molecular Weight ◽  
Nmr Spectroscopy ◽  
High Molecular Weight ◽  
Slow Dynamics ◽  
Methyl Trosy ◽  
High Molecular Weight Proteins ◽  
Methyl Trosy Nmr

scholarly journals Oligomeric assembly regulating mitochondrial HtrA2 function as examined by methyl-TROSY NMR

2021 ◽  
Vol 118 (11) ◽  
pp. e2025022118
Author(s):  
Yuki Toyama ◽  
Robert W. Harkness ◽  
Tim Y. T. Lee ◽  
Jason T. Maynes ◽  
Lewis E. Kay
Keyword(s):  
Active Sites ◽  
Transverse Relaxation ◽  
Catalytic Center ◽  
Protective Enzyme ◽  
Biochemical Assays ◽  
Methyl Trosy ◽  
Oligomeric Assembly ◽  
Domain Reorientation ◽  
Methyl Trosy Nmr ◽  
Trimeric Structure

Human High temperature requirement A2 (HtrA2) is a mitochondrial protease chaperone that plays an important role in cellular proteostasis and in regulating cell-signaling events, with aberrant HtrA2 function leading to neurodegeneration and parkinsonian phenotypes. Structural studies of the enzyme have established a trimeric architecture, comprising three identical protomers in which the active sites of each protease domain are sequestered to form a catalytically inactive complex. The mechanism by which enzyme function is regulated is not well understood. Using methyl transverse relaxation optimized spectroscopy (TROSY)-based solution NMR in concert with biochemical assays, a functional HtrA2 oligomerization/binding cycle has been established. In the absence of substrates, HtrA2 exchanges between a heretofore unobserved hexameric conformation and the canonical trimeric structure, with the hexamer showing much weaker affinity toward substrates. Both structures are substrate inaccessible, explaining their low basal activity in the absence of the binding of activator peptide. The binding of the activator peptide to each of the protomers of the trimer occurs with positive cooperativity and induces intrasubunit domain reorientations to expose the catalytic center, leading to increased proteolytic activity. Our data paint a picture of HtrA2 as a finely tuned, stress-protective enzyme whose activity can be modulated both by oligomerization and domain reorientation, with basal levels of catalysis kept low to avoid proteolysis of nontarget proteins.

Bringing Dynamic Molecular Machines into Focus by Methyl-TROSY NMR

2014 ◽  
Vol 83 (1) ◽  
pp. 291-315 ◽  
Author(s):  
Rina Rosenzweig ◽  
Lewis E. Kay
Keyword(s):  
Molecular Machines ◽  
Methyl Trosy ◽  
Methyl Trosy Nmr

scholarly journals Ligand modulation of sidechain dynamics in a wild-type human GPCR

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Lindsay D Clark ◽  
Igor Dikiy ◽  
Karen Chapman ◽  
Karin EJ Rödström ◽  
James Aramini ◽  
...  
Keyword(s):  
Human Physiology ◽  
Structural Changes ◽  
Inverse Agonist ◽  
Methyl Groups ◽  
Signaling Proteins ◽  
Wild Type ◽  
Biophysical Studies ◽  
Methyl Trosy ◽  
Conformational Regulation ◽  
Methyl Trosy Nmr

GPCRs regulate all aspects of human physiology, and biophysical studies have deepened our understanding of GPCR conformational regulation by different ligands. Yet there is no experimental evidence for how sidechain dynamics control allosteric transitions between GPCR conformations. To address this deficit, we generated samples of a wild-type GPCR (A2AR) that are deuterated apart from 1H/13C NMR probes at isoleucine δ1 methyl groups, which facilitated 1H/13C methyl TROSY NMR measurements with opposing ligands. Our data indicate that low [Na+] is required to allow large agonist-induced structural changes in A2AR, and that patterns of sidechain dynamics substantially differ between agonist (NECA) and inverse agonist (ZM241385) bound receptors, with the inverse agonist suppressing fast ps-ns timescale motions at the G protein binding site. Our approach to GPCR NMR creates a framework for exploring how different regions of a receptor respond to different ligands or signaling proteins through modulation of fast ps-ns sidechain dynamics.

scholarly journals Propogation of the Allosteric Signal in Bacillus Stearothermophilus Phosphofructokinase Examined by Methyl-TROSY NMR

2015 ◽  
Vol 108 (2) ◽  
pp. 30a-31a
Author(s):  
Amy M. Whitaker ◽  
Mandar T. Naik ◽  
Gregory D. Reinhart
Keyword(s):  
Bacillus Stearothermophilus ◽  
Methyl Trosy ◽  
Methyl Trosy Nmr

Measurement of Active Site Ionization Equilibria in the 670 kDa Proteasome Core Particle Using Methyl-TROSY NMR

2013 ◽  
Vol 135 (25) ◽  
pp. 9259-9262 ◽  
Author(s):  
Algirdas Velyvis ◽  
Lewis E. Kay
Keyword(s):  
Active Site ◽  
Core Particle ◽  
Methyl Trosy ◽  
Ionization Equilibria ◽  
Methyl Trosy Nmr

scholarly journals Conformational dynamics of the HIV Vif protein complex

2018 ◽  
Author(s):  
K. A. Ball ◽  
L. M. Chan ◽  
D. J. Stanley ◽  
E. Tierney ◽  
S. Thapa ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Protein Complex ◽  
Conformational Dynamics ◽  
Intrinsically Disordered Protein ◽  
Md Simulations ◽  
Global Dynamics ◽  
Intrinsically Disordered ◽  
Ring E3 Ligase ◽  
Methyl Trosy ◽  
Methyl Trosy Nmr

AbstractHIV-1 viral infectivity factor (Vif) is an intrinsically disordered protein responsible for the ubiquitination of the APOBEC3 antiviral proteins. Vif folds when it binds the Cullin-RING E3 ligase CRL5 and the transcription cofactor CBF-β. A five-protein complex containing the substrate receptor (Vif, CBF-β, Elongin-B, Elongin-C) and Cullin5 (CUL5) has a published crystal structure, but dynamics of this VCBC-CUL5 complex have not been characterized. Here, we use Molecular Dynamics (MD) simulations and NMR to characterize the dynamics of the VCBC complex with and without CUL5 and APOBEC3 bound. Our simulations show that the VCBC complex undergoes global dynamics involving twisting and clamshell opening of the complex, while VCBC-CUL5 maintains a more static conformation, similar to the crystal structure. This observation from MD is supported by methyl-transverse relaxation optimized spectroscopy (methyl-TROSY) NMR data, which indicates that the entire VCBC complex without CUL5 is dynamic on the μs-ms timescale. Vif binds APOBEC3 to recruit it to the complex, and methyl-TROSY NMR shows that the VCBC complex is more conformationally restricted when bound to APOBEC3F, consistent with our MD simulations. Vif contains a flexible linker region located at the hinge of the VCBC complex, which changes conformation in conjuction with the global dynamics of the complex. Like other ubiquitin substrate receptors, VCBC can exist alone or in complex with CUL5 in cells. Accordingly, the VCBC complex could be a good target for therapeutics that would inhibit full assembly of the ubiquitination complex by stabilizing an alternate VCBC conformation.

scholarly journals Biphasic activation of β-arrestin 1 upon interaction with a GPCR revealed by methyl-TROSY NMR

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yutaro Shiraishi ◽  
Yutaka Kofuku ◽  
Takumi Ueda ◽  
Shubhi Pandey ◽  
Hemlata Dwivedi-Agnihotri ◽  
...  
Keyword(s):  
Nuclear Magnetic Resonance ◽  
Nmr Spectroscopy ◽  
Magnetic Resonance ◽  
Conformational Changes ◽  
G Protein Coupled Receptors ◽  
Binding Modes ◽  
Partial Activation ◽  
Methyl Trosy ◽  
G Protein Coupled ◽  
Methyl Trosy Nmr

Abstractβ-arrestins (βarrs) play multifaceted roles in the function of G protein-coupled receptors (GPCRs). βarrs typically interact with phosphorylated C-terminal tail (C tail) and transmembrane core (TM core) of GPCRs. However, the effects of the C tail- and TM core-mediated interactions on the conformational activation of βarrs have remained elusive. Here, we show the conformational changes for βarr activation upon the C tail- and TM core-mediated interactions with a prototypical GPCR by nuclear magnetic resonance (NMR) spectroscopy. Our NMR analyses demonstrated that while the C tail-mediated interaction alone induces partial activation, in which βarr exists in equilibrium between basal and activated conformations, the TM core- and the C tail-mediated interactions together completely shift the equilibrium toward the activated conformation. The conformation-selective antibody, Fab30, promotes partially activated βarr into the activated-like conformation. This plasticity of βarr conformation in complex with GPCRs engaged in different binding modes may explain the multifunctionality of βarrs.

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