scholarly journals Detection of Selenium/Sulphur substitution in the heterologous expression of Thioredoxin isoform 1 (Trx1) from Saccharomyces cerevisiae.

2021 ◽  
Author(s):  
Humberto Antunes de Almeida Filho ◽  
Fabio C. L. Almeida
Keyword(s):  
Chemical Shift ◽  
Active Site ◽  
Quantum Coherence ◽  
Conformational Dynamics ◽  
Broad Band ◽  
Multiple Quantum ◽  
Spectral Window ◽  
Maldi Tof ◽  
Peptide Mass ◽  
Thioredoxin 1

Thioredoxins are ubiquous proteins with 2 cysteines at the active site. The isoform 1 of Thioredoxin from Saccharomyces cerevisae (Trx1) has six sulphur aminoacids, two cysteines and four methionines. In this work we performed the replacement of cysteines by selenocysteines by growth of a transformed celular expression vector E. coli BL21-DE3 in selenocysteine containing culture medium. The Maldi-TOF spectra of Seleno/Sulphur substituted Trx1 revealed six component peaks with 46-48 Da range between them, that is the isotopic Seleno-Sulfur difference, showing the replacement of the Cysteines and Methionines to Selenocysteines and Selenomethionines. The Maldi-TOF spectra of the peptides derived from Trypsin digestion of the purified Thioredoxin (peptide mass fingerprint) show Selenocysteine and Selenomethionine containing peptides. Therefore we are demonstrating that cystein can be replaced by selenocystein and be metabolically converted to selenomethionine during Trx1 heterologous translation. Furthermore, the Maldi-TOF spectra are showing the presence of the most abundant isotopes of selenium inserted in the peptides containing cysteine and methionine, derived from the Trx1 digestion. The one dimensional 77 Se − 1 H heteronuclear multiple quantum coherence NMR spectroscopy (1D-HMQC) for reduced Seleno substituted Trx1 (Se-Trx1), revealed three ressonance lines for 1 H β 1 from Selenocysteines 30 and 33, between 1.6 and 2,0 ppm. The bidimensional HMQC spectra (2D-HMQC) of the reduced Se Trx1 show the 77 Se ressonance signal in 178 ppm, coupled with 1 H β 1 and 1 H β 2 lines between 2.1 and 1.8 ppm. The 1D-HMQC for oxidized Trx1 revealed the only one broad resonance in 2.6 ppm probably relative to the 1 H β 1 protons. The 2D-HMQC spectrum of oxidized protein shows a higher chemical shift of selenocysteine 77 Se (832 ppm) if compared to reduced state (178 ppm). Together these data are showing that the protocol of Se − S substitution developed here is a efficient method to label the active site of Thioredoxin 1 with a broad band chemical shift atom 77 Se. Furthermore the large spectral window of the 77 Se NMR detected between reduced and oxidized states of the Thioredoxin 1 shows that this atom is an excellent probe for accessing oxidative states and probably the conformational dynamics of the active site of the Se-Trx1.

scholarly journals Lactate imaging with Hadamard-encoded slice-selective multiple quantum coherence chemical-shift imaging

2008 ◽  
Vol 60 (2) ◽  
pp. 299-305 ◽  
Author(s):  
Stephen Pickup ◽  
Seung-Cheol Lee ◽  
Anthony Mancuso ◽  
Jerry D. Glickson
Keyword(s):  
Chemical Shift ◽  
Quantum Coherence ◽  
Chemical Shift Imaging ◽  
Multiple Quantum ◽  
Multiple Quantum Coherence

scholarly journals Proton-Detected Solid-State NMR Spectroscopy of Spin-1/2 Nuclei with Large Chemical Shift Anisotropy

2021 ◽  
Author(s):  
Amrit Venkatesh ◽  
Frédéric Perras ◽  
Aaron Rossini
Keyword(s):  
Chemical Shift ◽  
Quantum Coherence ◽  
Double Resonance ◽  
Chemical Shift Anisotropy ◽  
Pulse Sequences ◽  
Constant Time ◽  
Magic Angle ◽  
Multiple Quantum ◽  
Transverse Relaxation ◽  
Adiabatic Pulses

<p>Constant-time (CT) dipolar heteronuclear multiple quantum coherence (D-HMQC) has previously been demonstrated as a method for proton detection of high-resolution wideline NMR spectra of spin-1/2 nuclei with large chemical shift anisotropy (CSA). However, <sup>1</sup>H transverse relaxation and <i>t</i><sub>1</sub>-noise often reduce the sensitivity of D-HMQC experiments, preventing the theoretical gains in sensitivity provided by <sup>1</sup>H detection from being realized. Here we demonstrate a series of improved pulse sequences for <sup>1</sup>H detection of spin-1/2 nuclei under fast MAS, with <sup>195</sup>Pt SSNMR experiments on cisplatin as an example. First, a new <i>t</i><sub>1</sub>-incrementation protocol for D-HMQC dubbed Arbitrary Indirect Dwell (AID) is demonstrated. AID allows the use of arbitrary, rotor asynchronous <i>t</i><sub>1</sub>-increments, but removes the constant time period from CT D-HMQC, resulting in improved sensitivity by reducing transverse relaxation losses. Next, we show that short high-power adiabatic pulses (SHAPs), which efficiently invert broad MAS sideband manifolds, can be effectively incorporated into <sup>1</sup>H detected symmetry-based resonance echo double resonance (S-REDOR) and <i>t</i><sub>1</sub>-noise eliminated D-HMQC experiments. The S-REDOR experiments with SHAPs provide approximately double the dipolar dephasing, as compared to experiments with rectangular inversion pulses. We lastly show that sensitivity and resolution can be further enhanced with the use of swept excitation pulses as well as adiabatic magic angle turning.</p>

scholarly journals Dual allosteric activation mechanisms in monomeric human glucokinase

2015 ◽  
Vol 112 (37) ◽  
pp. 11553-11558 ◽  
Author(s):  
A. Carl Whittington ◽  
Mioara Larion ◽  
Joseph M. Bowler ◽  
Kristen M. Ramsey ◽  
Rafael Brüschweiler ◽  
...  
Keyword(s):  
Active Site ◽  
Quantum Coherence ◽  
Limited Proteolysis ◽  
Enzyme Activation ◽  
Hyperinsulinemic Hypoglycemia ◽  
General Strategy ◽  
Multiple Quantum ◽  
Glucose Binding ◽  
Activation Mechanisms ◽  
Hmqc Spectrum

Cooperativity in human glucokinase (GCK), the body’s primary glucose sensor and a major determinant of glucose homeostatic diseases, is fundamentally different from textbook models of allostery because GCK is monomeric and contains only one glucose-binding site. Prior work has demonstrated that millisecond timescale order-disorder transitions within the enzyme’s small domain govern cooperativity. Here, using limited proteolysis, we map the site of disorder in unliganded GCK to a 30-residue active-site loop that closes upon glucose binding. Positional randomization of the loop, coupled with genetic selection in a glucokinase-deficient bacterium, uncovers a hyperactive GCK variant with substantially reduced cooperativity. Biochemical and structural analysis of this loop variant and GCK variants associated with hyperinsulinemic hypoglycemia reveal two distinct mechanisms of enzyme activation. In α-type activation, glucose affinity is increased, the proteolytic susceptibility of the active site loop is suppressed and the 1H-13C heteronuclear multiple quantum coherence (HMQC) spectrum of 13C-Ile–labeled enzyme resembles the glucose-bound state. In β-type activation, glucose affinity is largely unchanged, proteolytic susceptibility of the loop is enhanced, and the 1H-13C HMQC spectrum reveals no perturbation in ensemble structure. Leveraging both activation mechanisms, we engineer a fully noncooperative GCK variant, whose functional properties are indistinguishable from other hexokinase isozymes, and which displays a 100-fold increase in catalytic efficiency over wild-type GCK. This work elucidates specific structural features responsible for generating allostery in a monomeric enzyme and suggests a general strategy for engineering cooperativity into proteins that lack the structural framework typical of traditional allosteric systems.

Use of1H−15N Heteronuclear Multiple-Quantum Coherence NMR Spectroscopy To Study the Active Site of Aspartate Aminotransferase†

Biochemistry ◽  
1997 ◽  
Vol 36 (3) ◽  
pp. 615-625 ◽  
Author(s):  
Emilia T. Mollova ◽  
David E. Metzler ◽  
Agustin Kintanar ◽  
Hiroyuki Kagamiyama ◽  
Hideyuki Hayashi ◽  
...  
Keyword(s):  
Nmr Spectroscopy ◽  
Active Site ◽  
Aspartate Aminotransferase ◽  
Quantum Coherence ◽  
Multiple Quantum ◽  
Heteronuclear Multiple Quantum Coherence ◽  
Multiple Quantum Coherence

scholarly journals Proton-Detected Solid-State NMR Spectroscopy of Spin-1/2 Nuclei with Large Chemical Shift Anisotropy

2021 ◽  
Author(s):  
Amrit Venkatesh ◽  
Frédéric Perras ◽  
Aaron Rossini
Keyword(s):  
Chemical Shift ◽  
Quantum Coherence ◽  
Double Resonance ◽  
Chemical Shift Anisotropy ◽  
Pulse Sequences ◽  
Constant Time ◽  
Magic Angle ◽  
Multiple Quantum ◽  
Transverse Relaxation ◽  
Adiabatic Pulses

<p>Constant-time (CT) dipolar heteronuclear multiple quantum coherence (D-HMQC) has previously been demonstrated as a method for proton detection of high-resolution wideline NMR spectra of spin-1/2 nuclei with large chemical shift anisotropy (CSA). However, <sup>1</sup>H transverse relaxation and <i>t</i><sub>1</sub>-noise often reduce the sensitivity of D-HMQC experiments, preventing the theoretical gains in sensitivity provided by <sup>1</sup>H detection from being realized. Here we demonstrate a series of improved pulse sequences for <sup>1</sup>H detection of spin-1/2 nuclei under fast MAS, with <sup>195</sup>Pt SSNMR experiments on cisplatin as an example. First, a new <i>t</i><sub>1</sub>-incrementation protocol for D-HMQC dubbed Arbitrary Indirect Dwell (AID) is demonstrated. AID allows the use of arbitrary, rotor asynchronous <i>t</i><sub>1</sub>-increments, but removes the constant time period from CT D-HMQC, resulting in improved sensitivity by reducing transverse relaxation losses. Next, we show that short high-power adiabatic pulses (SHAPs), which efficiently invert broad MAS sideband manifolds, can be effectively incorporated into <sup>1</sup>H detected symmetry-based resonance echo double resonance (S-REDOR) and <i>t</i><sub>1</sub>-noise eliminated D-HMQC experiments. The S-REDOR experiments with SHAPs provide approximately double the dipolar dephasing, as compared to experiments with rectangular inversion pulses. We lastly show that sensitivity and resolution can be further enhanced with the use of swept excitation pulses as well as adiabatic magic angle turning.</p>

scholarly journals Crystal Structure of Escherichia coli Agmatinase: Catalytic Mechanism and Residues Relevant for Substrate Specificity

2021 ◽  
Vol 22 (9) ◽  
pp. 4769
Author(s):  
Pablo Maturana ◽  
María S. Orellana ◽  
Sixto M. Herrera ◽  
Ignacio Martínez ◽  
Maximiliano Figueroa ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Conformational Dynamics ◽  
High Specificity ◽  
Arginine Decarboxylase ◽  
Space Groups ◽  
X Ray Crystallography ◽  
High Dynamics ◽  
Dynamics Simulations

Agmatine is the product of the decarboxylation of L-arginine by the enzyme arginine decarboxylase. This amine has been attributed to neurotransmitter functions, anticonvulsant, anti-neurotoxic, and antidepressant in mammals and is a potential therapeutic agent for diseases such as Alzheimer’s, Parkinson’s, and cancer. Agmatinase enzyme hydrolyze agmatine into urea and putrescine, which belong to one of the pathways producing polyamines, essential for cell proliferation. Agmatinase from Escherichia coli (EcAGM) has been widely studied and kinetically characterized, described as highly specific for agmatine. In this study, we analyze the amino acids involved in the high specificity of EcAGM, performing a series of mutations in two loops critical to the active-site entrance. Two structures in different space groups were solved by X-ray crystallography, one at low resolution (3.2 Å), including a guanidine group; and other at high resolution (1.8 Å) which presents urea and agmatine in the active site. These structures made it possible to understand the interface interactions between subunits that allow the hexameric state and postulate a catalytic mechanism according to the Mn2+ and urea/guanidine binding site. Molecular dynamics simulations evaluated the conformational dynamics of EcAGM and residues participating in non-binding interactions. Simulations showed the high dynamics of loops of the active site entrance and evidenced the relevance of Trp68, located in the adjacent subunit, to stabilize the amino group of agmatine by cation-pi interaction. These results allow to have a structural view of the best-kinetic characterized agmatinase in literature up to now.

scholarly journals The Active Site of a Prototypical “Rigid” Drug Target is Marked by Extensive Conformational Dynamics

2020 ◽  
Vol 59 (51) ◽  
pp. 22916-22921
Author(s):  
Himanshu Singh ◽  
Chandan K. Das ◽  
Suresh K. Vasa ◽  
Kristof Grohe ◽  
Lars V. Schäfer ◽  
...  
Keyword(s):  
Active Site ◽  
Drug Target ◽  
Conformational Dynamics

NMR studies on Fraser fir Abies fraseri (Pursh) Poir. lignins

Holzforschung ◽  
2005 ◽  
Vol 59 (5) ◽  
pp. 488-496 ◽  
Author(s):  
Mikhail Yu. Balakshin ◽  
Ewellyn A. Capanema ◽  
Barry Goldfarb ◽  
John Frampton ◽  
John F. Kadla
Keyword(s):  
Quantum Coherence ◽  
Lignin Content ◽  
Lower Amount ◽  
Multiple Quantum ◽  
Normal Wood ◽  
Nmr Studies ◽  
Oh Groups ◽  
Fraser Fir ◽  
Heteronuclear Multiple Quantum Coherence ◽  
Balsam Woolly Adelgid

Abstract The composition of mature, juvenile uninfested and juvenile infested Fraser fir wood (Rotholz) and the structures of lignins isolated from these woods were elucidated to verify differences between juvenile and mature wood and the effect of balsam woolly adelgid (BWA) infestation. Milled wood lignin (MWL) isolated from mature, juvenile and Rotholz wood were comprehensively characterized using heteronuclear multiple quantum coherence (HMQC) and quantitative 13C NMR techniques. The Rotholz wood was found to have ∼13% higher lignin content and more than five-fold the amount of galactans than that of the uninfested wood. Rotholz lignin possesses higher amounts of p-hydroxyphenyl units and aliphatic OH groups and a lower amount of alkyl-O-alkyl linkages and dibenzodioxocin moieties. The degree of condensation of the Rotholz lignin was rather similar to that of normal wood. Only small differences in the structure of mature and juvenile wood components were found.

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