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Direct assignment of 13 C solid-state NMR signals of TF o F 1 ATP synthase subunit c -ring in lipid membranes and its implication for the ring structure
Dec 03, 2017 - 4:52 AM - by nmrlearner
nmrlearner's Avatar Direct assignment of 13 C solid-state NMR signals of TF o F 1 ATP synthase subunit c -ring in lipid membranes and its implication for the ring structure

Abstract

FoF1-ATP synthase catalyzes ATP hydrolysis/synthesis coupled with a transmembrane H+ translocation in membranes. The Fo c-subunit ring plays a major role in this reaction. We have developed an assignment strategy for solid-state 13C NMR (ssNMR) signals of the Fo c-subunit ring of thermophilic Bacillus PS3 (TFo c-ring, 72 residues), carrying one of the basic folds of membrane proteins. In a ssNMR spectrum of uniformly 13C-labeled sample, the signal overlap has been a major bottleneck because most amino acid residues are hydrophobic. To overcome signal overlapping, we developed a method designated as COmplementary Sequential assignment with MInimum Labeling Ensemble (COSMILE). According to this method, we generated three kinds of reverse-labeled samples to suppress signal overlapping. To assign the carbon signals sequentially, two-dimensional Cα(i+1)??C?Cα(i) correlation and dipolar assisted rotational resonance (DARR) experiments were performed under magic-angle sample spinning. On the basis of inter- and intra-residue 13C??13C chemical shift correlations, 97% of Cα, 97% of Cβ and 92% of C? signals were assigned directly from the spectra. Secondary structure analysis predicted a hairpin fold of two helices with a central loop. The effects of saturated and unsaturated phosphatidylcholines on TFo c-ring structure were examined. The DARR spectra at 15*ms mixing time are essentially similar to each other in... [Read More]
0 Replies | 21 Views
Discovery puts the brakes on HIV's ability to infect: Researchers find ... - Science Daily
Dec 03, 2017 - 4:52 AM - by nmrlearner
nmrlearner's Avatar
Science Daily


Discovery puts the brakes on HIV's ability to infect: Researchers find ...
Science Daily
Researchers have discovered a 'brake' that interferes with HIV's development into an infectious agent. This mechanism prevents the capsid -- the protein shell covering the virus -- from forming.

and more »

Discovery puts the brakes on HIV's ability to infect: Researchers find ... - Science Daily
... [Read More]
0 Replies | 21 Views
Genetically encoded amino acids with tert -butyl and trimethylsilyl groups for site-selective studies of proteins by NMR spectroscopy
Dec 02, 2017 - 2:54 PM - by nmrlearner
nmrlearner's Avatar Genetically encoded amino acids with tert -butyl and trimethylsilyl groups for site-selective studies of proteins by NMR spectroscopy

Abstract

The amino acids 4-(tert-butyl)phenylalanine (Tbf) and 4-(trimethylsilyl)phenylalanine (TMSf), as well as a partially deuterated version of Tbf (dTbf), were chemically synthesized and site-specifically incorporated into different proteins, using an amber stop codon, suppressor tRNA and the broadband aminoacyl-tRNA synthetase originally evolved for the incorporation of p-cyano-phenylalanine. The 1H-NMR signals of the tert-butyl and TMS groups were compared to the 1H-NMR signal of tert-butyltyrosine (Tby) in protein systems with molecular weights ranging from 8 to 54*kDa. The 1H-NMR resonance of the TMS group appeared near 0*ppm in a spectral region with few protein resonances, facilitating the observation of signal changes in response to ligand binding. In all proteins, the R 2 relaxation rate of the tert-butyl group of Tbf was only little greater than that of Tby (less than two-fold). Deuteration of the phenyl ring of Tbf made only a relatively small difference. The effective T 2 relaxation time of the TMS signal was longer than 140*ms even in the 54*kDa system.



Source: Journal of Biomolecular NMR
0 Replies | 20 Views
Selective labeling and unlabeling strategies in protein solid-state NMR spectroscopy
Dec 02, 2017 - 2:54 PM - by nmrlearner
nmrlearner's Avatar Selective labeling and unlabeling strategies in protein solid-state NMR spectroscopy

Abstract

Selective isotope labeling is central in NMR experiments and often allows to push the limits on the systems investigated. It has the advantage to supply additional resolution by diminishing the number of signals in the spectra. This is particularly interesting when dealing with the large protein systems which are currently becoming accessible to solid-state NMR studies. Isotope labeled proteins for NMR experiments are most often expressed in E. coli systems, where bacteria are grown in minimal media supplemented with 15NH4Cl and 13C-glucose as sole source of nitrogen and carbon. For amino acids selective labeling or unlabeling, specific amino acids are supplemented in the minimal medium. The aim is that they will be incorporated in the protein by the bacteria. However, E. coli amino-acid anabolism and catabolism tend to interconnect different pathways, remnant of a subway system. These connections lead to inter conversion between amino acids, called scrambling. A thorough understanding of the involved pathways is thus important to obtain the desired labeling schemes, as not all combinations of amino acids are adapted. We present here a detailed overview of amino-acid metabolism in this context. Each amino-acid pathway is described in order to define accessible combinations for 13C or 15N specific labeling or unlabeling. Using as example the ABC transporter BmrA, a membrane protein of 600 residues, we demonstrate how these strategies can be applied. Indeed, even though there is no size limit in solid-state NMR, large (membrane) proteins are still a challenge due to heavy signal... [Read More]
0 Replies | 27 Views
Magic-angle-spinning NMR used to probe protein/microtubule assembly at atomic scale - Phys.Org
Dec 02, 2017 - 2:54 PM - by nmrlearner
nmrlearner's Avatar Magic-angle-spinning NMR used to probe protein/microtubule assembly at atomic scale - Phys.Org


Phys.Org


Magic-angle-spinning NMR used to probe protein/microtubule assembly at atomic scale
Phys.Org
"Just as we are always moving our arms and legs about, proteins are very dynamic. They do not stand still," Polenova says. "These motions are essential to their biological function, and NMR spectroscopy is the only technique that can record such ...

... [Read More]
0 Replies | 19 Views
[NMR paper] NMR analysis of the backbone dynamics of the small GTPase Rheb and its interaction with the regulatory protein FKBP38.
Dec 02, 2017 - 2:54 PM - by nmrlearner
nmrlearner's Avatar NMR analysis of the backbone dynamics of the small GTPase Rheb and its interaction with the regulatory protein FKBP38.

NMR analysis of the backbone dynamics of the small GTPase Rheb and its interaction with the regulatory protein FKBP38.

FEBS Lett. 2017 Nov 30;:

Authors: De Cicco M, Kiss L, Dames SA

Abstract
Ras homologue enriched in brain (Rheb) is a small GTPase that regulates mammalian/mechanistic target of rapamycin complex 1 (mTORC1) and, thereby, cell growth and metabolism. Cycling between the inactive GDP- and the active GTP-bound state modulates the backbone dynamics of a C-terminal truncated form, Rheb?CT, which is suggested to influence its interactions. Here, we investigated the interactions between Rheb?CT and the proposed Rheb-binding domain of the regulatory protein FKBP38. The weak interactions with the GTP-analogue- (GppNHp-) but not the GDP-bound state observed here, appear to accelerate the GDP to GTP exchange, but only very weakly compared to a genuine GEF. Thus, FKBP38 is most likely not a GEF, but a Rheb effector that may function in membrane targeting of Rheb. This article is protected by copyright. All rights reserved.


PMID: 29194576 [PubMed - as supplied by publisher]



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0 Replies | 25 Views
[NMR paper] In situ characterization of advanced glycation end products (AGEs) in collagen and model extracellular matrix by solid state NMR.
Dec 02, 2017 - 2:54 PM - by nmrlearner
nmrlearner's Avatar In situ characterization of advanced glycation end products (AGEs) in collagen and model extracellular matrix by solid state NMR.

In situ characterization of advanced glycation end products (AGEs) in collagen and model extracellular matrix by solid state NMR.

Chem Commun (Camb). 2017 Dec 01;:

Authors: Li R, Rajan R, Wong WCV, Reid DG, Duer MJ, Somovilla VJ, Martinez-Saez N, Bernardes GJL, Hayward R, Shanahan CM

Abstract
Non-enzymatic glycation of extracellular matrix with (U-13C5)-d-ribose-5-phosphate (R5P), enables in situ 2D ssNMR identification of many deleterious protein modifications and crosslinks, including previously unreported oxalamido and hemiaminal (CH3-CH(OH)NHR) substructures. Changes in charged residue proportions and distribution may be as important as crosslinking in provoking and understanding harmful tissue changes.


PMID: 29192920 [PubMed - as supplied by publisher]



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0 Replies | 23 Views
[NMR paper] Cracking the allosteric code of NMR chemical shifts.
Dec 01, 2017 - 9:24 PM - by nmrlearner
nmrlearner's Avatar Cracking the allosteric code of NMR chemical shifts.

Related Articles Cracking the allosteric code of NMR chemical shifts.

Proc Natl Acad Sci U S A. 2016 08 23;113(34):9407-9

Authors: VanSchouwen B, Melacini G

PMID: 27512035 [PubMed - indexed for MEDLINE]



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0 Replies | 19 Views
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