2022
DOI: 10.1021/jacs.2c09692
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Nanobody GPS by PCS: An Efficient New NMR Analysis Method for G Protein Coupled Receptors and Other Large Proteins

Abstract: NMR chemical shift changes can report on the functional dynamics of biomacromolecules in solution with sizes >1 MDa. However, their interpretation requires chemical shift assignments to individual nuclei, which for large molecules often can only be obtained by tedious point mutations that may interfere with function. We present here an efficient pseudocontact shift NMR method to assign biomacromolecules using bound antibodies tagged with lanthanoid DOTA chelators. The stability of the antibody allows positioni… Show more

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Cited by 11 publications
(7 citation statements)
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“…Taking advantage of the identified glutamine nitrogen scrambling routes in insect cells, any protein can be selectively labeled in alanine, aspartate, glutamate, and glutamine from the input of labeled glutamine. We show here this particular type of selective labeling for the β 1 -adrenergic G protein-coupled receptor that we used in previous NMR studies ( 28 , 29 , 30 , 31 ). β 1 AR was expressed in 4 L of insect cell growth medium supplemented with 0.25 g/l synthesized 15 N 2 -glutamine-α,β,β-d 3 yielding 0.8 mg/l purified, detergent-solubilized receptor.…”
Section: Resultsmentioning
confidence: 96%
See 1 more Smart Citation
“…Taking advantage of the identified glutamine nitrogen scrambling routes in insect cells, any protein can be selectively labeled in alanine, aspartate, glutamate, and glutamine from the input of labeled glutamine. We show here this particular type of selective labeling for the β 1 -adrenergic G protein-coupled receptor that we used in previous NMR studies ( 28 , 29 , 30 , 31 ). β 1 AR was expressed in 4 L of insect cell growth medium supplemented with 0.25 g/l synthesized 15 N 2 -glutamine-α,β,β-d 3 yielding 0.8 mg/l purified, detergent-solubilized receptor.…”
Section: Resultsmentioning
confidence: 96%
“…The good resolution indicates that this very simple and selective labeling approach may be useful for obtaining assignments and other information for a considerable part of amino acids in large proteins. The sparseness of the spectrum is also very well suited for the recently developed nanobody GPS–PCS spectral assignment method ( 31 ).
Figure 4 1 H- 15 N TROSY NMR spectrum of selectively isotope-labeled β 1 AR using 15 N 2 -glutamine-α,β,β-d 3 .
…”
Section: Resultsmentioning
confidence: 99%
“…Non-native metal substitution with paramagnetic systems [131,132], as well as the use of paramagnetic tags attached to the protein post-expression [124,[133][134][135][136], has also allowed for the characterization of structure and dynamics in several biomolecular systems. The reader is referred to [6,8,9,134,[137][138][139][140].…”
Section: Whymentioning
confidence: 99%
“…As a consequence, complementary information are obtained from C α to C’ and C’ to C α transfer pathways (Machonkin et al 2002 ; Bertini et al 2005b ), contributing to characterize the first coordination sphere of a metal center. In the absence of magnetic anisotropies of the metal ions, when no structural information can be obtained from chemical shifts (Pintacuda et al 2007 ; Zhu et al 2023 ; Wu et al 2022 ; Herath et al 2021 ; Muntener et al 2022 ; Parker et al 2020 ), the proximity of the metal center can be monitored only by mapping the paramagnetic relaxation of nuclear spins nearby and each individual spin system has a different behaviour, depending on its topology vis-à-vis the metal center (Trindade et al 2021a ).…”
Section: Introductionmentioning
confidence: 99%