BEST and SOFAST experiments for resonance assignment of histidine and tyrosine side chains in 13C/15N labeled proteins

Christou, Nina Eleni; Brutscher, Bernhard

doi:10.1007/s10858-018-0216-z

“…The relaxation rates were measured at four different temperatures: 15, 25, 35 and 45 °C. The magnetization decay was sampled at (0, 100, 200, 400, 600, 800, 1,100, 1,500 and 1,900) milliseconds (ms) for longitudinal and at (10,30,50,70,90,130,170, 210 and 250) ms for transverse relaxation. Technical replicates of one or two of these delays were acquired to estimate the uncertainty on the relaxation rates using a Monte Carlo approach.…”

Section: N Relaxation Measurements Of Jip1-sh3mentioning

confidence: 99%

“…The 13 Cε-1 Hε tyrosine resonances were assigned at 45 °C by acquiring a two-dimensional (2D) plane of a BEST-TROSY intra-residue HNCACB experiment 44 , an aromatic BEST constant-time 1 H- 13 C HSQC experiment 50 and a (Hβ)Cβ(CγCδCε)Hε experiment 51 linking the Cβ chemical shifts directly to the Hε chemical shifts. The spectra were manually peak-picked using NMRFAM-Sparky 45 and 13 Cε- 1 Hε tyrosine resonances were assigned manually (Supplementary Fig.…”

Section: Tyrosine Assignments and 13 C Cpmg Relaxation Dispersion Of ...mentioning

confidence: 99%

Visualizing protein breathing motions associated with aromatic ring flipping

Pérez

¹

,

Ielasi

²

,

Bessa

³

et al. 2022

Nature

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Aromatic residues cluster in the core of folded proteins, where they stabilize the structure through multiple interactions. Nuclear magnetic resonance (NMR) studies in the 1970s showed that aromatic side chains can undergo ring flips—that is, 180° rotations—despite their role in maintaining the protein fold1–3. It was suggested that large-scale ‘breathing’ motions of the surrounding protein environment would be necessary to accommodate these ring flipping events1. However, the structural details of these motions have remained unclear. Here we uncover the structural rearrangements that accompany ring flipping of a buried tyrosine residue in an SH3 domain. Using NMR, we show that the tyrosine side chain flips to a low-populated, minor state and, through a proteome-wide sequence analysis, we design mutants that stabilize this state, which allows us to capture its high-resolution structure by X-ray crystallography. A void volume is generated around the tyrosine ring during the structural transition between the major and minor state, and this allows fast flipping to take place. Our results provide structural insights into the protein breathing motions that are associated with ring flipping. More generally, our study has implications for protein design and structure prediction by showing how the local protein environment influences amino acid side chain conformations and vice versa.

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“…The chromophore contains 5 C-H pairs (Figure 8c) in the tyrosine (Y) part of the chromophore that is made up from the A66-Y67-G68 tripeptide in the rsFolder sequence. The 5 corresponding correlation peaks detected in the spectrum of Figure 8d were identified and unambiguously assigned by a set of 2D correlation spectra shown in Figure S9, recorded with pulse sequences that were specifically tailored for tyrosine side chains (31). These NMR data allowed us to assign all 1 H and 13 C resonances (except for CO) of the tyrosine moiety of the chromophore ( Table 1).…”

Section: Fold Stability and Water Accessibilitymentioning

confidence: 88%

“…Additional side chain assignments of aromatic tyrosine and histidine residues, including the tyrosine moiety of the p-HBI chromophore, was achieved using a series of specifically tailored 2D correlation experiments as described recently (31). Partial methyl ( 1 H, 13 C) assignments were obtained by a combination of 3D CcoNH-TOCSY, 3D HccoNH-TOCSY, and 3D H met C met C experiments, complemented by amino-acid-edited SOFAST 1 H- 13 C correlation spectra (32,33) 15 N relaxation experiments (T 1 , T 2 , HETNOE) were performed using standard pulse schemes implemented in NMRlib (27).…”

Section: Nmr Measurementsmentioning

confidence: 99%

NMR Reveals Light-Induced Changes in the Dynamics of a Photoswitchable Fluorescent Protein

Christou

¹

,

Ayala

²

,

Giandoreggio-Barranco

³

et al. 2019

Biophysical Journal

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The availability of fluorescent proteins with distinct phototransformation properties is crucial for a wide range of applications in advanced fluorescence microscopy and biotechnology. To rationally design new variants optimized for specific applications, a detailed understanding of the mechanistic features underlying phototransformation is essential. At present, little is known about the conformational dynamics of fluorescent proteins at physiological temperature, and how these dynamics contribute to the observed phototransformation properties. Here, we apply high-resolution NMR spectroscopy in solution combined with in-situ sample illumination at different wavelengths to investigate the conformational dynamics of rsFolder, a GFP-derived protein that can be reversibly switched between a green fluorescent state and a non-fluorescent state. Our results add a dynamic view to the static structures obtained by X-ray crystallography. Including NMR into the analytical toolbox used for fluorescent protein research provides new opportunities for investigating the effect of mutations or changes in the environmental conditions on the conformational dynamics of phototransformable fluorescent proteins, and their correlation with the observed photochemical and photophysical properties.

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“…Additional side chain assignments of aromatic tyrosine and histidine residues, including the tyrosine moiety of the p-HBI chromophore, was achieved using a series of specifically tailored 2D correlation experiments as described recently (31). Partial methyl ( 1 H, 13 C) assignments were obtained by a combination of 3D CcoNH-TOCSY, 3D HccoNH-TOCSY, and 3D H met C met C experiments, complemented by amino-acid-edited SOFAST 1 H-13 C correlation spectra (32,33) 15 N relaxation experiments (T 1 , T 2 , HETNOE) were performed using standard pulse schemes implemented in NMRlib (27).…”

Section: Nmr Measurementsmentioning

confidence: 99%

NMR reveals light-induced changes in the dynamics of a photoswitchable fluorescent protein

Christou

¹

,

Ayala

²

,

Giandoreggio-Barranco

³

et al. 2019

Preprint

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The availability of fluorescent proteins with distinct phototransformation properties is crucial for a wide range of applications in advanced fluorescence microscopy and biotechnology. To rationally design new variants optimized for specific applications, a detailed understanding of the mechanistic features underlying phototransformation is essential. At present, little is known about the conformational dynamics of fluorescent proteins at physiological temperature, and how these dynamics contribute to the observed phototransformation properties. Here, we apply high-resolution NMR spectroscopy in solution combined with in-situ sample illumination at different wavelengths to investigate the conformational dynamics of rsFolder, a GFP-derived protein that can be reversibly switched between a green fluorescent state and a non-fluorescent state. Our results add a dynamic view to the static structures obtained by X-ray crystallography. Including NMR into the analytical toolbox used for fluorescent protein research provides new opportunities for investigating the effect of mutations or changes in the environmental conditions on the conformational dynamics of phototransformable fluorescent proteins, and their correlation with the observed photochemical and photophysical properties.Significance Photo-transformable Fluorescent Proteins (PTFPs) are essential tools for superresolution (SR) microscopy. In practical applications, however, researchers often encounter problems when using PTFPs in a particular cellular context, because the environmental conditions (pH, temperature, redox potential, oxygen level, viscosity, …) affect their brightness, photostability, phototransformation kinetics, etc. Rational fluorescent protein engineering exploits the mechanistic information available from structural studies, mainly Xray crystallography, in order to design new PTFP variants with improved properties for particular applications. Here we apply NMR spectroscopy in solution to investigate the lightinduced changes in conformational dynamics of rsFolder, a reversibly switchable fluorescent protein. The dynamic view offered by NMR highlights protein regions that comprise potentially interesting mutation points for future mutagenesis campaigns.

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BEST and SOFAST experiments for resonance assignment of histidine and tyrosine side chains in 13C/15N labeled proteins

Cited by 8 publications

References 34 publications

Visualizing protein breathing motions associated with aromatic ring flipping

Visualizing protein breathing motions associated with aromatic ring flipping

NMR Reveals Light-Induced Changes in the Dynamics of a Photoswitchable Fluorescent Protein

NMR reveals light-induced changes in the dynamics of a photoswitchable fluorescent protein

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