High resolution 13C-detected solid-state NMR spectroscopy of a deuterated protein

Tang, Ming; Comellas, Gemma; Mueller, Leonard J.; Rienstra, Chad M.

doi:10.1007/s10858-010-9442-8

Cited by 23 publications

(14 citation statements)

References 36 publications

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“…Furthermore, deuteration at a carbon site induces an isotope effect that manifests as a chemical shift difference relative to that of proton-attached carbon (Tang et al 2010). In our case of fractional (~70%) deuteration, broadening of the 13 C line widths is expected to arise from the mixed contributions of 1 H and 2 H attached to each carbon site.…”

Section: Resultsmentioning

confidence: 99%

High resolution solid-state NMR spectroscopy of the Yersinia pestis outer membrane protein Ail in lipid membranes

et al. 2017

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The outer membrane protein Ail (Adhesion invasion locus) is one of the most abundant proteins on the cell surface of Yersinia pestis during human infection. Its functions are expressed through interactions with a variety of human host proteins, and are essential for microbial virulence. Structures of Ail have been determined by X-ray diffraction and solution NMR spectroscopy, but those samples contained detergents that interfere with functionality, thus, precluding analysis of the structural basis for Ail’s biological activity. Here, we demonstrate that high-resolution solid-state NMR spectra can be obtained from samples of Ail in detergent-free phospholipid liposomes, prepared with a lipid to protein molar ratio of 100. The spectra, obtained with 13C or 1H detection, have very narrow line widths (0.40–0.60 ppm for 13C, 0.11–0.15 ppm for 1H, and 0.46–0.64 ppm for 15N) that are consistent with a high level of sample homogeneity. The spectra enable resonance assignments to be obtained for N, CO, CA and CB atomic sites from 75 out of 156 residues in the sequence of Ail, including 80% of the transmembrane region. The 1H-detected solid-state NMR 1H/15N correlation spectra obtained for Ail in liposomes compare very favorably with the solution NMR 1H/15N TROSY spectra obtained for Ail in nanodiscs prepared with a similar lipid to protein molar ratio. These results set the stage for studies of the molecular basis of the functional interactions of Ail with its protein partners from human host cells, as well as the development of drugs targeting Ail.

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Section: Resultsmentioning

confidence: 99%

High resolution solid-state NMR spectroscopy of the Yersinia pestis outer membrane protein Ail in lipid membranes

et al. 2017

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“…Table 3 compares the values of the n ΔC(D) obtained for to those previously determined for solution-state α-synuclein (Maltsev et al 2012), Human carbonic anhydrase (Venters et al 1996), and SH2 C-terminal domain (Gardner et al 1997). In order to determine if there is any clear distinction between the isotope effect in the solution-state and the solidstate, we also calculate the n ΔC(D) using previously published data for Ubiquitin (Penzel et al 2015;Schubert et al 2006) and GB1 (Tang et al 2010). In fact, one finds that the values for the different proteins cannot be said to be different at the 95% confidence level (with the exception that 3 ΔC(D) for HET-s and Human carbonic anhydrase are significantly different).…”

Section: Resultsmentioning

confidence: 99%

“…Calculated from deuterated Cα and Cβ shifts reported by (Penzel et al 2015) and protonated shifts by (Schubert et al 2006) e Calculated from deuterium isotope effect on aliphatic carbons reported by (Tang et al 2010) f Note that one standard deviation was originally reported, which is multiplied by two to give the 95% confidence interval here…”

Section: Resultsmentioning

confidence: 99%

Partially-deuterated samples of HET-s(218–289) fibrils: assignment and deuterium isotope effect

et al. 2017

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Fast magic-angle spinning and partial sample deuteration allows direct detection of H in solid-state NMR, yielding significant gains in mass sensitivity. In order to further analyze the spectra,H detection requires assignment of the H resonances. In this work, resonance assignments of backbone H and Hα are presented for HET-s(218-289) fibrils, based on the existing assignment of Cα, Cβ, C', and N resonances. The samples used are partially deuterated for higher spectral resolution, and the shifts in resonance frequencies of Cα and Cβ due to the deuterium isotope effect are investigated. It is shown that the deuterium isotope effect can be estimated and used for assigning resonances of deuterated samples in solid-state NMR, based on known resonances of the protonated protein.

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“…Stronger 1 H decoupling lengthens the T 2 and decreases the homogeneous linewidths 82,83 . As noted above, 13 C- 13 C J-coupling and residual dipolar coupling also contribute a fixed amount to the homogeneous linewidths of these uniformly 13 C-labeled residues.…”

Section: Discussionmentioning

confidence: 99%

Conformational Disorder of Membrane Peptides Investigated from Solid-State NMR Line Widths and Line Shapes

Hong

2011

J. Phys. Chem. B

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A challenge in the application of solid-state NMR spectroscopy to membrane peptides and proteins is the relatively broad linewidths compared to solution NMR spectra. To understand the linewidth contributions to membrane protein NMR spectra, we have measured the inhomogeneous and homogeneous linewidths of several well-studied membrane peptides under immobilized conditions. 13C T2 relaxation times of uniformly 13C-labeled residues show that the homogeneous linewidths of the peptides are comparable to those of crystalline model compounds under identical 1H decoupling and magic-angle-spinning conditions, indicating that the homogeneous linewidths are determined by conformation-independent factors, including residual dipolar coupling, J coupling and intrinsic T2 relaxation. However, the membrane peptides exhibit larger apparent linewidths than the crystalline compounds, indicating conformational disorder. A cationic cell-penetrating peptide, the human immunodeficiency virus TAT, exhibits the largest apparent linewidths, which are about 5-fold larger than the homogeneous linewidths, while the transmembrane helix of the influenza M2 peptide and the β-hairpin antimicrobial peptide PG-1 show moderately larger apparent linewidths than the crystalline compounds. These results are consistent with the random coil nature of the TAT peptide, which contrasts with the intramolecularly hydrogen-bonded M2 and PG-1. Cross peak lineshapes of 2D double-quantum correlation spectra show that the conformational disorder can occur at the residue level and can result from three origins: lipid-peptide interaction, intrinsic conformational disorder encoded in the amino acid sequence, and sidechain rotameric averaging. A particularly important lipid-peptide interaction for cationic membrane peptides is guanidinium-phosphate ion pair interaction. Thus, NMR linewidths and lineshapes are useful for understanding the conformational disorder of membrane peptides and proteins.

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High resolution 13C-detected solid-state NMR spectroscopy of a deuterated protein

Cited by 23 publications

References 36 publications

High resolution solid-state NMR spectroscopy of the Yersinia pestis outer membrane protein Ail in lipid membranes

High resolution solid-state NMR spectroscopy of the Yersinia pestis outer membrane protein Ail in lipid membranes

Partially-deuterated samples of HET-s(218–289) fibrils: assignment and deuterium isotope effect

Conformational Disorder of Membrane Peptides Investigated from Solid-State NMR Line Widths and Line Shapes

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