Quantification of protein secondary structure by 13C solid-state NMR

Andrade, Fabiana Diuk; Forato, L. A.; Filho, Rubens Bernardes; Colnago, Luiz Alberto

doi:10.1007/s00216-016-9484-1

Cited by 4 publications

(2 citation statements)

References 22 publications

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“…S1), which provides similar CP excitation of the C@O groups if the same cross-polarization time is used. 52 These features allow us to relate the relative C@O intensities of the 13 C CPMAS spectra to probe changes in the secondary structure of the Gli and Glu-glycerol blends, keeping in mind the limitations due to the non-quantitative nature of the 13 C CPMAS technique.…”

Section: Resultsmentioning

confidence: 99%

An insight into molecular motions and phase composition of gliadin/glutenin glycerol blends studied by ¹³C solid‐state and ¹H time‐domain NMR

Andrade

Newson

Bernardinelli

et al. 2018

J Polym Sci B Polym Phys

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Monitoring of the molecular motions and secondary structures of gliadin (Gli) and glutenin (Glu) in blends with 10, 20, 30, and 40% glycerol was performed by solid‐state (SS) and time domain (TD) NMR spectroscopy. Increasing the glycerol content increased the relative amount of β‐sheets and disordered structures, while decreasing α‐helices in Gli/Glu–glycerol blends studied by 13C CPMAS NMR. For ≥20% glycerol samples, the protein side‐chain mobility increased similarly for Gli and Glu. A higher proportion of α‐helices versus β‐sheets was found in Gli‐glycerol blends compared with Glu–glycerol blends. Glycerol acted as “immobilized” in 10–20% glycerol Gli samples and was found mainly “free” in 30 and 40% glycerol Gli/Glu samples. During temperature experiments, 30 and 40% glycerol amounts impacted the dynamic molecular behavior of the Gli and Glu proteins differently than lipids, as observed by TD‐NMR. The combination of TD‐NMR together with SS‐NMR showed details of the dynamic molecular variations in Gli/Glu protein structure and are promising techniques to monitor the molecular dynamics of plasticized proteins. © 2018 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2018, 56, 739–750

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Section: Resultsmentioning

confidence: 99%

An insight into molecular motions and phase composition of gliadin/glutenin glycerol blends studied by ¹³C solid‐state and ¹H time‐domain NMR

Andrade

Newson

Bernardinelli

et al. 2018

J Polym Sci B Polym Phys

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“…As expected, dendritic cells stimulating assay in vitro and adjuvant evaluation assay in BALB/c mice using OVA demonstrated that both TFPR-hd and TFPR-ta have similar activity to activate dendritic cells and adjuvanticity to TFPR1, indicating that the integrity of conformational structure is essential for proteins to maintain their immunologic activity [49][50][51], α1-helix and β4-fold should not be removed at the same time. The secondary structural analysis by CD-spectrum preliminarily proved the conformational changes of different fragments; in order to better interpret the relationship between the conformation and function, the more accurate conformational structures of these proteins need to be dissolved by other more precise method, such as nuclear magnetic resonance (NMR) [52] and X-ray [53] diffraction in the future.…”

Section: Discussionmentioning

confidence: 99%

The Integrity of α-β-α Sandwich Conformation Is Essential for a Novel Adjuvant TFPR1 to Maintain Its Adjuvanticity

Ning

Wang

et al. 2019

Biomolecules

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TFPR1 is a novel peptide vaccine adjuvant we recently discovered. To define the structural basis and optimize its application as an adjuvant, we designed three different truncated fragments that have removed dominant B epitopes on TFPR1, and evaluated their capacity to activate bone marrow-derived dendritic cells and their adjuvanticity. Results demonstrated that the integrity of an α-β-α sandwich conformation is essential for TFPR1 to maintain its immunologic activity and adjuvanticity. We obtained a functional truncated fragment TFPR-ta ranging from 40-168 aa of triflin that has similar adjuvanticity as TFPR1 but with 2-log fold lower immunogenicity. These results demonstrated a novel approach to evaluate and improve the activity of protein-based vaccine adjuvant.CAP signature motifs, just as the other members in the CAP superfamily [24]. Our previous studies indicated that recombinant protein TFPR1 augmented the immunogenicity of protein (OVA and recombinant HBsAg) and peptide (HIV-1 pep5 envelope) antigens, and induced Th1-biased antibodyand cell-mediated immune responses. Its adjuvant activity may be related to its capacity of activating antigen-processing cells, such as dendritic cells [25], B cells and macrophages, and promoting the secretion of Th1-biased proinflammatory and immunoregulatory cytokines [26]. However, as a protein adjuvant, TFPR1 has relatively strong immunogenicity [27], the structural basis of its function is not yet defined.In this study, we not only confirmed that TFPR1 acts as an effective adjuvant for peptide antigen, but also identified its minimal functional region. By rationally design functional fragment with minimum immunogenicity, through deleting B cell dominant epitopes while retaining its main structure and motifs, and expressed these truncated forms in E.coli protein expression system, we obtained a functional truncated fragment TFPR-ta ranging from 40-168 aa of triflin that has similar adjuvanticity as TFPR1 but with 2-log fold lower immunogenicity. Our results suggest that the integrity of the typical α-β-α sandwich conformational structure is essential for TFPR1 protein to maintain its adjuvanticity, and α1-helix and β4-fold cannot be deleted at the same time.

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Utilization of 13C-labeled amino acids to probe the α-helical local secondary structure of a membrane peptide using electron spin echo envelope modulation (ESEEM) spectroscopy

Bottorf

Sahu

McCarrick

et al. 2018

Biochimica et Biophysica Acta (BBA) - Biomembranes

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Electron spin echo envelope modulation (ESEEM) spectroscopy in combination with site-directed spin labeling (SDSL) has been established as a valuable biophysical technique to provide site-specific local secondary structure of membrane proteins. This pulsed electron paramagnetic resonance (EPR) method can successfully distinguish between α-helices, β-sheets, and 3-helices by strategically using H-labeled amino acids and SDSL. In this study, we have explored the use ofC-labeled residues as the NMR active nuclei for this approach for the first time. C-labeled d-valine (Val) or C-labeled d-leucine (Leu) were substituted at a specific Val or Leu residue (i), and a nitroxide spin label was positioned 2 or 3 residues away (denoted i-2 and i-3) on the acetylcholine receptor M2δ (AChR M2δ) in a lipid bilayer. The C ESEEM peaks in the FT frequency domain data were observed for the i-3 samples, and noC peaks were observed in the i-2 samples. The resulting spectra were indicative of the α-helical local secondary structure of AChR M2δ in bicelles. This study provides more versatility and alternative options when using this ESEEM approach to study the more challenging recombinant membrane protein secondary structures.

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Quantification of protein secondary structure by 13C solid-state NMR

Cited by 4 publications

References 22 publications

An insight into molecular motions and phase composition of gliadin/glutenin glycerol blends studied by ¹³C solid‐state and ¹H time‐domain NMR

An insight into molecular motions and phase composition of gliadin/glutenin glycerol blends studied by ¹³C solid‐state and ¹H time‐domain NMR

The Integrity of α-β-α Sandwich Conformation Is Essential for a Novel Adjuvant TFPR1 to Maintain Its Adjuvanticity

Utilization of 13C-labeled amino acids to probe the α-helical local secondary structure of a membrane peptide using electron spin echo envelope modulation (ESEEM) spectroscopy

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Quantification of protein secondary structure by 13C solid-state NMR

Cited by 4 publications

References 22 publications

An insight into molecular motions and phase composition of gliadin/glutenin glycerol blends studied by 13C solid‐state and 1H time‐domain NMR

An insight into molecular motions and phase composition of gliadin/glutenin glycerol blends studied by 13C solid‐state and 1H time‐domain NMR

The Integrity of α-β-α Sandwich Conformation Is Essential for a Novel Adjuvant TFPR1 to Maintain Its Adjuvanticity

Utilization of 13C-labeled amino acids to probe the α-helical local secondary structure of a membrane peptide using electron spin echo envelope modulation (ESEEM) spectroscopy

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An insight into molecular motions and phase composition of gliadin/glutenin glycerol blends studied by ¹³C solid‐state and ¹H time‐domain NMR

An insight into molecular motions and phase composition of gliadin/glutenin glycerol blends studied by ¹³C solid‐state and ¹H time‐domain NMR