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Residue-specific structures and membrane locations of pH-low insertion peptide by solid-state nuclear magnetic resonance.

Shu NS, Chung MS, Yao L, An M, Qiang W - Nat Commun (2015)

Bottom Line: Here, we show the first study on membrane-associated pHLIP using solid-state NMR spectroscopy.The critical membrane-adsorbed state is more complex than previously envisioned.At pH 6.4, for the major unstructured population, the peptide sinks deeper into the membrane in a state II' that is distinct from the adsorbed state II observed at pH 7.4, which may enable pHLIP to sense slight change in acidity even before insertion.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, State University of New York, Binghamton, New York 13902, USA.

ABSTRACT
The pH-low insertion peptide (pHLIP) binds to a membrane at pH 7.4 unstructured but folds across the bilayer as a transmembrane helix at pH∼6. Despite their promising applications as imaging probes and drug carriers that target cancer cells for cytoplasmic cargo delivery, the mechanism of pH modulation on pHLIP-membrane interactions has not been completely understood. Here, we show the first study on membrane-associated pHLIP using solid-state NMR spectroscopy. Data on residue-specific conformation and membrane location describe pHLIP in various surface-bound and membrane-inserted states at pH 7.4, 6.4 and 5.3. The critical membrane-adsorbed state is more complex than previously envisioned. At pH 6.4, for the major unstructured population, the peptide sinks deeper into the membrane in a state II' that is distinct from the adsorbed state II observed at pH 7.4, which may enable pHLIP to sense slight change in acidity even before insertion.

No MeSH data available.


Related in: MedlinePlus

Representative 13C–31P fsREDOR spectra taken at 17.8 ms dephasing time.The dashed lines on top of each spectra pair (S0 and S1) highlight the peak intensities in S0. All the spectra were processed with 10 Hz Gaussian line broadening. Each NMR spectrum was collected with ∼40 k scans (that is, ∼24 h).
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f5: Representative 13C–31P fsREDOR spectra taken at 17.8 ms dephasing time.The dashed lines on top of each spectra pair (S0 and S1) highlight the peak intensities in S0. All the spectra were processed with 10 Hz Gaussian line broadening. Each NMR spectrum was collected with ∼40 k scans (that is, ∼24 h).

Mentions: The 13C–31P frequency-selective rotational-echo double-resonance (fsREDOR) NMR47 spectroscopy was used to quantify the membrane locations of labelled amino acids. The fsREDOR pulse sequence is developed to provide specific observation of a certain spectral region, which is particularly useful for our samples as the 13C signals are from both labelled amino acids and naturally abundant lipids. The 31P nuclei naturally exist at the membrane/water interface in the phosphate diester moieties of POPC lipid head groups. We selected the Ala 13Cβ signals for REDOR analysis because they do not overlap with lipids or Leu 13C signals. The Gaussian selective pulse was set to 6.0 ms to provide a ±300 Hz (that is, ∼4.0 p.p.m. in 13C spectra) observation window, according to previous studies47. This observation range covered the chemical shifts for Ala Cβ based on our 2D spin diffusion experiments (Table 1). The pulsed-spin locking (PSL) technique48 was applied during the acquisition time to increase the signal-to-noise ratio thus facilitating quantification. The representative REDOR full (S0) and reduced (S1) 13C spectra with the longest dephasing time (that is, 17.8 ms) are shown in Fig. 5, and the plots of dephasing (ΔS/S0) as a function of dipolar evolution time are provided in Fig. 6, along with the theoretical fitting curves. A detailed description of the calculation of ΔS/S0 is provided in the Supplementary Methods.


Residue-specific structures and membrane locations of pH-low insertion peptide by solid-state nuclear magnetic resonance.

Shu NS, Chung MS, Yao L, An M, Qiang W - Nat Commun (2015)

Representative 13C–31P fsREDOR spectra taken at 17.8 ms dephasing time.The dashed lines on top of each spectra pair (S0 and S1) highlight the peak intensities in S0. All the spectra were processed with 10 Hz Gaussian line broadening. Each NMR spectrum was collected with ∼40 k scans (that is, ∼24 h).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4518304&req=5

f5: Representative 13C–31P fsREDOR spectra taken at 17.8 ms dephasing time.The dashed lines on top of each spectra pair (S0 and S1) highlight the peak intensities in S0. All the spectra were processed with 10 Hz Gaussian line broadening. Each NMR spectrum was collected with ∼40 k scans (that is, ∼24 h).
Mentions: The 13C–31P frequency-selective rotational-echo double-resonance (fsREDOR) NMR47 spectroscopy was used to quantify the membrane locations of labelled amino acids. The fsREDOR pulse sequence is developed to provide specific observation of a certain spectral region, which is particularly useful for our samples as the 13C signals are from both labelled amino acids and naturally abundant lipids. The 31P nuclei naturally exist at the membrane/water interface in the phosphate diester moieties of POPC lipid head groups. We selected the Ala 13Cβ signals for REDOR analysis because they do not overlap with lipids or Leu 13C signals. The Gaussian selective pulse was set to 6.0 ms to provide a ±300 Hz (that is, ∼4.0 p.p.m. in 13C spectra) observation window, according to previous studies47. This observation range covered the chemical shifts for Ala Cβ based on our 2D spin diffusion experiments (Table 1). The pulsed-spin locking (PSL) technique48 was applied during the acquisition time to increase the signal-to-noise ratio thus facilitating quantification. The representative REDOR full (S0) and reduced (S1) 13C spectra with the longest dephasing time (that is, 17.8 ms) are shown in Fig. 5, and the plots of dephasing (ΔS/S0) as a function of dipolar evolution time are provided in Fig. 6, along with the theoretical fitting curves. A detailed description of the calculation of ΔS/S0 is provided in the Supplementary Methods.

Bottom Line: Here, we show the first study on membrane-associated pHLIP using solid-state NMR spectroscopy.The critical membrane-adsorbed state is more complex than previously envisioned.At pH 6.4, for the major unstructured population, the peptide sinks deeper into the membrane in a state II' that is distinct from the adsorbed state II observed at pH 7.4, which may enable pHLIP to sense slight change in acidity even before insertion.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, State University of New York, Binghamton, New York 13902, USA.

ABSTRACT
The pH-low insertion peptide (pHLIP) binds to a membrane at pH 7.4 unstructured but folds across the bilayer as a transmembrane helix at pH∼6. Despite their promising applications as imaging probes and drug carriers that target cancer cells for cytoplasmic cargo delivery, the mechanism of pH modulation on pHLIP-membrane interactions has not been completely understood. Here, we show the first study on membrane-associated pHLIP using solid-state NMR spectroscopy. Data on residue-specific conformation and membrane location describe pHLIP in various surface-bound and membrane-inserted states at pH 7.4, 6.4 and 5.3. The critical membrane-adsorbed state is more complex than previously envisioned. At pH 6.4, for the major unstructured population, the peptide sinks deeper into the membrane in a state II' that is distinct from the adsorbed state II observed at pH 7.4, which may enable pHLIP to sense slight change in acidity even before insertion.

No MeSH data available.


Related in: MedlinePlus