1. Determination of the Membrane Translocation pK of the pH-Low Insertion Peptide 
Haden L. Scott, Justin M. Westerfield, Francisco N. Barrera 
Biophysical Journal 
Volume 113, Issue 4, Pages (August 2017)
DOI: /j.bpj
Copyright © 2017 Biophysical Society Terms and Conditions

2. Figure 1
CD monitors helical formation during the membrane insertion of pHLIP. (A) Given here are CD spectra showing the membrane surface-bound State II (solid trace) and the inserted transmembrane State III (shaded trace) of pHLIP in POPC vesicles. (B) The difference in molar ellipticity ([θ]) between 222 and 262 nm was plotted against the pH to determine pKCD, the midpoint of helical formation. (Inset) Given here is titration CD spectra, showing that increasing acidity leads to α-helix formation, and thus more negative molar ellipticity at 222 nm.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2017 Biophysical Society Terms and Conditions

3. Figure 2
Three different analyses of W fluorescence pH titrations. (A) Sample acidification (shaded arrow) results in an increase in W fluorescence intensity and spectral blue shift. A similar pK was obtained by following changes in CM (B) and SM (C) upon acidification. (D) W intensity changes were also analyzed by following the FI at 335 nm. Lines in (B–D) show the fits of Eq. 2 to the data. Solid arrows mark the approximate pH values for the start and end of the titrations.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2017 Biophysical Society Terms and Conditions

4. Figure 3
The pHLIP does not have a unique pK value. Bars correspond to the pK determined by W fluorescence SM, spectral CM, or FI, by CD, or by Nt-NBD or Ct-NBD of pHLIP. Mean values are shown, for experiments repeated three to six times, and error bars are the SD. Color shading represents groups with no statistical difference. ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.005; NS; no significance.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2017 Biophysical Society Terms and Conditions

5. Figure 4
Membrane exit of pHLIP yields similar pKCM, pKSM and pKFI values. (A) The increase in pH (shaded arrow) results in a decrease in the tryptophan fluorescence intensity and a red shift in spectral maximum in the emission spectra. (B) The three membrane exit pK values determined by W fluorescence (CM, SM, and FI) are not statistically significant (p > 0.05). NS; no significance. Error bars are the SD.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2017 Biophysical Society Terms and Conditions

6. Figure 5
NBD conjugates of pHLIP display different pK values based on location. (A and B) Representative emission spectra of pHLIP-NBD (A) and NBD-pHLIP (B) show an increase in fluorescence intensity as acidity is increased (shaded arrows). (C and D) Normalized fluorescence intensity at 540 nm is plotted versus pH to determine pKCt-NBD (C) and pKNt-NBD (D). Lines in (C) and (D) show the fits of Eq. 2 to the data. Solid arrows mark the approximate pH values for the start and end of the titrations.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2017 Biophysical Society Terms and Conditions

7. Figure 6
Intrinsic W, Nt-NBD, and Ct-NBD fluorescence display similar kinetics during pHLIP membrane insertion. Given here are average spectra of W (dark shaded), Nt-NBD (dashed light shaded), and Ct-NBD (solid) in POPC.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2017 Biophysical Society Terms and Conditions

8. Figure 7
Qualitative comparison of fluorescence pK titrations with ssNMR pKa titrations for the individual aspartate residues. The individual titrations for D14 (magenta), D25 (blue), D31 (green), and D34 (red) obtained by Hanz et al. (10) are overlaid with the fluorescence data obtained following tryptophan SM (A) and FI (B), and pHLIP-NBD (C). The ssNMR data were represented by the fitting to the Boltzmann equation (10). Normalized chemical shifts are shown for a better comparison, and values were inverted in (B) and (C) to align with the fluorescence data changes.
Biophysical Journal  , DOI: ( /j.bpj )
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9. Figure 8
Tentative model showing the transition from State II to State III during the membrane insertion of pHLIP, highlighting the insertion step/s that each pK value reports on. The solid arrows represent the transitions between each step in the membrane insertion process, with the corresponding pH midpoints. The change of the D residues from shaded to solid represents the protonation. CD, and potentially SM and CM, reports on the insertion process encompassing D33, D31, and D25, and are then shown labeling the first three transitions. FI and Nt-NBD follow the protonation of D14, and to a lesser extent D25 and D33 (not shown in figure). Finally, Ct-NBD reports on the last insertion step, where the C-terminal end translocates across the bilayer.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2017 Biophysical Society Terms and Conditions