Electrostatic Environment of Proteorhodopsin Affects the pKa of Its Buried Primary Proton Acceptor

Abstract

The protonation state of embedded charged residues in transmembrane proteins (TMPs) can control the onset of protein function. It is understood that interactions between an embedded charged residue and other charged or polar residues in the moiety would influence its pKa, but how the surrounding environment in which the TMP resides affects the pKa of these residues is unclear. Proteorhodopsin (PR), a light-responsive proton pump from marine bacteria, was used as a model to examine externally accessible factors that tune the pKa of its embedded charged residue, specifically its primary proton acceptor D97. The pKa of D97 was compared between PR reconstituted in liposomes with different net headgroup charges and equilibrated in buffer with different ion concentrations. For PR reconstituted in net positively charged compared to net negatively charged liposomes in low-salt buffer solutions, a drop of the apparent pKa from 7.6 to 5.6 was observed, whereas intrinsic pKa modeled with surface pH calculated from Gouy-Chapman predictions found an opposite trend for the pKa change, suggesting that surface pH does not account for the main changes observed in the apparent pKa. This difference in the pKa of D97 observed from PR reconstituted in oppositely charged liposome environments disappeared when the NaCl concentration was increased to 150 mM. We suggest that protein-intrinsic structural properties must play a role in adjusting the local microenvironment around D97 to affect its pKa, as corroborated with observations of changes in protein side-chain and hydration dynamics around the E-F loop of PR. Understanding the effect of externally controllable factors in tuning the pKa of TMP-embedded charged residues is important for bioengineering and biomedical applications relying on TMP systems, in which the onset of functions can be controlled by the protonation state of embedded residues.

Figure 1

Difference optical absorbance at 570 nm under various bulk pH from WT PR reconstituted in POPC/POPG (80/20, mol/mol) liposomes and in a 10 mM HEPES buffer. The difference absorbance at 570 nm under different pH was normalized to the difference between the maximum and the minimum. The pH-dependent absorbance curve was fitted by the Henderson-Hasselbalch equation to get the apparent pKa_D97.

Figure 2

(A) Apparent pKa_D97 of WT PR reconstituted in negatively charged POPC/POPG (80/20, mol/mol) liposomes (blue triangle) and positively charged (80/20, mol/mol) liposomes (red triangle) in a 10 mM HEPES buffer. The HEPES buffer contained different concentrations of NaCl between 0 and 150 mM. (B) Apparent pKa_D97 of WT PR reconstituted in negatively charged POPC/POPG (80/20, mol/mol) liposomes in the HEPES buffer containing different concentrations of NaCl (triangle, same as in A), KCl (diamond), CaCl₂ (circle), and MgCl₂ (square) is shown. (C) Intrinsic pKa_D97 of WT PR reconstituted in negatively charged POPC/POPG (80/20, mol/mol) liposomes (blue triangle) and positively charged (80/20, mol/mol) liposomes (red triangle) in a HEPES buffer containing different concentrations of NaCl is shown.

Figure 3

(A) PR structure and a schematic diagram of the α-helical structure of its E-F loop. The spin-labeled 174 was shown to be located at the interface between the region with fast water dynamics (blue shading) and the region with slow water dynamics (red shading). The chemical structure of the MTSL spin label is also shown along the side. (B) cw EPR spectra of PR spin labeled at E-F loop site 174 reconstituted in negatively charged POPC/POPG (80/20, mol/mol) liposomes (blue spectra) or positively charged POPC/DOTAP (80/20, mol/mol) liposomes (red spectra) in 10 mM HEPES buffers with 0 mM NaCl or with 150 mM NaCl are shown. All the samples were equilibrated at pH 8.5.

Figure 4

Cross-relaxivities k_σ that reflect the hydration dynamics (approximately tens of picoseconds timescale) around MTSL-labeled site 174 measured by ODNP on PR reconstituted in negatively charged POPC/POPG (80/20, mol/mol) liposomes or positively charged POPC/DOTAP (80/20, mol/mol) liposomes, in HEPES buffers with 0 mM NaCl or with 150 mM NaCl. The intrinsic pKa_D97 under each condition is also plotted along the side as a reference (triangle). To see this figure in color, go online.