TY - JOUR

T1 - IKMTSL-PTE, a Phospholipid-Based EPR Probe for Surface Electrostatic Potential of Biological Interfaces at Neutral pH

T2 - Effects of Temperature and Effective Dielectric Constant of the Solvent

AU - Voinov, Maxim A.

AU - Scheid, Christina T.

AU - Kirilyuk, Igor A.

AU - Trofimov, Dmitrii G.

AU - Smirnov, Alex I.

PY - 2017/3/23

Y1 - 2017/3/23

N2 - The synthesis and characterization of a lipidlike electrostatic spin probe, (S)-2,3-bis(palmitoyloxy)propyl 2-((4-(4-(dimethylamino)-2-ethyl-1-oxyl-5,5-dimethyl-2,5-dihydro-1H-imidazol-2-yl)benzyl)disulfanyl)ethyl phosphate (IKMTSL-PTE), are being reported. The intrinsic pKa0 of IKMTSL-PTE was determined by X-band (9.5 GHz) electron paramagnetic resonance (EPR) titration of a water-soluble model compound, 4-(dimethylamino)-2-ethyl-2-(4-(((2-hydroxyethyl)disulfanyl)methyl)phenyl)-5,5-dimethyl-2,5-dihydro-1H-imidazol-1-oxyl (IKMTSL-ME), an adduct of methanethiosulfonate spin label IKMTSL and 2-mercaptoethanol. The pKa0 of IKMTSL-ME in bulk aqueous solutions was found to be significantly higher than that of 4-(((2-hydroxyethyl)disulfanyl)methyl)-2,2,3,5,5-pentamethylimidazolidin-1-oxyl (IMTSL-ME), an adduct of the corresponding methanethiosulfonate spin label IMTSL and 2-mercaptoethanol (17°C, pKa0 = 6.16 ± 0.03 vs 20°C, pKa0 = 3.33 ± 0.03, respectively). A series of EPR titration experiments with IKMTSL-ME in aqueous solutions containing 0-60% v/v isopropanol have been carried out at 17 and 48°C to determine the effects of temperature and bulk dielectric permittivity constant, ε, on the probe pKa. A linear relationship between the probe pKa and ε has been established and found to be essentially the same at 17 and 48°C. The polarity term contributing to the pKa of IKMTSL-PTE at an uncharged lipidlike interface was determined by incorporating the probe into electrically neutral micelles formed from nonionic detergent Triton X-100, and it was found, similar to IMTSL-PTE, to be negative. In negatively charged DMPG lipid bilayers, IKMTSL-PTE exhibits ionization transitions with significantly higher pKa values than those previously reported for IMTSL-PTE (e.g., at 17°C, pKai = 7.80 ± 0.03 vs pKa0 = 5.70 ± 0.05). The surface electrostatic potentials of DMPG lipid bilayers calculated using IKMTSL-PTE titration data were found to be somewhat lower than those calculated using IMTSL-PTE. The lower values measured by IKMTSL-PTE are the likely consequences of the structure of the linker that positions the reporter nitroxide further away from the bilayer plane into aqueous phase. Overall, the ionization transitions of IKMTSL-PTE with pKa values close to the neutral pH range make this lipidlike molecule a valuable spectroscopic EPR probe for studying the electrostatic phenomena at biological interfaces, including lipid bilayer/membrane protein systems, that could be unstable in the acidic pH range accessible by the previously available probes. (Figure Presented).

AB - The synthesis and characterization of a lipidlike electrostatic spin probe, (S)-2,3-bis(palmitoyloxy)propyl 2-((4-(4-(dimethylamino)-2-ethyl-1-oxyl-5,5-dimethyl-2,5-dihydro-1H-imidazol-2-yl)benzyl)disulfanyl)ethyl phosphate (IKMTSL-PTE), are being reported. The intrinsic pKa0 of IKMTSL-PTE was determined by X-band (9.5 GHz) electron paramagnetic resonance (EPR) titration of a water-soluble model compound, 4-(dimethylamino)-2-ethyl-2-(4-(((2-hydroxyethyl)disulfanyl)methyl)phenyl)-5,5-dimethyl-2,5-dihydro-1H-imidazol-1-oxyl (IKMTSL-ME), an adduct of methanethiosulfonate spin label IKMTSL and 2-mercaptoethanol. The pKa0 of IKMTSL-ME in bulk aqueous solutions was found to be significantly higher than that of 4-(((2-hydroxyethyl)disulfanyl)methyl)-2,2,3,5,5-pentamethylimidazolidin-1-oxyl (IMTSL-ME), an adduct of the corresponding methanethiosulfonate spin label IMTSL and 2-mercaptoethanol (17°C, pKa0 = 6.16 ± 0.03 vs 20°C, pKa0 = 3.33 ± 0.03, respectively). A series of EPR titration experiments with IKMTSL-ME in aqueous solutions containing 0-60% v/v isopropanol have been carried out at 17 and 48°C to determine the effects of temperature and bulk dielectric permittivity constant, ε, on the probe pKa. A linear relationship between the probe pKa and ε has been established and found to be essentially the same at 17 and 48°C. The polarity term contributing to the pKa of IKMTSL-PTE at an uncharged lipidlike interface was determined by incorporating the probe into electrically neutral micelles formed from nonionic detergent Triton X-100, and it was found, similar to IMTSL-PTE, to be negative. In negatively charged DMPG lipid bilayers, IKMTSL-PTE exhibits ionization transitions with significantly higher pKa values than those previously reported for IMTSL-PTE (e.g., at 17°C, pKai = 7.80 ± 0.03 vs pKa0 = 5.70 ± 0.05). The surface electrostatic potentials of DMPG lipid bilayers calculated using IKMTSL-PTE titration data were found to be somewhat lower than those calculated using IMTSL-PTE. The lower values measured by IKMTSL-PTE are the likely consequences of the structure of the linker that positions the reporter nitroxide further away from the bilayer plane into aqueous phase. Overall, the ionization transitions of IKMTSL-PTE with pKa values close to the neutral pH range make this lipidlike molecule a valuable spectroscopic EPR probe for studying the electrostatic phenomena at biological interfaces, including lipid bilayer/membrane protein systems, that could be unstable in the acidic pH range accessible by the previously available probes. (Figure Presented).

KW - SENSITIVE SPIN PROBES

KW - NUCLEAR-MAGNETIC-RESONANCE

KW - LIPID-BILAYERS

KW - HYDROGEN-BOND

KW - ACID

KW - DEPENDENCE

KW - NITROXIDE

KW - MEMBRANES

KW - POLARITY

KW - SPECTRA

UR - http://www.scopus.com/inward/record.url?scp=85019166335&partnerID=8YFLogxK

U2 - 10.1021/acs.jpcb.7b00592

DO - 10.1021/acs.jpcb.7b00592

M3 - Article

AN - SCOPUS:85019166335

VL - 121

SP - 2443

EP - 2453

JO - Journal of Physical Chemistry B

JF - Journal of Physical Chemistry B

SN - 1520-6106

IS - 11

ER -