Lipid-Dependent Titration of Glutamic Acid at a Bilayer Membrane Interface

Abstract

The ionization properties of protein side chains in lipid-bilayer membranes will differ from the canonical values of side chains exposed to an aqueous solution. While the propensities of positively charged side chains of His, Lys, and Arg to release a proton in lipid membranes have been rather well characterized, the propensity for a negatively charged Glu side chain to receive a proton and achieve the neutral state in a bilayer membrane has been less well characterized. Indeed, the ionization of the glutamic acid side chain has been predicted to depend on its depth of burial in a lipid membrane but has been difficult to verify experimentally. To address the issue, we incorporated an interfacial Glu residue at position 4 of a distinct 23-residue transmembrane helix and used 2 H NMR to examine the helix properties as a function of pH. We observe that the helix tilt and azimuthal rotation vary little with pH, but the extent of helix unraveling near residues 3 and 4 changes as the Glu residue E4 titrates. Remarkably, the 2 H quadrupolar splitting for the side chain of alanine A3 responds to pH with an apparent pK a of 4.8 in 1,2-dilauroyl-snglycero-3-phosphocholine (DLPC) and 6.3 in 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC), but is unchanged up to pH 8.0 in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) in the presence of residue E4. With bilayers composed of alkali-stable ether-linked lipids, the side chain of A3 responds to pH with an apparent pK a of 11.0 in the ether analogue of DOPC. These results suggest that the depth dependence of Glu ionization in lipid-bilayer membranes may be steeper than previously predicted or envisioned.

Illuminating Disorder Induced by Glu in a Stable Arg-Anchored Transmembrane Helix

“…As the pH increases, more signals appear until an apparent endpoint is reached near pH 8 involving a disordered aggregate of immobilized states indicated by a spectral Pake pattern. Partial unwinding of the helix terminals is anticipated, even for the parent R14 helix, with the extent of fraying notably depending on the identities and ionization states of particular residues near the membrane interface. ,− While small changes in the local fraying are detected easily by the highly sensitive 2 H NMR methods, such changes involving helix terminal disorder may not necessarily be reflected in the circular dichroism spectra. , Notably, the helix disorder, whether arising from changes in end fraying or orientations of the core helix, is lipid-dependent as well as pH-dependent as the detailed behavior varies among bilayers of DLPC, DMPC, and DOPC. These features will be important for understanding the plasticity of protein functional domains for which, notably, the cholesterol content also is a significant regulatory factor. ,, …”

D-to-E and T19V Variants of the pH-Low Insertion Peptide and Their Doxorubicin Conjugates Interact with Membrane at Higher pH Ranges Than WT

“…The insertion of pHLIP is unidirectionalonly the C-terminus is translocated across the membrane. ,,, In the final TM state, the D14 and D25 residues are located in the hydrophobic core of the membrane, while the C-terminal D31, D33, and E34 residues are positioned in the headgroup region of the trans lipid leaflet . These features make pHLIP an intriguing model for understanding how the protonation states of Asp/Glu residues can influence the folding and topogenesis of a TM α-helixan important topic that has been explored in the context of other TM or membrane-bound helices such as pLeu­(D), , GALA, − GWALP23­(E), − MelP5­(Δ4/6), pHD, − macrolittins, PLP TM2, CFTR TM4, diphtheria toxin T-domain TH8-TH9, − and others. , Interestingly, the pH-dependent membrane insertion behavior of pHLIP can even be obtained by grafting the key Asp/Glu residues onto completely different background TM sequences, such as in the ATRAM peptide, and the TYPE7 peptide that can activate the receptor EphA2 …”

Surface Properties of Synaptosomes in the Presence of L-Glutamic and Kainic Acids: In Vitro Alteration of the ATPase and Acetylcholinesterase Activities

“…Glutamate is localized in the cytoplasm without selective accumulation in the synaptic areas. The propensity for a negatively charged glutamate side chain to receive a proton and achieve a neutral state in bilayer membranes has been studied [21]. Glutamate can be found in the interior of certain membrane proteins and, when charged, can alter protein folding [22] and strongly influences the helix tilt [21].…”

“…The propensity for a negatively charged glutamate side chain to receive a proton and achieve a neutral state in bilayer membranes has been studied [21]. Glutamate can be found in the interior of certain membrane proteins and, when charged, can alter protein folding [22] and strongly influences the helix tilt [21]. The ionization of the glutamic chain predicts a dependance on its depth of burial in a lipid membrane [21].…”

“…Glutamate can be found in the interior of certain membrane proteins and, when charged, can alter protein folding [22] and strongly influences the helix tilt [21]. The ionization of the glutamic chain predicts a dependance on its depth of burial in a lipid membrane [21]. Analytical tools for quantitative measurements of glutamate show that an isolated single synaptic vesicle encapsulates about 8000 glutamate molecules and is comparable to the measured exocytotic quantal glutamate release in amperometric glutamate sensing in the nucleus accumbens of mouse brain tissue [23].…”