J Biol Chem.2016 Sep;291(36):19146-19156

Ionization Properties of Histidine Residues in the Lipid-Bilayer Membrane Environment

Ashley N. Martfeld, Denise V. Greathouse and Roger E. Koeppe II

From the Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, Arkansas 72701

Running title:  Histidine imidazole ionization in bilayer membranes

To whom correspondence should be addressed:  Prof. Roger E. Koeppe II, Department of Chemistry and Biochemistry, 119 Chemistry Building, University of Arkansas, Fayetteville, Arkansas 72701, Telephone: (479) 575 4976; FAX (479) 575 4049; E-mail: rk2@uark.edu



We address the critically important ionization properties of histidine side chains of membrane proteins, when exposed directly to lipid acyl chains within lipid bilayer membranes. The problem is important for addressing general principles that may underlie membrane protein function. To this end, we have employed a favorable host peptide framework provided by GWALP23 (acetyl-GGALW(5)LALALALALALALW(19)LAGA-amide). We inserted His residues into position 12 or 14 of GWALP23 (replacing either Leu(12) or Leu(14)) and incorporated specific [(2)H]Ala labels within the helical core sequence. Solid-state (2)H NMR spectra report the folding and orientation of the core sequence, revealing marked differences in the histidine-containing transmembrane helix behavior between acidic and neutral pH conditions. At neutral pH, the GWALP23-H12 and GWALP23-H14 helices exhibit well defined tilted transmembrane orientations in dioleoylphosphatidylcholine (DOPC)and dilauroylphosphatidylcholine (DLPC) bilayer membranes. Under acidic conditions, when His(12) is protonated and charged, the GWALP23-H12 helix exhibits a major population that moves to the DOPC bilayer surface and a minor population that occupies multiple transmembrane states. The response to protonation of His(14) is an increase in helix tilt, but GWALP23-H14 remains in a transmembrane orientation. The results suggest pKa values of less than 3 for His(12) and about 3-5 for His(14) in DOPC membranes. In the thinner DLPC bilayers, with increased water access, the helices are less responsive to changes in pH. The combined results enable us to compare the ionization properties of lipid-exposed His, Lys, and Arg side chains in lipid bilayer membranes.

PMID: 27440045; DOI: 10.1074/jbc.M116.738583



Charged amino-acid residues are critical for the functions of membrane proteins in the nervous system.  For example, charged functional groups are needed for the opening and closing of membrane channels in response to changes in voltage, and for the regulation of ionic currents through these channels when signals are sent by means of the nervous system.  When trying to understand biological function, nevertheless, it is difficult to measure and difficult to predict the status (charged or neutral) of particular protein functional groups in the non-polar lipid environment provided by a cell membrane.  Our recent publication addresses this issue for histidine residues in lipid-bilayer membranes.

While most of the twenty common amino-acid building blocks for proteins are electrically neutral, several of them can accept or release protons (H+) and thereby transition between a neutral state and a state that is either positively or negatively charged.  Among these proton-transferring or “titrating” amino acids, histidine is unique in having about equal populations of a neutral state and a positively charged state in physiological solution at neutral pH.  As such, histidine is a significant player in many biological proton transfer reactions and is prominent at the active sites of many important enzymes.

Though sparse in membrane proteins, potentially charged amino-acid residues such as histidine are highly conserved and often serve important biological functions.  For example, proton transfer reactions and charged/neutral transitions are critical for the voltage responses of membrane channels in the nervous system and for the ionic currents that pass through these channels during signal transduction.



Figure 1.  The model transmembrane helical peptide GWALP23 as a framework for hosting particular histidine residues (H12 and H14) in lipid bilayer membranes.  The interfacial tryptophan residues W5 and W19 provide stability.  The choices for placement of the diagnostic deuterium labels are indicated as space filling atoms.  The results from deuterium magnetic resonance experiments indicate that the helices are never oriented straight across the membrane, as shown in the figure, but rather are always tilted in the lipid bilayer membrane, as reported in J. Biol. Chem. 291, 19146-19156 (2016).  The relative extent of the helix tilt reflects and reports the status of the histidine functional group, whether charged or neutral.


It is difficult to assess particular ionic properties in lipid membranes.  As opposed to the polar environment provided by a physiological solution, membrane proteins reside in the non-polar environment of a lipid bilayer membrane.  As a result, the balance between a neutral state and a positively charged state for histidine in a membrane protein is likely to be quite different from the situation in physiological solution.  The actual charged state of histidine in a biological membrane can be difficult to measure and is furthermore difficult to predict [1].  We addressed the problem fundamentally by using designed helical peptides (Figure 1) that would insert into lipid membranes and keep the histidine side chain somewhat buried and close to non-polar lipid molecules.  We incorporated deuterium atoms (“heavy” hydrogen or 2H) as probes at some of the locations indicated by space-filling atoms in Figure 1.  The magnetic properties of the deuterium probes revealed that each model helix occupies a particular tilted transmembrane orientation when surrounded by lipids.  Remarkably, the helix tilt changes when the status of the histidine residue changes from charged to neutral [2].  The tilt of each helix can therefore be measured as a function of the experimental acidity or pH.

The results of our experiments reveal changes in the charge status and titration behavior of histidine in lipid membranes compared to the situation in physiological solution.  Indeed the lipid membrane environment favors the neutral state, and the magnitude of the change depends upon the exact location of the histidine residue that is tested (position 12 or position 14 in Figure 1).  The midpoint for the charged/neutral transition is shifted by 2 to 4 units on the logarithmic pH scale, meaning a shift of 102 to 104 for the ionization behavior of histidine in the lipid membrane environment.

In addition to studies of histidine, we have completed similar experiments to characterize the status (charged versus neutral) of other ionizable amino acids, namely lysine [3], arginine [3] and glutamic acid [4] in lipid bilayer membranes.



[1]  J.L. MacCallum, W.F.D. Bennett, D.P. Tieleman, Distribution of amino acids in a lipid bilayer from computer simulations, Biophys. J. 94 (2008) 3393-3404.

[2]  A.N. Martfeld, D.V. Greathouse, R.E. Koeppe, II Ionization properties of histidine residues in the lipid-bilayer membrane environment, J. Biol. Chem. 291 (2016) 19146-19156.

[3]  N.J. Gleason, V.V. Vostrikov, D.V. Greathouse, R.E. Koeppe, II, Buried lysine, but not arginine, titrates and alters transmembrane helix tilt, Proc. Natl. Acad. Sci. U. S. A. 110 (2013) 1692-1695.

[4]  V. Rajagopalan, D.V. Greathouse, R.E. Koeppe, II, Influence of glutamic acid residues and pH on the properties of transmembrane helices, BBA – Biomembranes 1859 (2017) in press.  http://www.sciencedirect.com/science/article/pii/S0005273617300068 .



Figure 2.  The authors with other lab members.  From left:  Denise Greathouse, Armin Mortazavi, Ryan Wendt, Venkatesan Rajagopalan, Alexandrea Kim, Jordana Thibado, Roger Koeppe, Ashley Martfeld, Karli Lipinski, Matthew McKay, Jenny Afrose.






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