The role of Trp side chains in tuning single proton conduction through gramicidin channels

ABSTRACT We present an extensive set of measurements of proton conduction through gramicidin A (gA), B (gB), and M (gM) homodimer channels which have 4, 3, or 0 Trp residues at each end of the channel, respectively. In gA we find a shoulder separating two domains of conductance increasing with concentration, confirming the results of Eisenman, G., B. Enos, J. Hagglund, and J. Sandblom. 1980. Ann. NY Acad. Sci. 339:8-20. In gB, the shoulder is shifted by ~1/2 pH unit to higher H^sup +^ concentrations and is very sharply defined. No shoulder appears in the gM data, but an associated transition from sublinear to superlinear I-V values occurs at a 100-fold higher [H^sup +^] in gM than in gA. The data in the low concentration domain are analyzed using a configuration space model of single-proton conduction, assuming that the difference in the proton potential of mean force (PMF) between gA and its analogs is constant, similar to the results of Anderson, D., R. B. Shirts, T. A. Cross, and D. D. Busath. 2001. Biophys. J. 81:1255-1264. Our results suggest that the average amplitudes of the calculated proton PMFs are nearly correct, but that the water reorientation barrier calculated for gA by molecular dynamics using the PM6 water model (Pomes, R., and B. Roux. 1997. Biophys. J. 72:246a) must be reduced in amplitude by 1.5 kcal/mol or more, and is not rate-limiting for gA.

INTRODUCTION

The gramicidin A (gA) monomer is a pentadecapeptide consisting of alternating L and D amino acids. The conducting form of the channel is an N-terminal to N-terminal dimer composed of two identical beta^sup 6.5^ helices. The channel is 25 Angstrom long, with a central pore diameter of 4 Angstrom (Arsenyev et al., 1990; Ketchem et al., 1997). The amino acid side chains extend radially outward, away from the helical backbone. These include four pairs of tryptophans at positions 9, 11, 13, and 15 positioned near the lipid-water interface. The tryptophan indole ring system has a dipole moment of -2.1 Debye (Gotten et al., 1999b), comparable to that of a water molecule (Duca and Jordan, 1998). The orientations and dynamics of the indoles in the membrane and their dipole moment have been studied by solid-state NMR (Hu et al., 1993, 1995; Hu and Cross, 1995; Gotten et al., 1999b) and molecular modeling (Woolf and Roux, 1997; Dorigo et al., 1999; Anderson et al., 2001). The indole dipole moments contribute substantially to the electrical potential in the pore region. There is reasonable agreement (Hu and Cross, 1995) between the approximate magnitude of the dipole potential from the atomistic force field computations (Woolf and Roux, 1997; Dorigo et al., 1999; Anderson et al., 2001) and the effects of Trp-to-Phe changes on the Na^sup +^ conductance measured experimentally (Becker et al., 1991). However, the shape of the axial potential profile from the Trp side chains depends on the force field used. The results of Anderson et al. (2001), based upon an ab initio force field, are particularly simple. They find that the indoles of each tryptophan pair stabilize cations in the pore by -0.6 kcal/ mol. This change is approximately constant throughout the channel, independent of the spatial coordinate parallel to the pore axis, and presumably is extinguished by bulk electrolyte shielding just outside the channel.

An extensive set of gramicidin analogs have been developed and investigated by Andersen, Busath, Cukierman, Cross, Heitz, Koeppe, Woolley, and others (e.g., Andersen et al., 1998; Busath et al., 1998; Cotten et al., 1999b; Quigley et al., 2000; Jaikaran and Woolley, 1995; for reviews of earlier work, see Woolley and Wallace, 1992; Busath, 1993). In gramicidin B (gB), the tryptophans at position 11 are replaced by phenylalanine. The indole residues of the tryptophans are located outside the beta helix -7 or 8 Angstrom from the pore axis (Gotten et al., 1999b). The phenylalanine side chain is not expected to have a significant dipole moment. Therefore, it appears likely that differences between the conductance properties of gB and gA are mainly due to the change in the electrical potential in the pore region due to the decreased dipole moment of phenylalanine. Replacement of Trp by Phe provides a mechanism for tuning the electrostatic environment of the pore. In gramicidin M (gM), all four pairs of Trp are replaced by Phe (Heitz et al., 1982). The electrical potential of the gramicidin pore can also be modified by fluorinating the indole ring (Gotten et al., 1999b). In contrast to the effect of replacing Trp by Phe, 5-fluorination increases the side chain dipole moment according to both experiment (Andersen et al., 1998; Busath et al., 1998; Thompson et al., 2001) and computation (Anderson et al., 2001). Lipids also make an important contribution to the electrical potential within the pore interior. De Godoy and Cukierman (2001) report a recent study of this influence on proton conduction through dioxolane-linked gramicidin analogs. Measurements of proton conduction through gA in diphytanoylphosphatidylcholine (Rokitskaya et al., 2002) provide an intriguing comparison with the results in glycerolmonooleate reported here.

The authors thank Sam Cukierman and Regis Pomes for helpful discussions. M.F.S. thanks Amd Roth for providing an updated version of his ActivityCoefficients package for Mathematics.

This work was supported by National Science Foundation Grant 9630475 (to M.F.S.) and National Institutes of Health Grant ROI A123007 (to D.B.).

[Reference]

REFERENCES

[Reference]

Andersen, 0. S. 1983. Ion movement through gramicidin A channels. Interfacial polarization effects on single-channel current measurements. Biophys. J. 41:135-146.

Andersen, 0. S. 1999. Graphic representation of the results of kinetic analyses. J. Gen. Physiol. 114:589-590.

Andersen, 0. S., D. V. Greathouse, L. L. Providence, M. D. Becker, and R. E. Koeppe If. 1998. Importance of tryptophan dipoles for protein function: 5-fluorination of tryptophans in gramicidin A channels. J. Am. Chem. Soc. 120:5142-5146.

Anderson, D., R. B. Shirts, T. A. Cross, and D. D. Busath. 2001. Noncontact dipole effects on channel permeation. V. Computed potentials for fluorinated gramicidin. Biophys. J. 81:1255-1264.

Arsenyev, A. S., A. L. Lomize, 1. L. Barsukov, and V. F. Bystrov. 1990. Gramicidin A transmembrane channel. Three-dimensional structural rearrangement based on NMR spectroscopy and energy refinement. Biol. Mem. 3:1723-1778.

Becker, M. D., D. V. Greathouse, R. E. Koeppe II, and 0. S. Andersen. 1991. Amino acid sequence modulation of gramicidin channel function: effects of tryptophan-to-phenylalanine substitutions on the singlechannel conductance and duration. Biochemistry. 30:8830-8839.

Becker, M. D., R. E. Koeppe II, and 0. S. Andersen. 1992. Amino acid substitutions and ion channel function: model-dependent conclusions. Biophys. J. 62:25-27.

Busath, D. D. 1993. The use of physical methods in determining gramicidin channel structure and function. Annu. Rev. Physiol. 55:473-501. Busath, D. D., C. D. Thulin, R. W. Hendershot, L. R. Phillips, P. Maughan,

C. D. Cole, N. C. Bingham, S. Morrison, L. C. Baird, R. J. Hendershot, M. Gotten, and T. A. Cross. 1998. Non-contact dipole effects on channel permeation. I. Experiments with (SF-indole)Trp-13 gramicidin A channels. Biophys. J. 75:2830-2844.

Chiu, S.-W., E. Jakobsson, S. Subramanian, and J. Andrew McCammon. 1991. Time-correlation analysis of simulated water motion in flexible and rigid gramicidin channels. Biophys. J. 60:273-285.

Chiu, S.-W., S. Subramanian, and E. Jakobsson. 1999. Simulation study of a gramicidin/lipid bilayer system in excess water and lipid. II. Rates and mechanisms of water transport. Biophys. J. 76:1939-1950.

Corry, B., S. Kuyucak, and S. H. Chung. 2000. Tests of continuum theories as models of ion channels. II. Poisson-Nernst-Planck theory versus Brownian dynamics. Biophys. J. 78:2364-2381.

Gotten, M., R. Fu, and T. A. Cross. 1999a. Solid-state NMR and hydrogendeuterium exchange in a bilayer-solubilized peptide: structural and mechanistic implications. Biophys. J. 76:1179-1189.

Gotten, M., C. Tian, D. D. Busath, R. B. Shirts, and T. A. Cross. 1999b. Modulating dipoles for structure-function correlations in the gramicidin A channel. Biochemistry. 38:9185-9197.

Cukierman, S. 2000. Proton mobilities in water and in different stereoisomers of covalently linked gramicidin A channels. Biophys. J. 78: 1825-1834.

DeCoursey, T. E., and V. V. Cherny. 1999. An electrophysiological comparison of voltage-gated proton channels, other ion channels, and other proton channels. Israel J. Chem. 39:409-418.

[Reference]

De Godoy, C. M. G., and S. Cukierman. 2001. Modulation of proton transfer in the water wire of dioxolane-linked gramicidin channels by lipid membranes. Biophys. J. 81:1430-1438.

Dorigo, A. E., D. G. Anderson, and D. D. Busath. 1999. Noncontact dipole effects on channel permeation. II. Trp conformations and dipole potentials in gramicidin A. Biophys. J. 76:1897-1908.

Duca, K. A., and P. C. Jordan. 1998. Comparison of selectively polarizable force fields for ion-water-peptide interactions: ion translocation in a gramicidin-like channel. J. Phys. Chem. B. 102:9127-9138.

Eisenman, G., B! Enos, J. Hagglund, and J. Sandblom. 1980. Gramicidin as an example of a single-filing ionic channel. Ann. NY. Acad. Sci. 339: 8-20.

Heitz, F., G. Spach, and Y. Trudelle. 1982. Single channels of 9,11,13,15destryptophyl-phenylalanyl-gramicidin A. Biophys. J. 40:87-89. Hladky, S. B. 1999. Can we use rate constants and state models to describe

ion transport through gramicidin channels? Novartis Found. Symp. 225: 93-107.

Hu, W., and T. A. Cross. 1995. Tryptophan hydrogen bonding and electrical dipole moments: functional roles in the gramicidin channel and implications for membrane proteins. Biochemistry. 34:14147-14155.

Hu, W., N. D. Lazo, and T. A. Cross. 1995. Tryptophan dynamics and structural refinement in a lipid bilayer environment: solid state NMR of the gramicidin channel. Biochemistry. 34:14138-14146.

Hu, W., K.-C. Lee, and T. A. Cross. 1993. Tryptophans in membrane proteins: indole ring orientations in the gramicidin channel. Biochemistry. 32:7035-7047.

Jaikaran, D. C. J., and G. A. Woolley. 1995. Characterization of thermal isomerization at the single molecule level. J. Phys. Chem. 99: 13352-13355.

Jordan, P. C. 1982. Electrostatic modeling of ion pores. Energy barriers and electric field profiles. Biophys. J. 39:157-164.

Ketchem, R. R., B. Roux, and T. A. Cross. 1997. High-resolution polypeptide structure in a lamellar phase lipid environment from solid state NMR derived orientational constraints. Structure. 5:1655-1669.

Koeppe, II, R. E., and L. B. Weiss. 1981. Resolution of linear gramicidins by preparative reversed-phase high-performance liquid chromatography. J. Chromatagr. 208:414-418.

Mapes, E., and M. F. Schumaker. 2001. Mean first passage times across a potential barrier in the lumped state approximation. J. Chem. Phys. 114:76-83.

Markham, J. C., J. A. Gowen, T. A. Cross, and D. D. Busath. 2001. Comparison of gramicidin A and gramicidin M channel conductance dispersities. Biochim. Biophys. Acta. 1513:185-192.

Miller, C. 1999. Ionic hopping defended. J. Gen. Physiol. 113:783-787. Moy, G., B. Corry, S. Kuyucak, and S. H. Chung. 2000. Tests of continuum theories as models of ion channels. I. Poisson-Boltzmann theory versus Brownian dynamics. Biophys. J. 78:2349-2363.

Neher, E., J. Sandblom, and G. Eisenman. 1978. Ionic selectivity, saturation, and block in gramicidin A channels. II. Saturation behavior of single channel conductances and evidence for the existence of multiple binding sites in the channel. J. Membr. Biol. 40:97-116.

Phillips, L. R., C. D. Cole, R. J. Hendershot, M. Cotten, T. A. Cross, and D. D. Busath. 1999. Noncontact dipole effects on channel permeation. III. Anomalous proton conductance effects in gramicidin. Biophys. J. 77:2492-2501.

Pomes, R. 1999. Theoretical studies of the Grotthus mechanism in biological proton wires. Israel J. Chem. 39:387-395.

Pomes, R., and B. Roux. 1996. Structure and dynamics of a proton wire: a theoretical study of H+ translocation along the single-file water chain in the gramicidin A channel. Biophys. J. 71:19-39.

Pomes, R., and B. Roux. 1997. Free energy profiles governing H+ conductance in proton wires. Biophys. J. 72:246a (Abstr.).

Pomes, R., and B. Roux. 1998. Free energy profiles for H+ conduction along hydrogen-bonded chains of water molecules. Biophys. J. 75: 33-40.

Pomes R., and B. Roux. 2002. Molecular mechanism of H+ conduction in the single-file water chain of the gramicidin channel. Biophys. J. 82: 2304-2316.

[Reference]

Press, W. H., S. A. Teukolsky, W. T. Vetterling, and B. P. Flannery. 1992. Numerical Recipes in Fortran. Cambridge University Press, New York. 653-655; 749-751.

Quigley, E. P., D. S. Crumrine, and S. Cukierman. 2000. Gating and permeation in ion channels formed by gramicidin A and its dioxolanelinked dimer in Na+ and Cs+ solutions. J. Membr. Biol. 174:207-212.

Rokitskaya, T. L, E. A. Kotova, and Y. N. Antonenko. 2002. Membrane dipole potential modulates proton conductance through gramicidin channel: movement of negative ionic defects inside the channel. Biophys. J. 82:865-873.

Roux, B. 1997. Influence of the membrane potential on the free energy of an intrinsic protein. Biophys. J. 73:2980-2989.

Roux, B. 1999. Statistical mechanical equilibrium theory of selective ion channels. Biophys. J. 77:139-153.

Ruskeepii, H. 1999. Mathematica Navigator. Academic Press, New York. 690-691.

Schumaker, M. F., R. PomBs, and B. Roux. 2000. A combined molecular dynamics and diffusion model of single proton conduction through gramicidin. Biophys. J. 79:2840-2857.

[Reference]

Schumaker, M. F., R. Pomps, and B. Roux. 2001. A framework model for single proton conductance through gramicidin. Biophys. J. 80:12-30.

Thompson, N., G. Thompson, C. D. Cole, M. Cotten, T. A. Cross, and D. D. Busath. 2001. Non-contact dipole effects on channel permeation. IV. Kinetic model of 5F-Trp*3 gramicidin A currents. Biophys. J. 81: 1245-1254.

Urry, D. W., M. C. Goodall, J. D. Glickson, and D. F. Mayers. 1971. The gramicidin A transmembrane channel: characteristics of headto-head dimerized Tr(L,D) helices. Proc. Natl. Acad. Sci. U.S.A. 68: 1907-1911.

Wallace, B. A. 1990. Gramicidin channels and pores. Annu. Rev. Biophys. Biophys. Chem. 19:127-157.

Woolf, T. B., and B. Roux. 1997. The binding site of sodium in the gramicidin A channel: comparison of molecular dynamics with solidstate NMR data. Biophys. J. 72:1930-1945.

Woolley, G. A., and B. A. Wallace. 1992. Model ion channels: gramicidin and alamethicin. J. Membr. BioL 129:109-136.

[Author Affiliation]

Joseph A. Gowen,* Jeffrey C. Markham,* Sara E. Morrison,* Timothy A. Cross,^ David D. Busath,* Eric J. Mapes,^^ and Mark F. Schumaker^^

*Zoology Department and Center for Neuroscience, Brigham Young University, Provo, Utah 84602; ^Center for Interdisciplinary Magnetic Resonance at the National High Field Laboratory, Institute of Molecular Biophysics and Department of Chemistry, Florida State University, Tallahassee, Florida 32306; and ^^Department of Mathematics, Washington State University, Pullman, Washington 99164 USA

[Author Affiliation]

Submitted July 31, 2001, and accepted for publication May 21, 2002. Address reprint requests to Dr. Mark F. Schumaker, Dept. of Pure and Applied Mathematics, Washington State University, Pullman, WA 99164. Tel.: 509-335-7273; Fax: 509-335-1188; E-mail: schumaker@wsu.edu.