Proceedings

Conference onNinth InternationalNinth International

Conference on

FlowDynamicsFlowDynamics

Global COE Program “World Center of Education and Research for Trans-disciplinary Flow Dynamics”Tohoku University

Hotel Metropolitan Sendai, JapanHotel Metropolitan Sendai, JapanSeptember 19-21,2012September 19-21,2012

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OS8: Membrane Micro Channel for Health Care

FUJISeptember 20, 2012

Chair: Makoto Ohta (Tohoku University, Japan) OS8-18:00-8:20

OS8-28:20-8:40

OS8-38:40-9:00

Structural Elements which Control the Signal Transduction Pathway of GPCR.(Invited)Minoru Sugihara (Aoyamagakuin University, Japan), Makiko Suwa (Aoyamagakuin University, Japan / National Institute of Advanced Industrial Science and Technology, Japan)

Computational Detection of Organelle Sorting Signals in Membrane Localized Proteins (Invited) Yuri Mukai, Hirotaka Tanaka, Tatsuya Konishi, Hiromu Takata, Naoyuki Takachio (Meiji University, Japan), Masami Ikeda (Aoyama Gakuin University, Japan) and Takanori Sasaki (Meiji University, Japan)

Electrophysiological Properties of Bacterial Membrane Channel Proteins Depending on Lipid Component and Bilayer Stability (Invited) Noriko Tomita (Tohoku University, Japan), Liviu Movileanu (Syracuse University, USA) and Makoto Ohta (Tohoku University, Japan)

FUJISeptember 21, 2012

Chair: Noriko Tomita (Tohoku University, Japan) OS8-48:00-8:30

OS8-58:30-9:00

Engineering Membrane Protein Nanopores for Biomolecular Detection (Invited)Mohammad M. Mohammad, Raghuvaran Iyer, Khalil R. Howard (Syracuse University, USA), Mark P. McPike (AptaMatrix, Inc., USA), Philip N. Borer (Syracuse University, USA / AptaMatrix, Inc., USA) and Liviu Movileanu (Syracuse University, USA)

Crosstalk between Helicobacter pylori and Host Cells (Invited)Hitomi Mimuro (The University of Tokyo, Japan)

September 19, 2012 in US

OS8-1 7:00-7:20 pm in US OS8-2 7:20-7:40 pm in US OS8-3 7:40-8:00 pm in US

September 20, 2012 in US

OS8-4 7:00-7:30 pm in US OS8-5 7:30-8:00 pm in US

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Electrophysiological Properties of Bacterial Membrane Channel Proteins Depending on Lipid Component and Bilayer Stability

Noriko Tomita1, Liviu Movileanu2, 3, 4, and Makoto Ohta1

1. Institute of Fluid Science, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai, Japan2. Department of Physics, Syracuse University, Syracuse, New York, USA

3. Structural Biology, Biochemistry, and Biophysics Program, Syracuse University, Syracuse, New York, USA4. Syracuse Biomaterials Institute, Syracuse University, Syracuse, New York 13244, USA

tomita@biofluid.ifs.tohoku.ac.jp

ABSTRACTActivities and structures of membrane proteins are affected by the lipid bilayer environment. We herein show diversity in channel properties of engineered ferric hydroxamate protein uptake component A (FhuA ∆C/∆4L), an outer membrane receptor of Escherichia coli, on planar lipid bilayers consisting of various types of phospholipids. Single-channel electrical analysis revealed that the shape of phospholipids affects the sensitivity of the FhuA ∆C/∆4L channel to closure under high-voltages. This study expands the versatility of the engineered FhuA ∆C/∆4L channel, making the channel suitable for biosensors and nanodevices.

1. IntroductionFhuA (Ferric hydroxamate protein uptake component

A) is a ligand-gated channel in the outer membrane of Escherichia coli, which forms a monomeric 22-stranded β-barrel protein [1]. Based on the versatile properties of the FhuA channel, including dynamics, robustness, and thermal stability, focus has been placed on its application as a stochastic biosensing element [2]. Mohammad et al. have created new engineered FhuA ∆C/∆4L by a removal of almost one-third of the total number of amino acids of the wild-type FhuA (Fig. 1) [3], and revealed that FhuA ∆C/∆4L forms a functional, open pore in planar lipid bilayers with a more stable and greater conductance than the other engineered FhuA proteins recorded previously [3], which implied FhuA ∆C/∆4L has promising potential to be applied as a more useful stochastic sensor for protein and DNA

While protein engineering techniques have been applied to improve its utility and advantages as stochastic sensor, electrophysiological properties of the FhuA ∆C/∆4L affecting the lipid bilayer environment,such as gating with conformational change and voltage-dependency, had yet to be elucidated. In this study, we investigate the effect of the phospholipid component on the voltage-dependent gating of the FhuA ∆C/∆4L channel by using single-channel electrical analysis in planar lipid bilayer. We selected either neutral or negative-charge, cylindrical or cone-shape, and different lipid mixtures to explore a wide variety of lipid types that might effect gating. This work should contribute not only to providing new insights into the FhuA ∆C/∆4L channel properties and their dependence on the membrane environment, but also the expansion of the availability of the FhuA ∆C/∆4L channel as a nanodevice that can be regulated by phospholipid component.

2. Method2.1. Preparation on planar lipid bilayers

For preparation of the planar lipid bilayer, we used five types of phospholipid, 1,2-diphytanoyl-sn-glycerophosphatidylcholine(DPhPC), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine, (DPhPE), 1,2-diphytanoyl-sn-glycero-3-phospho-L-serine, (DPhPS),

N-palmitoyl-D-erythro-sphingosylphosphorylcholine (SM) and E. coli Polar Lipid Extract (ECTPL) containing of PE (phosphatidylethanolamine):PG (phosphatidylglycerol):CL (cardiolipin) in a volume ratio of 6.7:2.3:1.0. These phospholipids were purchased from Avanti Polar Lipids (Alabaster, AL). Powders of DPhPC, DPhPE, DPhPS and ECTPL were solubilized with pentane at a concentration of 10 mg/mL. SM powder was solubilized with a mixture of chloroform and methanol (2:1, v/v). Phospholipid solutions for bilayer formation were prepared by mixing DPhPE and DPhPC in a volume ratio of 3:7 and 1:1, and SM:DPh in a volume ratio of 1:1. To investigate an effect of charge, DPhPS was used without being mixed with DPhPC..

2.2 Preparation of the FhuA C/ 4L proteinThe construction of the plasmid for the expression of

the engineered FhuA ∆C/∆4L protein nanopores and modifications of the protocol for obtaining the FhuA ∆C/∆4L protein nanopore through rapid-dilution refolding have been performed as described previously [3]. Briefly, 40 L of His+-tag purified, denatured FhuA ∆C/∆4L was diluted 50-fold into a 1.5 % n-Dodecyl--D-maltopyranoside (DDM), in 50 mM Tris-HCl, containing 200 mM NaCl, 1 mM EDTA, pH 8.0. The diluted protein samples were left overnight at 23°C to complete the refolding of FhuA C/∆4L protein.

2.3. Electrical recordings on planar lipid bilayersThe cis and trans chambers (1.5 ml each) of the

apparatus were separated by a 25-µm-thick Teflon septum (Goodfellow Corp., Malvern, PA). An aperture in the septum, ~ 80–120 µm in diameter, was pretreated with hexadecane (Sigma-Aldrich Co. LLC. St. Louis, MO) dissolved in highly purified pentane (Fisher HPLCgrade, Fair Lawn, NJ) at a concentration of 10% (v/v). DPhPE: DPhPC = 3:7, DPhPE: DPhPC = 1:1, SM: DPhPC = 1:1, ECTPL and DPhPS bilayers were formed across the aperture. Single channels of the engineered FhuA ∆C/∆4L protein were obtained by adding purifiedand refolded protein into the cis chamber at a final amount of 150-300 ng. Electrical recordings were carried out an Axon 200B patch clamp amplifier (Axon

Ninth International Conference on Flow DynamicsSeptember 19-21, 2012, Sendai, Miyagi, Japan

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Instruments, Foster City, CA) in the voltage-clamp mode. Data was collected by an Intel Core Duo PC (Dell, Austin, TX) connected to a Digidata 1440A (Axon Instruments). Output was filtered using a Frequency Devices Model 900B 8-pole Bessel filter (Frequency Devices, Ottawa, IL) at 10 kHz. The acquisition rate was 50 kHz.

2.4. Analysis of voltage-dependent channel propertiesA first closure time (FCT) of the channel is defined as

the number of seconds taken for the channel to enter its first closure state, normalized to the total duration of the experiment, 60 seconds. If the channel did not close within 60 seconds, the normalized FCT (NFCT) was calculated as 1. The three distinct single-channel experiments were performed for analyzing the NSS, the number of states, and NFCT on each kind of lipid bilayer.

4. ResultsWe investigated the sensitivity of FhuA C/ 4L

channel for closure under high-voltage when reconstituted in lipid bilayers that include cylindrical phospholipids (Bcyl) and cone-shaped phospholipid (Bcon). Figure 5A show representative traces of FhuA

C/ 4L single channel at + 180 mV on Bcyl (upper panel) and Bcon (lower panel). As shown in Fig. 5B, the NFCTs of the channels on the SM: DPhPC = 1:1 and DPhPS bilayers, which are Bcyl bilayer, are significantly greater than those on the DPhPE: DPhPC = 3:7, DPhPE: DPhPC = 1:1 and ECTPL bilayer, which are Bcon bilayer. On the contrary, there are no significant differences between the Bcon bilayers and between the Bcyl bilayers (Tukey-Kramer method). While, channel closing did not take place on any of the bilayer at + 40 mV.

Taken together, the FhuA ∆C/∆4L channels are susceptible to high voltage for closing and the degree of sensitivity will be influenced by the shape of phospholipid rather than by lipid charge.

4. DiscussionBy adding cone-shaped phospholipids into a Bcyl,

spontaneous curvature stress is produced within the bilayer and lateral pressure is imparted on the embedded membrane protein [4]. Actually, previous study reported that thermodynamic stability and folding efficacy of the Escherichia coli -barrel outer membrane proteins (OMPs) increased by insertion of PE into PC bilayer [5], and the frequency of gating of the E. coli OmpG channel was changed by lipid charge [6]. Our results in the present study, in which the conductance, the NSS and the number of state of FhuA C/ 4L channel were similar but the sensitivities to high-voltage for closure were significantly different between Bcon and Bcyl, indicate the shape of the phospholipid can alter the sensitivity of the FhuA C/ 4L channel without any substantial change in the structure. Because there are no significant differences in the NFCTs between DPhPE:DPhPC = 3:7 and 1:1 bilayers, the ratio of cone-shaped

phospholipid to cylindrical-shaped phospholipid does not appear to affect high-voltage sensitivity of the channel. Our result indicates a little difference in the number of states for closure between SM: DPhPC and DPhPS bilayers. However, further analysis is needed to evince whether the significant difference could be induced by lipid charge.

To our knowledge, this is the first experimental study suggesting that the electrophysiological properties of -barrel membrane protein could be affected by lipid shape. The phospholipid shape-dependent nature of FhuA ∆C/∆4L channel can expand the versatility of the FhuA ∆C/∆4L channel for nanopore engineering. Thefeature of FhuA ∆C/∆4L channel formed on Bcon, which can keep opening for a longer time than that on Bcyl at the high-volatge, should be useful for future stochastic sensing applications, allowing for the measurements to be taken at high voltage, thus improving the signal to noise ratio, for long periods of time.

AcknowledgementsThe authors thank Jiaming Liu, Belete R. Cheneke

and Aaron J. Wolfe from Syracuse University for their technical assistance. This work was partially supported by a Grant-in-Aid for JSPS Fellows (RPD), Young Researcher Overseas Visits Program for Vitalizing Brain Circulation (IFS, JSPS) and a Global COE Program Grant of the World Center of Education and Research for Trans-disciplinary Flow Dynamics from Tohoku University.

N. Tomita is the recipient of a postdoctoral fellowship from JSPS.

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