Iván Bárcena Uribarri†1, Marcus Thein1, Elke Maier1, Mari Bonde2, Ignas Bunikis2, Sven Bergström2 and Roland Benz1

1 Biozentrum University of Würzburg, D- 97974 Würzburg, Germany.

2 Department of Molecular Biology, Umeå University, Sweden. †Corresponding author: ivan.barcenauribarri@uni-wuerzburg.de

Channel size and oligomeric constitution

of the Borrelia burgdorferi P66 porin

Borrelia species are obligate parasites transmitted to mammals by ticks. Borrelia have limited biosynthetic capacity [1] and, therefore, they are dependent on nutrients provided by their hosts. The first step of

nutrient availability is accomplished by water-filled channels, so called porins, across the outer membrane of these bacteria. P66 is so far the best studied porin in Borrelia showing dual function, acting not only

as a porin but also as an adhesin [2,3]. P66 form pores in planar lipid bilayers with a conductance of 11 nS in 1 M KCl [2] which is an atypical and rare high single channel conductance for Gram-negative

bacterial porins [4]. Previous estimations of P66 channel diameter led to a diameter estimation of 2.6 nS [2]. This calculation of the P66 channel diameter was based on the assumption that the conductance of

the channel is equal to the conductivity of a simple cylinder of aqueous salt solution. Therefore, the calculated value of the P66 diameter appears to be somewhat preliminary and its apparent size and structure

remain unclear. An applied method using nonelectrolytes with known hydrodynamic radii [5] was used to calculate the real diameter of P66. This method should provide a more accurate estimate of the P66

channel diameter using a biophysical approach.

Planar Lipid Bilayer Assay Use of Nonelectrolytes to Determine the Channel Diameter

Results (1): P66 Pore Diameter

Results (2): Blockage of P66

and Oligomeric Structure

Nonelectrolyte G

(nS)

r

(nm)

χ

(mS

cm-1)

F%

None 11 - 110.3 -

Ethylene glycol 6.5 0.26 57.2 96.8

Glycerol 5.5 0.31 49.1 106.7

Arabinose 7 0.34 63.7 100.6

Sorbitol 7.5 0.39 57.8 65.2

PEG 200 7.5 0.50 46.1 64.1

PEG 300 7.5 0.60 45.5 42.6

PEG 400 1 0.70 46.4 -

PEG 600 1 0.80 54.1 -

PEG 1000 12 0.94 49.5 9.0

PEG 3000 14 1.44 48.9 5.2

PEG 6000 10.5 2.50 50.5 5.2

0

20

40

60

80

100

120

0,0 0,5 1,0 1,5 2,0

Hydrodynamic radius (nm)

Co

nd

uc

tan

ce

(%

)

NE inside

the channel

NE too big to be

inside the channel

NE block the channel

What are nonelectrolytes?

-substances that are not charged when dissolved in

aqueous solutions. They should have a spherical

shape with a particular radius when dissolved in

water (Table 1).

Principles:

- NEs, when added to salt solutions, increase the

viscosity and thus decrease their conductivity (20%

NEs reduce the aqueous conductivity to 50-

60%)(Table 1).

- This decrease in the conductivity will only affect the

channel conductance when the NEs enter the

channel interior (Fig. 6).

K+

Cl-

Cl-

K+

K+

Cl-

Cl-

K+

Radius of the constriction zone:

should be equal to the radius of the smallest NE

that do not pass freely trough the channel and

therefore don’t fill it 100%.

Radius of the channel entrance:

should be equal to the radius of the smallest NEs that do not enter the pore.

Channel filling concept: Finding a inner constriction.

- The portion of a channel filled with an nonelectrolyte

can be determined using the following formulas:

F%=100

F%= 0

F%= 30-80

F = [(Go-Gi)/ Gi]/[(Χo -Χi)/ Χi]

F% = 2Fi/(F1+F2)*100%

Instrumentation:

-A Teflon chamber with two compartments containing a

1M KCl salt solution (Fig.1). The two compartments are

separated by a thin wall and connected by a 0.4 mm2

small circular hole. The membranes are formed

spreading a 1% (w/v) solution of diphytanoyl

phosphatidylcholine (PC) in n-decane over the hole

(Fig.2).

-Ag/AgCl electrodes (Fig.1)

-Voltage source (Fig.3)

-Amplifier (Fig.3)

-PC/Recorder (Fig.3)

F%

100%

60%

0%

Large nonpermeant NEs with hydrodynamic radii between 0.94

and 2.50 nm did not enter the P66 channel and showed no

effect on its conductance. However, in the presence of small

NEs with hydrodynamic radii up to 0.60 nm, the P66 single-

channel conductance decreased proportional to that of the bulk

solution conductivity (Fig.8).

Surprisingly, the presence of PEG 400 and PEG 600 resulted in

an exceptional low single-channel conductance of 0.9 nS that

was not proportional to the bulk aqueous conductivity (Fig.8).

To determine a possible constriction the results of the dependence

of F% on the hydrodynamic radii of the NEs are shown in figure 9.

The estimation of the P66 pore size based on our single-channel

measurements with different NEs indicated an entrance pore

diameter of approximately 1.9 nm with a 0.8 nm inner constriction.

References:

Membrane experiments in the presence of 20 % PEG

400 or PEG 600 resulted in drastically reduced single-

channel conductance during multi-channel

measurements which revealed that the P66

conductance could be blocked by 80-90% after the its

addition (Fig.10).

The size of the channel as derived from measurement with

NEs does not agree with its extremely high single-channel

conductance of about 11 nS in 1 M KCl. Furthermore, the

stepwise block of a single P66 unit with certain NEs

occurred in seven substates (Fig. 11). All these results

suggested that the P66 channel may be formed by a bundle

of pores.

To support this view, purified P66 was investigated by Blue

native PAGE, a method that allows the determination of native

protein masses and oligomeric states of protein complexes. A

460 kDa band agree with the oligomeric theory as a P66

heptamer would have a molecular mass of 462 kDa.

Fig. 1:Teflon chamber

and Ag/AgCl electrodes. Fig. 3: Black Lipid assay set.

Fig. 2: Bilayer Formation. Multicolor multilayers lead to the

formation of only one bilayer (black)

Fig. 4: Porin inserted in a

lipid bilayer

Fig. 5: Step-like record from

a pore forming sample.

Principles:

- When added to a KCl solution,

protein samples with pore-forming

activity get inserted in the PC

membranes increasing its

conductance (Fig.4). Each

insertion is registered by the

recorder as a step (Fig.5).

Fig. 6: Effects of NEs in the

conductance of a porin.

Fig. 7: Channels filled by NEs to different degrees.

Fig. 8: Effects of different NEs in the conductance of P66.

Fig. 9: P66 channel filling with different NEs .

Table 1: Conductance of P66 in presence of

different NEs (G), NEs hydrodynamic radius (r),

solution conductance (χ) and channel filling in terms

of percentage (F%)

Fig. 10: Blockage of approximately hundred P66 channels with PEG600.

Fig. 11: Blockage of a single P66 unit with PEG400.

Fig. 12: BN Page and WB for native P66.

[1] Fraser et al. Genomic sequence of a Lyme disease spirochaete, Borrelia burgdorferi, Nature 390 (1997) 580-586.

[2] Skare et al. The Oms66 (p66) protein is a Borrelia burgdorferi porin, Infect Immun 65 (1997) 3654-3661.

[3] Coburn et al. Characterization of a candidate Borrelia burgdorferi beta3-chain integrin ligand identified using a phage display library, Mol Microbiol 34 (1999) 926-940.

[4] Benz, R. Porin from bacterial and mitochondrial outer membranes. CRC Crit Rev Biochem (1985) 19:145-190.

[5] Krasilnikov et al. A novel approach to study the geometry of the water lumen of ion channels: colicin Ia channels in planar lipid bilayers. J Membr Biol (1998)161:83-92.

Conclusions The extremely high conductance of P66 gives an idea

of a big channel that would allow a free molecule

exchange between the environment and the periplasmic

space. This fact could impair the defense function of the

outer membrane and it makes it difficult to understand

why small pores like Oms38 are next to such big

channels.

Using the nonelectrolytes method, a constriction zone

could be determined with a diameter of 0.8 nm. Such

an estimation is much smaller than a previous one of

2.6 nm which was based on theoretical considerations.

Single P66 blockage experiments with some NEs lead

to the idea of P66 being an association of smaller

channels and not a big hole in the outer membrane.

This fact could explain the high conductance of P66.

If that is the case, P66 could be the first known example

of a porin constituted by a bundle of seven independent

channels in a protein complex. Such a structure is until

today only observed in Borrelia, but not in any other

bacterium or any other living organisms