Engineering Enhanced Pore Sizes Using FhuA Î1-160 from E. coli

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Engineering enhanced pore sizes using FhuA #1-160 from E. coli outer membrane as template Zhanzhi Liu, Ishan Ghai, Mathias Winterhalter, and Ulrich Schwaneberg ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.7b00481 • Publication Date (Web): 20 Oct 2017 Downloaded from http://pubs.acs.org on October 22, 2017

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Engineering enhanced pore sizes using FhuA ∆1-160 from E. coli outer membrane as template Zhanzhi Liu1, Ishan Ghai2, Mathias Winterhalter2*, Ulrich Schwaneberg1,3 1

Institute of Biotechnology, RWTH Aachen University, Worringer Weg 3, 52074, Aachen,

Germany 2

Department of Life Sciences and Chemistry, Jacobs University Bremen, 28719, Bremen,

Germany 3

DWI - Leibniz-Institute for Interactive Materials, Forckenbeckstrasse 50, D-52074 Aachen,

Germany *Corresponding author: Prof. Dr. Mathias Winterhalter Department of Life Sciences and Chemistry, Jacobs University Bremen, Campus Ring 1, 28759 Bremen, Germany. E-Mail: [email protected]

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Abstract Biological membranes are the perfect example of a molecular filter using membrane channels to control the permeability of small water-soluble molecules. To allow filtering of larger hydrophilic molecules we started from the known mutant channel FhuA ∆1-160 in which the ´´cork´´ domain closing the channel had been removed. Here we further expand the pore diameter by copying the amino acid sequence of two ß-strands in a step-wise manner increasing the total number of ß-strands from 22 to 34. The pore size of the respective expanded channel protein was characterized by single-channel conductance. Insertion of additional ß-strands increased the pore conductance but also induced more ion current flickering on the millisecond scale. Further, polymer exclusion measurements were performed by analyzing single-channel conductance in the presence of differently sized polyethylene glycol of known polymer random coil radii. The conclusion from channel conductance of small channel penetrating polymers versus larger excluded ones suggested an increase in pore radii from 1.6 nm for FhuA ∆1-160 up to a maximum of about 2.7 nm for + 8 ß insertion. Integration of more ß-strand caused instability of the channel and exclusion of smaller sized polymer. FhuA ∆1-160 + 10 ß and FhuA ∆1-160 + 12 ß effective radius decreased to 1.4 and 1.3 nm respectively showing the limitations of this approach.

Keywords: membrane protein, molecular filters, FhuA, ß-strands, nanopore, permeability

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Membrane proteins fulfill a variety of functions as diffusion pores, substrate transporters, signal transduction and catalytic conversion 1. Table 1 shows examples of channel forming ß-barrel membrane proteins listed according to their increasing size ranging from 8 to 22 ßstrands. One of the largest known ß-barrel membrane proteins with an open aqueous channel is the ferric hydroxamate uptake protein component A (FhuA) containing 22 ßstrands

2-4

. This ß-barrel channel as nanopore has been extensively been employed in

various fields including single-molecule detection, nanoreactors for conversions and DNA sequencing

5-16

.

Table 1. Representative outer membrane proteins with ß-strands structure. Protein

ß-strands

Number of pores

Organism

Residues

OmpX17

8

1

Escherichia coli

148

20 Å * (32 Å ~ 50 Å)

OmpA18

8

1

Escherichia coli

171

26 Å * 57 Å

NspA19

8

1

Neisseria meningitidis

155

20 Å * 50 Å

OmpT20

10

1

Escherichia coli

297

32 Å * 40 Å

OpcA21

10

1


253

15 Å ~ 23 Å

Tsx22

12

1

Escherichia coli

272

(10 Å ~ 12 Å) * (3 Å ~ 5 Å)

NalP23

12

1


308

10 Å ~ 12.5 Å

Hia24

12

3

Haemophilus influenzae

312*3

29 Å ~ 36 Å

CymA25-26

14

1

Klebsiella oxytoca

~300

13 Å ~ 15 Å

FadL27

14

1

Escherichia coli

427

50 Å

α-hemolysin 28

14

1

Staphylococcus aureus

293*7

26 Å * 100 Å

MspA29

16

1

Mycobacterium smegmatis

184*8

88 Å * 28 Å * 96 Å

OmpF30

16

3

Escherichia coli

340*3

38 Å * 27 Å * 30 Å

PhoE31

16

3

Escherichia coli

340*3

38 Å * 27 Å * 30 Å

Maltoporin32

18

3

Escherichia coli

427*3

5Å~6Å

ScrY33

18

3

Salmonella typhimurium

413*3

(50 Å ~ 28 Å) * (25 Å ~ 50 Å)

BtuB34

22

1

Escherichia coli

594

45 Å * 40 Å * 55 Å

FepA35

22

1

Escherichia coli

724

40 Å * 30 Å * 70 Å

FhuA2-3

22

1

Escherichia coli

714

46 Å * 39 Å * 69 Å

3


Cross*Height

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The outer membrane protein FhuA from Escherichia coli (E. coli) mediates the active transport of ferric siderophores across the outer membrane together with the energytransducing protein TonB 2-4. FhuA has an elliptical cross section of 39 ~ 46 Å, a height of 69 Å 2-3 and harbors two domains: the N-terminal cork domain (amino acids 1-160) blocking the channel and the 22 anti-parallel β-strands barrel domain (amino acids 161-714)2-4. FhuA can genetically be modified under a broad range of experimental conditions because of its remarkable resistance towards elevated temperature, alkaline pH, and organic solvent

5-7

.

After removing the plug, FhuA ∆1-160 became a passive diffusion channel (Figure 1A) 4,10,15

. FhuA ∆1-160 is also an ideal building block for the development of protein-polymer

conjugate in material science and biotechnology. Considerable progress has been made in ßbarrel membrane protein engineering.36-37 The research investigated includes but not least: covalent and non-covalent modification

38-39

, amino acid substitutions mutation

6, 16

, directed

40

evolution , re-design of ß-barrel structure, for example, extension, expansion and truncate of ß-barrel

7, 9, 41

and even artificial ß-barrel membrane proteins generation16,36. Some expansion

of FhuA ∆1-160 was accomplished via replicating the first two N terminal ß-sheets7. However, this modification was not sufficient to allow translocation of large substrates, such as peptide, protein and dsDNA, and further engineering is necessary. In the current work, we enlarged the FhuA ∆1-160 by duplicating stepwise two ß-strands. To quantify the effective change in pore diameter, we measured single ion channel conductance 42,43 in the absence and in the presence of a wide range of differently sized polyethylene glycol (PEG) to conclude on the pore radii by polymer exclusion.

Materials and Methods Materials: All chemicals used were purchased from Applichem (Darmstadt, Germany) or Sigma-Aldrich Chemie (Steinheim, Germany), if not stated otherwise. Spectra™ Multicolor High Range Protein Ladder was purchased from Thermo-Fisher (Massachusetts, USA). 1,2-diphytanoylsn-glycero-3-phosphocholine (DPhPC) was procured from Avanti Polar Lipids (Alabaster, Alabama, USA).

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Methods Cloning of Expanded FhuA ∆1-160 Variants The gene 2 ß encoding 2 ß sheets (primer: 2 ß-F: cgtgatgacaaaGGCGTTTATGTTCAG, 2 ßR:ccaggtaaactgTTTGTCATCACGTTTATC; Lower case letters indicated phosphorothioated bonds) and Back FhuA ∆1-160 encoding backbone of FhuA ∆1-160 (primer: Back-F: cagtttacctggCGTGGTGGTGTTAAC,

Back-R:

tttgtcatcacgTTTATCGGTCGTC)

were

amplified separately by PCR using pPR-IBA1-FhuA ∆1-160 as template. FhuA ∆1-160 + 2 ß was generated by construction of 2 ß and Back FhuA ∆1-160 by PLICing44. After PCR amplification of Back FhuA ∆1-160 + 2 ß encoding backbone of FhuA ∆1-160 + 2 ß using pPR-IBA1-FhuA ∆1-160 + 2 ß as template, FhuA ∆1-160 + 4 ß was established by PLICing of 2 ß and Back FhuA ∆1-160 + 2 ß. By repeating these steps, FhuA ∆1-160 + 6 ß, FhuA ∆1160 + 8 ß, FhuA ∆1-160 + 10 ß and FhuA ∆1-160 + 12 ß were generated (figure 1C) (Amino acids sequence in SI). Further expression and extraction was done as previously described (see SI for details).

Planar Lipid Bilayer and Electrical Recording Planar lipid bilayer was made according to Montal and Mueller and described in detail elsewhere42,43,45,46. Briefly, an aperture in a Teflon septum with a diameter of 80−120 µm was prepainted with hexadecane dissolved in n-hexane at 1-3% (v/v) and the chambers were dried for 20-25 min, to remove the solvent. Bilayers were made with DPhPC at a concentration of 5 mg/ml in n-pentane. Stock solutions of FhuA ∆1-160 variants (0.5-1 µl 1mg protein/ml) were added to the cis side for all the measurements. Standard Ag/AgCl or calomel electrodes were used to detect the ionic current. Note that under the asymmetric condition we used either homemade salt bridges or commercial calomel electrodes (Metrohm AG, Germany)48, 54. The cis side electrode of the cell was grounded, whereas the trans side electrode was connected to the headstage of an Axopatch 700B amplifier (Axon Instruments, Sunnyvale, CA), used for the conductance measurements in the voltage clamp mode. Signals were filtered by an on board low pass Bessel filter at 10 kHz and recorded onto a computer hard drive with a sampling frequency of 50 kHz. Analysis of the current recordings was performed using Clampfit (Axon Instruments, Sunnyvale, CA). Standard solutions contained 1 M KCl, 20 mM HEPES, pH 7.4. 5


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The single channel was reconstituted in the planar lipid bilayer, and the ion current was recorded, providing an indirect conclusion on the channel size. The single-channel conductance recordings were repeated with buffers containing differently sized polymers. If the channel conductance, scales similar than the buffer conductance the polymer is expected to penetrate. Larger polymers are excluded causing high channel conductance as expected from the bulk values 42. We added 10 % (w/v) of an appropriate non-electrolyte (NE). As suggested earlier by Krasilnikov

42, 47-51

48-49

, differently sized polyethylene glycol (PEG) were

used as the polymer of choice as they show little interaction with the channel interior and have spherical shape in aqueous solution. The bulk conductivity of each buffer solution was measured with a multi-range conductivity meter (Knick laboratory conductivity meter 702, Germany) using a 4-electrode sensor (Knick ZU 6985 conductivity sensor, Germany). To elucidate polymer size exclusion, the channel conductance in the presence of differently sized polymers were measured respectively (PEG 600, PEG 1000, PEG 2000, PEG 3000, PEG 4000, PEG 6000, PEG 8000, PEG 10,000 and PEG 12,000) 42, 47-51.

Results Generation of expanded FhuA ∆1-160 mutants FhuA ∆1-160 was selected as the start variant for the further expansion. The 2 ß-strands of FhuA ∆1-160 (G433 to K474, NH2-GVYVQDQAQWDKVLVTLGGRYDWADQESLNRV AGTTDKRDDK-COOH) (Figure 1B and SI) was chosen to serve as building block to be duplicated. This region contained a short loop to reduce the impact on the surrounding ß sheets. FhuA ∆1-160 + 2 ß, FhuA ∆1-160 + 4 ß, FhuA ∆1-160 + 6 ß, FhuA ∆1-160 + 8 ß, FhuA ∆1160 + 10 ß and FhuA ∆1-160 + 12 ß were generated by multi-duplicating of the gene encoding G433 to K474 (Figure 1C). FhuA ∆1-160 and the respective mutants were expressed, extracted and correctly refolded with the presence of hexylene glycol. The extraction of expanded FhuA ∆1-160 variants was analyzed by SDS-PAGE (Figure S1) and refolding of these variants was validated by circular dichroism spectrophotometry (Figure S2).

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Figure 1. (A) Comparison of FhuA WT and FhuA ∆1-160, the cork domain was marked in red and was removed in FhuA ∆1-160. (B) The structure of FhuA ∆1-160 and in green the duplicated 2 ß. (C) Cloning route to construct FhuA ∆1-160 + 2 ß, FhuA ∆1-160 + 4 ß, FhuA ∆1-160 + 6 ß, FhuA ∆1-160 + 8 ß, FhuA ∆1-160 + 10 ß, and FhuA ∆1-160 + 12 ß.

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Characterization of the channel diameter using single channel conductance Single channel conductance has often been used to estimate the pore size using a simplified estimation of an unstructured non-interacting cylinder 42-43. Within this approach, the channel conductance G (equation 1) is G= κ A/l = κ πd2/(4 l)

(1)

Where κ is the bulk conductance and in 1 M KCl it is about 11.2 S/m. A is the channel area and l is the length and is typically assumed to be around 4 nm. Although this equation is widely used to estimate the pore size the channel conductance depends strongly on the channel surface and in particular on the charge distribution and thus on salt concentration and pH. As correctly pointed out by one of our reviewer’s larger channel require corrections in form of an access resistance. However, here in this work, the goal is to conclude on the increase in channel size and such a separation will not provide more information. While waiting for a high-resolution structure the above equation provides a reasonable approximation. In Figure 2 A-D we show typical traces of single channel conductance of the respective FhuA mutants and the average pore conductance is summarized in Table 2. Addition of + 8 ß beta sheets increased the single channel conductance compared to FhuA ∆1160 whereas the conductance drastically reduces on further expansion for FhuA ∆1-160 + 10 ß and FhuA ∆1-160 + 12 ß in 1 M KCl solution. The ion current traces for FhuA ∆1-160 and expanded variants are very noisy and indicate a fluctuating behavior of the narrow channel that likely reflects movement of the extracellular loop region.

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Figure 2. Ion current traces of single FhuA mutants during reconstitution into a solvent free membrane of DPhPC and the corresponding ion current histograms. The single channel size is obtained from the step-wise increase in ion current (A) FhuA ∆1-160 at +150 mV applied voltage, on the right-hand side the ion current amplitude histogram shows a first maximum corresponding to ~2.6 nS for a single channel. Note that in Figure 2A the ion current trace shows initially one followed by two visible steps corresponding to finally 3 channels. (B) FhuA ∆1-160 + 8 ß at +125 mV applied voltage, the ion current amplitude histogram shows a first maximum corresponding to ~3 nS. The final trace contains 6 channels(C) FhuA ∆1-160 + 10 ß into a solvent free membrane of DPhPC at +125 mV applied voltage, the ion current amplitude histogram shows a first maximum corresponding to ~1.6 nS the final trace contains 6 channels (D) FhuA ∆1-160 + 12 ß into a solvent free membrane of DPhPC at +150 mV applied voltage, the ion current amplitude histogram shows a first maximum corresponding to ~1.9 nS. The final trace contains 7 channels Further experimental conditions have been 1 M KCl, 20 mM HEPES salt, pH 7.4 and T= 22 °C. Note that the average single channel conductance is obtain from an analysis of at least 25 steps. 9


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In the next step, we characterized the voltage dependence of the ion current for four mutants (Figure 3 A-D). Starting from FhuA ∆1-160 showing an almost Ohmic behavior without rectifying properties as expected for large channels. For the largest investigated channel (Fig 3D) the ion current saturates at higher applied voltages.

Figure 3. Selected I-V curves from bilayers containing multiple copies of the reconstituted FhuA ∆1-160 and expanded variants channels with symmetric conditions 1 M KCl, 20 mM HEPES, pH 7.4 cis/trans. (A) FhuA ∆1-160 (2 channel) (B) FhuA ∆1-160 + 8 ß (1 channel). (C) FhuA ∆1-160 + 10 ß (40 channel) (D) FhuA ∆1-160 + 12 ß (33 channel). Note that the IV traces were taken after protein insertion and carefully washing out remaining protein to avoid further protein insertion. Evaluation of the channel diameter using polymer exclusion A different approach to estimate the pore size is via polymer exclusion. Herein the pore size of a channel was estimated by polymer size exclusion visible by an apparent enhanced channel conductance. First, the channel conductance was recorded in the absence of polymers 10


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and used as reference. In the next step, the pore conductance was measured in buffer solution containing 10% of the respective PEG. In case of equal partitioning of the polymer into the channel the pore conductance would then be reduced accordingly

42

. Otherwise in case of

polymer exclusion from the channel interior the channel conductance corresponded to the polymer free conductance (Figure 4). Following this, the channel diameter can be estimated using published sizes of the respective polymer coil in the aqueous phase. According to this method, the radius of the constriction zone should be in the range of the radius of the smallest PEG that does not pass freely through the channel 42, 47-51.

Table 2. Average single-channel conductance of four FhuA mutants in the presence of different nonelectrolytes (NEs). Buffer Polymer composition radius

Solution Bulk

Single Channel Conductance G ± SD

r

X

(nm)

(mS cm-1 )

(nS)

Mutant

FhuA ∆1-160 FhuA ∆1-160 + 8 ß

1M KCl

FhuA ∆1-160 + 10 ß

FhuA ∆1-160 + 12 ß

106

2.6 ± 0.9

3 ± 1.3

1.1 ± 0.8

1.8 ± 0.9

PEG 600

0.751

81

1.2 ± 0.5

1.2 ± 0.6

0.5 ± 0.2

0.9 ± 0.5

PEG 1000

0.951

78

0.8 ± 0.4

1.6 ± 0.4

0.4 ± 0.2

1.2 ± 0.4

PEG 2000

1.351

75

1.2 ± 0.6

1.4 ± 0.5

0.5 ± 0.2

1.4 ± 0.6

PEG 3000

1.447

74

1.1 ± 0.4

1.4 ± 0.6

0.8 ± 0.3

2.9 ± 0.5

PEG 4000

1.651

77

2.6 ± 0.76

1.7 ± 0.6

1.1 ± 0.4

1.9 ± 0.6

PEG 6000

2.751

77

3.1± 0.9

1.25 ± 0.7

1.08 ± 0.3

1.9 ± 0.3

PEG 8000

349

75

2.7 ± 0.9

2.9 ± 0.6

1.8 ± 0.72

2.0 ± 0.8

PEG 10,000

3.549

73

3.0 ± 0.7

2.9 ± 0.6

1.6 ± 1.0

1.9 ± 0.8

PEG 12,000

3.949

73

2.5 ± 0.8

3.0 ± 0.8

1.9 ± 0.8

1.9 ± 1.0

11


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Average single-channel conductance G and its standard deviation SD was calculated from conductance steps. The aqueous phase contained 1 M KCl buffered with 20 mM HEPES and the corresponding nonelectrolyte at a concentration of 10 % (w/v). r = polymer radius obtained from the literature, or calculated from the fitting equation49 r = 0.508 + 0.37 10-3 Mw – 0.703 10-8 (Mw)2, X = Bulk conductivity of the aqueous solutions.

Figure 4. Ratio of the channel conductance in presence of 10 w/v vs. in absence of PEG in solution. (A) FhuA ∆1-160, (B) FhuA ∆1-160 + 8 ß, (C) FhuA ∆1-160 + 10 ß, (D) FhuA ∆112


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160 + 12 ß respectively. Experimental conditions are 1 M KCl, 20 mM HEPES, pH 7.4 and at 22°C. The bars indicate standard errors.

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As shown in Figure 4A FhuA ∆1-160 larger PEGs with radii above (>PEG 4000) did not affect the conductance suggesting to be excluded. In contrast, the presence of PEG 600, 1000, 2000, 3000 (radii of 0.7, 0.9, 1.3 and 1.4 nm, respectively) in the bathing solution resulted in a low single-channel conductance of 1.2, 0.8, 1.2, 1.1 nS. The interpretation for this effect is that smaller sized PEG may penetrate into the channel. In contrast, larger MW PEGs are excluded giving space for ions with an apparent higher ion concentration. Using this approach, a conclusion about the size of the FhuA ∆1-160 channel can be estimated around 1.4 ± 0.3nm. In Figure 4B FhuA ∆1-160 + 8 ß, channel showed no conductance drop for PEG 8000, 10,000, 12,000, respectively. In contrast, a conductance drop was observed for the PEG solution of 600, 1000, 2000, 3000, 4000, and 6000, respectively. From the data shown, it can be concluded that PEG 6000 can enter the channel and thus the size of the channel FhuA ∆1160 + 8 ß can be estimated around 2.7 ± 0.6 nm, which is substantially higher than the previous non-expanded variant. Figure 4C shows FhuA ∆1-160 + 10 ß having no reduction in the conductance values for PEG 4000, 6000, 8000, 10,000 and 12,000, respectively, where as in the presence of PEG solution of 600, 1000, 2000, 3000 a reduction in the current was observed hence from the values the size can be estimated in the range of 1.4 ± 0.3 nm. For FhuA ∆1-160 + 12 ß (Figure 4D) no reduction in the conductance values for PEG solution of 3000, 4000, 6000, 8000, 10,000 and 12,000 was observed and PEG solution of 600, 1000, 2000 showed significant reduction of conductance, respectively, which showed the approximate channel size in the range of about 1.3 ± 0.6 nm.

Discussion In this work, we investigated artificially expanded variants of FhuA ∆1-160. One challenge in membrane protein design is accomplishing extensive modifications of proteins without impairing their functionality. Starting from FhuA ∆1-160, we successfully increased the pore diameter stepwise by doubling the amino acid sequence of two ß-strands increasing the total number of ß strands from 22 to 30 to make a large conductance protein nanopore. FhuA ∆1160 + 8 ß, FhuA ∆1-160 + 10 ß and FhuA ∆1-160 + 12 ß were generated, and all of the expanded variants could be expressed in E.coli and refolded into ß-barrel in the presence of detergent. Then single-molecule electrophysiology and PEG size exclusion measurement were performed to determine the cut off size of channel diameter of expanded FhuA ∆1-160 variants. The result suggested that FhuA ∆1-160 and FhuA ∆1-160 + 8 ß channels showed the 14


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radius of 1.5 and 2.7 nm respectively, whereas for FhuA ∆1-160 + 10 ß and FhuA ∆1-160 + 12 ß the effective radius decreased to 1.4 and 1.3 nm respectively showing the limitations of this expansion process. Here we obtained an optimum for FhuA ∆1-160 + 8 ß. While waiting for a high-resolution structure typically channel conductance and polymer size exclusion are used to get an estimated size. Although both methods characterize only indirectly the pore diameter size and provide only an estimation due to the high flexibility of the polymer coil, this is the best what can be done.

Conclusion This study opens a direction for extensive engineering of a large monomeric β-barrel protein nanopore. The cut off sizes of expanded FhuA ∆1-160 variants were determined by PEG size exclusion and the FhuA ∆1-160 + 8 ß showed the largest channel allowing up to PEG 6000 to enter. The expanded FhuA variants could be employed to insert and functionalize the membrane and offers novel opportunities in the separation of large molecules and small peptides, and it sheds light on the sensor for the protein detection, DNA sequencing and even artificial tissue52-54. Associated Contents Supporting Information: Amino acids sequence of

FhuA variants, Expression and

Purification, CD curve of FhuA variants. Author Contributions: M.W., U.S. and I.G designed the work. Z.L. purified the proteins. Z.L and I.G. performed the experiments. All authors were involved in the writing the manuscript. Acknowledgment Z. L. is supported by a Ph.D. scholarship from China Scholarship Council (CSC No. 201306350039). The authors declare no competing financial interest. References

1. Koebnik, R.; Locher, K. P.; Van Gelder, P., Structure and function of bacterial outer membrane proteins: barrels in a nutshell. Mol Microbiol 2000, 37 (2), 239-253. 2. Locher, K. P.; Rees, B.; Koebnik, R.; Mitschler, A.; Moulinier, L.; Rosenbusch, J. P.; Moras, D., Transmembrane signaling across the ligand-gated FhuA receptor: crystal structures of free and ferrichrome-bound states reveal allosteric changes. Cell 1998, 95 (6), 771-778. 3. Ferguson, A. D.; Hofmann, E.; Coulton, J. W.; Diederichs, K.; Welte, W., Siderophoremediated iron transport: crystal structure of FhuA with bound lipopolysaccharide, 10.1126/science.282.5397.2215. Science 1998, 282 (5397), 2215-2220. 15


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Engineering Enhanced Pore Sizes Using FhuA Î1-160 from E. coli

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Engineering Enhanced Pore Sizes Using FhuA Î1-160 from E. coli