Title Page
1
Summary
2
Introduction
3
Experimental Procedures
Expression and Purification of P190His6 Mutants — The 190-residue hexahistidinetagged channel-forming domain of colicin E1 (p190His6) was purified from a lex A
strain of E. coli (IT3661), which contained the pSKHY plasmid that results in constitutive
production of the P190 protein. Mutant proteins were prepared as previously described
(1). Transformed cells were grown overnight (> 16 hours; OD600  1.2) in LB-media
with 100 g/mL ampicillin. Cells were harvested by centrifugation (5000 x g for 5 min),
resuspended in a loading buffer (50 mM sodium phosphate, 500 mM NaCl, 5 mM
imidazole, pH 7.2), and lysed at 15,000 p.s.i. using a French press. The lysate was spun
down at 31,000 x g for 30 min, filtered (2x with two Whatman #5 filter papers, followed
by 1x with Whatman #1 filter paper). The filtrate was further spun down at 31,000 x g
for 30 min and then loaded on to Zn2+-charged chelating Sepharose Fast Flow resin
(Amersham Pharmacia Biotech AB, Baie D'Urfé, Quebec, Canada) that was equilibrated
with the loading buffer. Upon loading, the column was washed with 20 bed volumes of
the loading buffer, followed by further washing with a wash buffer (50 mM sodium
phosphate, 500 mM NaCl, 15 mM imidazole, pH 7.2). The protein was eluted using an
elution buffer (50 mM sodium phosphate, 150 mM NaCl, 25 mM EDTA, pH 7.2) and
dialyzed against 50 mM sodium phosphate, 150 mM NaCl, pH 7.0 buffer. The purity of
the protein samples was assessed by SDS-PAGE, and all mutant proteins were at least
90% pure. The protein concentration was determined by UV absorption at 280 nm using
an extinction coefficient of 28,590 M–1 cm–1 (an 280 of 27, 310 M–1 cm–1 was used for
Y356C and Y363C).
Fluorescence Labeling of P190H Mutants — The fluorescent labeling of all mutants was
accomplished using a 20-fold molar excess of monobromobimane (mBBr; Molecular
Probes, Eugene, OR)) in 200 mM Tris-HCl, pH 8.1, at room temperature for 2 hours.
Prior to the addition of the fluorescent label, protein samples were reduced with a 5-fold
molar excess of dithiothreitol (DTT) for 30 minutes. At the end of the labeling reaction,
unreacted mBBr were quenched with an excess amount of DTT. The labelled protein was
separated from free label using a pre-packed BioRad Econo-Pac 10DG column (Bio-Rad
4
Laboratories, Mississauga, Ontario, Canada) equilibrated and eluted with pH 7.0 PBS
buffer. The absorbance spectrum of each labeled protein was obtained using a Cary 300
UV-Vis dual-beam spectrophotometer (Varian Instruments, Mississauga, Ontario,
Canada). The stoichiometry of the protein-mBBr conjugation reaction was calculated as
the molar ratio of conjugated mBBr and labeled proteins. The molar concentrations of the
conjugated mBBr and protein were determined from the absorbance at 380 and 280 nm
using the molar extinction coefficients () of 5,000 and 28,590 M–1 cm–1, respectively (an
280 of 27, 310 M–1 cm–1 was used for Y356C and Y363C). The contribution to the
absorbance at 280 nm from mBBr was accounted for prior to the calculation of the
protein concentrations. The labeling efficiency of all proteins was nearly 100%.
In addition to the introduced cysteine residues, all P190His6 possessed one
additional cysteine residue, C-505, which is buried within the core of the protein.
Labeling of the buried sulfhydryl side chain of C-505 has been shown previously to
require the partial unfolding of the protein (Musse and Merrill, 2003).
Incubation of
excess mBBr with WT p190His6 resulted in negligible background labeling (<1%).
Therefore, given the near 1:1 stoichiometry of the labeling reactions and the lack of
mBBr labeling of the WT protein, all the fluorophores were presumed to be attached to
the introduced cysteine residues and the buried C-505 did not participate in the labeling
reaction.
Preparation of Vesicles — Large unilamellar vesicles (LUVs) were prepared from 1,2dioleoyl-sn-glycero-3-phosphocholine
(DOPC)
and
1,2-dioleoyl-sn-glycero-3-
[phosphorac-(1-glycerol)] (DOPG) (Avanti Polar Lipids, Alabaster, AL) in 60:40% molar
ratio by extrusion through a 100-nm polycarbonate filter (Lipofast, Aventin Inc., Ottawa,
Ontario, Canada) in 20 mM DMG, 130 mM NaCl, at pH 4.0 as described earlier (Tory
and Merrill, 1999, Hope et al. 1985).
The phospholipid concentration was determined
using the Bartlett assay for phosphorus as described by New (New, R. R. C. 1990).
Steady-State Fluorescence Measurements of Mutants — All steady-state fluorescence
measurements were made with a PTI-Alphascan-2 spectrofluorometer (Photon
Technologies Inc., South Brunswick, NJ) equipped with a thermostated cell holder.
5
Unless otherwise stated, a 2.5 – 4.0 M protein sample in 20 mM DMG, 130 mM NaCl at
pH 4.0 - in the presence or absence of excess LUVs (800 M, final conc.) - was used And
for all measurements the temperature was maintained constant at 22 °C.
Structural and Functional Assessments — The folding properties of all mutants were
examined by monitoring the intrinsic tryptophan (Trp) fluorescence of each protein
compared to the WT. Trp fluorescence emission spectra of labeled and unlabeled mutant
proteins in pH 7.0 PBS buffer were obtained using 295-nm excitation light. The
excitation and emission slit-widths were 2 nm and 4 nm, respectively and the
fluorescence emission was scanned from 305 to 450 nm. Corrections were made for the
appropriate blanks and for the wavelength-dependent bias of the optical and detection
systems. The fluorescence emission maximum (λem max) values were determined from the
first derivative of the smoothed spectra. These data were used to assess the structural
integrity of the mutant proteins as compared with the WT protein. In addition, the in vitro
channel activity of all mutants was also assayed as described earlier (Musse and Merrill,
2003).
mBBr Fluorescence Emission Spectra - The steady-state fluorescence emission spectra,
in the presence or absence of LUVs, were measured from 395 to 600 nm while exciting at
381 nm. The excitation and emission slit-widths were kept at 2.0 nm and 4.0 nm,
respectively.
To minimize vesicle light scattering, a 390 nm cut-off filter (Oriel
Corporation, Rockford, IL) was placed in the emission light path. The signal from solvent
blank (buffer or buffer plus LUVs) was subtracted from the sample fluorescence, and the
spectra were corrected for the wavelength dependence of the instrument response.
Quantum Yield Measurements— The mBBr fluorescence quantum yields (QF), in the
presence and absence of LUVs, of all mutants were obtained using quinine sulfate (QF =
0.53 in 0.1 N H2SO4) as a standard reference (Fletcher, 1969). The excitation wavelength
was set at 360 nm while scanning the emission spectra from 370 to 600 nm in 1 nm
increments.
The excitation and emission slit-widths were 4.0 nm and 6.0 nm,
respectively. The absorbance at the excitation wavelength for the various samples ranged
6
between 0.05 and 0.1. To minimize light scattering effects, the excitation light was
vertically polarized and the emission was detected at 54.7o (magic angle). The signal
from the solvent blank (buffer or buffer plus LUVs) was subtracted from the sample
fluorescence, and the spectra were corrected for the wavelength dependence of the
instrument response. Using the wavelength-integrated fluorescence intensities and the
optical densities at the excitation wavelength of the sample and the reference, the
quantum yield was calculated as:
QF  QR
I AR
IR A
Where QR is the quantum yield of the reference sample; I and IR are the integrated
fluorescence intensities of the sample and the reference, respectively; A and AR are the
optical densities, at the excitation wavelength, of the sample and the reference,
respectively. The reported values represent the mean of at least three determinations.
Steady-State Fluorescence Anisotropy Measurements — The steady-state fluorescence
anisotropy (r) measurements were made using "T-format" detection by simultaneously
comparing the intensities of the vertically (IVV) and horizontally (IVH) polarized emitted
light when the sample was excited with a vertically polarized light. Using the IVV and IVH
fluorescence intensities, the anisotropy (r) was calculated as:
r
I VV  GIVH
I VV  2GIVH
The "G" instrumental factor, measured as IHV/IHH, was determined from the intensities of
the vertically (IHV) and horizontally (IHH) polarized emitted light from horizontally
polarized excitation light. For all measurements, the excitation was set at 381 nm (4 nm
slit-width) and emission was collected at 470 nm (10 nm slit-width) with a signal
integration time of 30s. Each anisotropy value is the mean of three determinations. A
solvent blank (either buffer alone or buffer plus LUVs) was subtracted from each
intensity reading prior to the calculation of the anisotropy value.
7
Fluorescence Lifetime Measurements — The fluorescence lifetimes of the labeled
mutants were performed using a PTI LaserStrobe model GL3300 (James et al., 1992).
The excitation source was a pulsed nitrogen laser, operating at 10 Hz, and a dye laser.
The fluorescence decay, following excitation at 381 nm, for each sample was measured
while monitoring the emission at 470 nm as well as through a long pass filter (>470 nm)
(identical results were obtained with either).
The emission slit-width was set to 4 nm.
Data were collected at 65 ps/channel in 500 channels in a 50 ns time window as the
average of 15 signals per channel. The instrument response function was determined
from a scattering solution of colloidal silica (Ludox.). All measurements were performed
in triplicate. The acquired data were analyzed with a 1-to-4 exponential fitting [the
commercial PTI software] involving iterative deconvolution and minimization of chisquare (χ2). The quality of the exponential decay fits was assessed by inspection of the
plots of weighted residuals and by the statistical parameters χ2 and SVR [serial variance
ratio]. The reduced χ2 for the selected exponential fit (χ2 = 1 for an ideal fit) ranged from
0.9 to 1.2 for all samples.
Sensitivity of the Fluorscence parameters of Bimane to Solvent Polarity — The sensitivity
of the mBBr fluorescence to solvent polarity was assessed using N-acetyl-cysteine
conjugated with mBBr (mBBr-Cys) as a probe. MBBr-Cys was produced by reacting
mBBr with 10-fold molar excess of N-acetyl-cysteine in 100 mM NH4HCO3, pH 8.1
buffer for 1 hr.
The reaction mixture was lyphophilized overnight and subsequently
resuspended in dioxane:water mixtures of 0 to 100% (v/v) dioxane. The fluorescence
emission and lifetime of mBBr-Cys (2 M mBBr-Cys) samples in dioxane:water
mixtures of different dielectric constants (  = 2.3 – 80 ) were determined.
Single Cysteine Synthetic Peptide: Depth Probes — Two single cysteine synthetic
peptides -Ac-K2ACALA2LA4(LA3)2LAK2–NH2 (pL4) and Ac-K2A2LA3LA2CA(LA3)2LAK2–
NH2
(pL12) – were used as a depth ladders in orders to determine the disposition of helix I
relative to surface of the membrane. The two peptides were synthesized from Queen's
University Peptide Synthesis Lab and were shown to be greater than 99% pure by mass
spectrometry (Queen's University, Kingston, ON). The designations pL4 and pL12
8
describe the positions (p) of the label (L) in each peptide relative to the N-termini (the 4th
and the 12th residue, respectively).
Peptides were labeled with mBBr following a
protocol similar to that used for the protein samples. However, 100 mM NH4HCO3, 20%
acetonitrile, pH 8.1 buffer was used. Also, the free dye was separated from the labeled
peptides using BioGel P2 resin (BioRad) that was equilibrated with the same reaction
buffer. On the basis of the absorbance profile of the collected fractions (1.0 mL), the
BioGel P2 resin separated the labelled peptides from the free dye completely. Fractions
containing the labeled peptides were pooled together and lyophilized overnight and the
resulting peptide powder redissolved in 80:20 ethanol:acetonitrile solution.
The
concentration of the labeled peptides was estimated from the absorbance at 380 nm using
an extinction coefficient of 5,000 M-1 cm-1.
Reconstitution of Labeled peptides in LUVs — Peptides dissolved in 80:20
ethanol/acetonitrile and lipids dissolved in chloroform were mixed and then dried under a
stream of N2 gas. The mixture were then dried under high vacuum for > 2 h. The dried
lipid:protein film was dissolved in 10 L of 100% ethanol and further resuspended in 690
L of 20 mM DMG, 130 mM NaCl at pH 4.0 while mixing on a vortex. The suspension
was then extruded 21 times through a 100-nm polycarbonate filter (Lipofast, Aventin
Inc., Ottawa, Ontario, Canada) to prepare a proteoliposomes containing the synthetic
peptides. The estimated final concentration of the labeled peptide in the proteoliposomes
for all fluorescence measurements was 2.5 M. The lipid:peptide molar ratio was 80:1
(mol:mol).
Analysis of the Solvent Accessible Surface Area — The accessibility of each residue in
helix 1 (347-365) to a 1.4 Å probe (water) was calculated using the Web-based program
GETAREA 1.1 (http://www.scsb.utmb.edu/getarea/area_man.html) (Frackiewicz and
Braun, 1998) and the crystal structure coordinates of the WT P190 colicin E1 (Elkins,
1997).
9
RESULTS
10
DISCUSSION
11
Acknowledgements
12
References
1. Tory, M. C., and Merrill, A. R. (1999) J. Biol. Chem. 274, 24539–24549
2. Hope, M. J., Bally, M. B., Webb, G., and Cullis, P. R. (1985) Biochim. Biophys.
Acta 812, 55–65
3. New, R. R. C. (1990) in Liposomes: A Practical Approach (New, R. R. C., ed) pp.
105–160, IRL Press at Oxford University Press, Oxford
4. James, D. R., Siemiarczuk, A., and Ware, W. R. (1992) Rev. Sci. Instrum., 63,
1710-1715.
5. Fletcher, A. N. ( 1969) Photochem. Photobiol. 9, 439-444.
6. Fraczkiewicz, R., and Braun, W. (1998) J. Comp. Chem., 19, 319 - 333
13
Figure Legends
Figure 1: Schematic representation of the channel domain of colicin E1. (A) The
sequence the hexahistidine-tagged channel domain (P190H6).
of the pH trigger
sequences within members of the channel-forming colicin family. Highlighted in bold
text and underlining are the residues corresponding to the consecutive Cys substitutions.
(B) The ribbon topology diagram of the crystal structure of the P190 peptide (Elkins et
al., 1997). The location of the cysteine mutation sites is highlighted with numbers and/or
black spheres that represent the -carbon of each substituted residue. The dark coloured
-helices are helices 8 and 9, which serve as a membrane-anchoring helical hairpin in the
membrane-associated closed channel state of the protein. Highlighted in side-chain stick
format are Y-356, Y-363, Y-367, and Y-434 (to be discussed later).
Figure 2: The fluorescence emission maximum (em max) of mBBr-labeled Cys mutants
of colicin E1 channel domain and the corresponding soluble state solvent accessibility of
each Cys substituted residue. (A) The mBBr em max values (●) of the soluble state Cys
mutants. (B) Side-chain solvent accessible surface area (SASA) of each Cys substituted
residues. Solvent accessible surface areas were calculated from the coordinates of the
crystal structure of soluble P190 peptide using 1.4 Å probe as described in the
Experimental Procedures. (C) The mBBrem max values (○) of the membrane-associated
Cys mutants. Also shown, as horizontal lines, are the em max values of mBBr-labeled the
pL4 () and pL12 (---) peptides in proteoliposomes. The insets in all panels illustrate
non-linear least square fittings of the corresponding profile to a harmonic wave function.
Figure 3: Solvent sensitivity of the em max of a modal mBBr-Cys and the calculated local
environment solvent polarities of the mBBr-labeled Cys mutants.
(A) The linear
dependence of the em max of mBBr-Cys on the dielectric constant (e) of the solvent. (B)
Comparison of the calculated soluble state side-chain solvent accessibility (○) and the
14
apparent local environment polarity (●) of the mBBr-labelled Cys at each substituted
site. The solvent accessibility of each residue was calculated as the ratio of the side-chain
surface area of each side-chain as estimated from the crystal structure (Fig. 2B) and the
expected theoretical value of each residue in the tripeptide Gly-X-Gly random coil.
Residues with solvent accessibility ratios above 50% () are considered to be surface
exposed, whereas residues with ratios  20% (---) are buried. (C) The apparent local
environment polarity values (○) of the mBBr-labeled Cys at each substituted site in the
membrane-associated state of all mutants. Also shown, as horizontal lines, are the
apparent polarity values of the mBBr-labeled pL4 () and pL12 (---) peptides in
proteoliposomes. The insets in panels B and C illustrate non-linear least square fittings
of the corresponding profile to a harmonic wave function. Apparent polarity values were
calculated from the em max of each Cys mutant (Fig. 2) according to the linear relations of
mBBr em max and solvent dielectric constants () illustrated in Fig. 3A.
Figure 4: The steady-state fluorescence anisotropy and probe mobility of mBBr-labeled
Cys mutants of colicin E1 channel domain. (A) The soluble state fluorescence anisotropy
(●) of the mBBr-labeled Cys mutants. (B) The membrane-associated state fluorescence
anisotropy (○) of the mBBr-laeled Cys mutants. (C) Comparison of the probe mobility
of the mBBr-labeled Cys mutants in the membrane-associated (○) and soluble state (●)
conformations.
Probe mobility of each site was calculated as the inverse of the
fluorescence anisotropy. The insets in panels A and B illustrate non-linear least square
fittings of the corresponding profile to a harmonic wave function.
Figure 5: Changes in fluorescence quantum yield (QF) of the mBBr-labeled Cys mutants
of colicin E1 channel domain upon binding to LUVs. (A) The relative fluorescence
quantum yield (QF) of the mBBr-labeled Cys mutants upon binding to LUVs. Relative
QF is calculated as the ratio of the membrane-associated state (QF-m) and the solution state
(QF-s) quantum yield values (Table 3) of the mBBr-labeled Cys substituted sites [Rel. QF
15
= QF-m/QF-s]. (B) The difference in the fluorescence quantum yield (QF) of the mBBrlabeled Cys mutants in the membrane-associated and solution states [QF = QF-m - QF-s].
The insets in all panels illustrate non-linear least square fittings of the corresponding
profile to a harmonic wave function.
Figure 6: Solvent sensitivity of the steady-state average lifetime (av-1) of a modal
mBBr-Cys and the av-1 values of the mBBr-labeled Cys mutants.
(A) The linear
dependence of the av-1 of mBBr-Cys on the dielectric constant () of the solvent. (B)
Comparison of the soluble (●) and membrane-associated state (○) av-1 values of mBBrlabeled Cys mutants. (C) Comparison of the membrane-associated state av-1 values of
mBBr-labeled Cys mutants (○) with the av-1 values of the pL4 () and pL12 (---)
peptides, in proteoliposomes.
av-1 was calculated according to av-1 = ∑ai i2/∑ai i
where ai and I are concentration and the individual decay time of ith lifetime component
calculated from the exponential decay fitting, respectively. The inset in panel C illustrates
harmonic wave function non-linear least square fit of the corresponding profile.
16
Table 1: Summary of the Trp em max values observed for the WT
and the mBBr-labeled/unlabeled Cys mutants of colicin E1 channel
peptide.
Sample
WT
D347C
A348C
V349C
D350C
A351C
T352C
V353C
S354C
F355C
Y356C
Q357C
T358C
L359C
T360C
E361C
K362C
Y363C
E365C
em max (nm)
Un-labeled
Labeled
318 ± 1
319 ± 1
319 ± 1
318 ± 1
318 ± 1
319 ± 1
318 ± 0
323 ± 1
318 ± 1
319 ± 1
319 ± 1
319 ± 1
318 ± 1
319 ± 1
318 ± 1
319 ± 1
318 ± 1
322 ± 1
318 ± 1
322 ± 2
319 ± 1
320 ± 1
317 ± 1
319 ± 1
319 ± 0
327 ± 0
323 ± 1
321 ± 1
321 ± 1
320 ± 0
319 ± 1
323 ± 0
321 ± 0
319 ± 1
318 ± 1
322 ± 1
319 ± 1
a
a
Values shown are the mean and the standard
deviations of at least triplicate measurements
17
Table 2: Summary of the fluorescence QF of mBBr-labeled
Cys mutants of colicin E1 channel peptide in the soluble
and membrane-associated states.
a
Sample
Soluble
Cys-mBBr
D347C
A348C
V349C
D350C
A351C
T352C
V353C
S354C
F355C
Y356C
Q357C
T358C
L359C
T360C
E361C
K362C
Y363C
E365C
0.18 ± 0.00
0.22 ± 0.00
0.24 ± 0.01
0.20 ± 0.00
0.12 ± 0.00
0.05 ± 0.00
0.21 ± 0.01
0.05 ± 0.00
0.23 ± 0.01
0.10 ± 0.00
0.11 ± 0.00
0.11 ± 0.00
0.21 ± 0.00
0.09 ± 0.00
0.19 ± 0.00
0.23 ± 0.00
0.18 ± 0.00
0.21 ± 0.00
0.18 ± 0.00
QF
Membraneassociated
0.35 ± 0.00
0.31 ± 0.01
0.52 ± 0.01
0.19 ± 0.00
0.12 ± 0.00
0.13 ± 0.00
0.12 ± 0.00
0.31 ± 0.01
0.32 ± 0.00
0.40 ± 0.01
0.13 ± 0.00
0.28 ± 0.01
0.28 ± 0.00
0.22 ± 0.00
0.20 ± 0.00
0.21 ± 0.00
0.57 ± 0.02
0.27 ± 0.01
a
QF were determined using quinine sulfate (QF =
0.53 in 0.1 N H2SO4). Values shown are the
mean and the standard deviations of at least
triplicate measurements.
18
Table 2: Summary of the non-linear least-square harmonic wave function analysis
of the observed fluorescence parameters.
Parameters
Soluble
a
p
(rpt)
3.8
3.7
3.6
3.9
b (deg.)
Residues
SASA
94.7
98.0
em max
App. Polarity
98.8
r
92.8
av-1
F
Rel. F
Average
3.7± 0.1 96.5 ± 3.3
350-362
350-362
350-362
350-362
c
Membrane-associated
p
Residues

(rpt)
(deg.)
3.7
96.1
347-362
3.8
95.8
347-362
3.6
101.2
350-362
3.8
94.1
351-358
3.8
94.6
354-362
3.6
100.3
353-362
3.7
96.1
347-362
3.7± 0.1 97± 3.0
a
c
The residue-per-turn (rpt) periodicity of the parameters; b angular frequency of the periodicity;
residues of which its parameters were used in the fitting process.
19
Fig. 1
A
347
HHHHHHETAE
SKMAQELADK
DRDAIFNALA
VSDILKIKDT
AGTTLGIWGI
NNLLNSQIKD
SKGKKIGNVN
SVKYDDWAKH
GDWKPLFLTL
AIVTGILCSY
365
AVDATVSFYQ
EALAAFEKYK
LDQFAKYLKI
EKKAADAGVS
IDKNKLNTIN
TLTEKYGEKY
DVLNKKFSKA
TGHVSFGYDV
YVVALLFSLL
EVLGI
B
365
363
361
357
359
354
350
355
352
Fig. 2
347
348
20
A
B
Rod, I tried to plot the delta
emmax and delta apparent
polarity; but the generated
figure looked very uninformative. So I excluded
it. I can forward both of
these figures if you like, so
you can see. The overall
appearance of the plot
looked similar to Fig. 4D
and E.
C
21
Fig. 3
A
B
C
22
Fig. 4
A
B
C
23
Fig. 4
D
Rod, I don’t know if these
two figures are that
informative. That is why I
didn’t include them in the
Figure legend section.
E
24
Fig. 5
A
B
25
Fig. 6
A
B
C
26
Fig. 7
27