High-Field EPR-Detected Shifts of Magnetic Tensor Components of

Appl. Magn. Reson. 21, 1#XXX (2001)
High-Field EPR-Detected Shifts of Magnetic Tensor
Components of Spin Label Side Chains Reveal Protein
Conformational Changes: the Proton Entrance Channel of
Bacteriorhodopsin
C. Wegener1, A. Savitsky2, M. Pfeiffer3, K. Möbius2, and H.-J. Steinhoff1
2
1
Max-Planck-Institut für molekulare Physiologie, Dortmund, Germany
Institut für Experimentalphysik, Freie Universität Berlin, Berlin, Germany
3
Max-Planck-Institut für Biochemie, Martinsried, Germany
Received August 27, 2001
Abstract. Continuous-wave high-field electron paramagnetic resonance (95 GHz, 3.4 T) is performed
on a spin label side chain located at residue position 171 in the proton entrance channel of
bacteriorhodopsin (BR). The conformational differences of three BR mutants, the single mutant F171C,
the double mutant D96G/F171C, and the triple mutant D96G/F171C/F219L, are reflected in different gxx and Azz tensor component shifts of the nitroxide side chain. The most polar microenvironment is found in the single mutant, whereas the open proton entrance channel reported for the triple
mutant allows a reorientation of the nitroxide group towards a microenvironment of lower polarity
and/or reduced hydrogen bonding. The experimental data of the double mutant are explained by a
light-independent equilibrium of two nitroxide orientations with different polarities of the local microenvironment. Upon illumination the spectrum of the single mutant reveals gxx and Azz tensor component shifts which resemble those determined for the triple mutant in the dark. This result provides
strong evidence for a light-induced opening of the proton entrance channel of the single mutant similar
to that found in the unilluminated triple mutant, in agreement with electron diffraction data.
1 Introduction
The membrane protein bacteriorhodopsin is a light driven pump which transports
protons across the cell membrane of the archaebacterium Halobacterium salinarum
(for reviews, see, e.g., refs. 1–4). Its seven transmembrane helices enclose the
chromophore retinal which is covalently attached to the amino acid lysine, K216,
via a protonated Schiff base. Absorption of a photon initiates the all-trans to
13-cis photoisomerization of the retinal. The Schiff base then releases a proton
to the extracellular medium and is subsequently reprotonated from the cytoplasm.
Intermediates of this catalytic cycle can be distinguished by the different absorption properties of the retinal and a sequence of intermediates J, K, L, M, N and
O has been characterized by time-resolved absorption spectroscopy [5]. Double
2
C. Wegener et al.
flash experiments divide the M intermediate into two substates, M1 and M2 [6,
7]. During this so-called photocycle a conformational change of the protein occurs, as has been detected by a variety of experimental techniques (for a review
see, e.g., ref. 4). Its physiological role is discussed to ensure that release and
uptake of protons do not occur from the same side of the membrane. To this
end, conformational changes associated with the M1 to M2 transition are suggested
to function as a “reprotonation switch” required for the vectorial proton transport. Hence, it is believed that during the life time of the M state the accessibility of the Schiff base for protons is switched from the extracellular to the cytoplasmic side of the membrane. Detailed analyses of the nature of the conformational changes with, for example, neutron diffraction [8, 9], electron microscopy
[10–13], X-ray diffraction [14, 15], solid-state nuclear magnetic resonance (NMR)
[16, 17], or electron paramagnetic resonance (EPR) spectroscopy [18–21], agree
in the major changes to be localized at the cytoplasmic moieties of helices F
and G. These helix movements, which can be observed to be present already in
the M intermediates with the Schiff base deprotonated, have been shown to provide an “opening” of the protein to protons on the cytoplasmic end of the transmembrane proton channel [12] and, thus, should allow proton transfer from the
internal aspartic acid proton donor, D96, to the Schiff base during the M to N
transition. The reprotonation of D96 from the cytoplasm occurs during the recovery of the BR initial state. A detailed inspection of the structure of the
unilluminated state of the protein reveals that the amino acid side chains at residue positions 41, 100, 170, 171 and 223 block the proton pathway from the
cytoplasm to D96 [12]. The region between D96 and the Schiff base is largely
nonpolar, packed with bulky amino acid residues. Hence, in this unilluminated
state the Schiff base is effectively inaccessible to protons from the cytoplasm,
and the pK value of D96 is high. In contrast, in the functionally intact triple
mutant D96G/F171C/F219L1 [22], there is a clearly visible opening of the upper
cytoplasmic region in the vicinity of D96, which is discussed in the recent literature to provide the full extent of the light driven conformational change already in the unilluminated state [11, 12]. The observed helix movements appear
to be sufficiently large to allow entry of water molecules into the channel region and provide a path for proton conduction from the cytoplasmic boundary
to the Schiff base. Simultaneously, and as a consequence, the pK value of D96
must be decreased to allow proton release to the Schiff base. However, the extent of the conformational change in the wild type protein is still under debate,
since the published models differ in the details, most probably due to different
methods of stabilization of the intermediate in question.
In the present work we apply site-directed spin labeling and high-field EPR
spectroscopy to study the magnetic tensors of nitroxide spin labels attached to
position 171 at the cytoplasmic end of helix F (see Fig. 1) in the single mutant
1
To designate mutants, we give the single-letter code for the original amino acid, then the position
of the amino acid and finally the single-letter code for the replacement. Single-letter codes: C, Cys;
D, Asp; G, Gly, F, Phe; L, Leu.
Conformational Changes of Spin-Labeled BR
3
F171C, in the double mutant D96G/F171C and in the triple mutant D96G/F171C/
F219L. The study of the spin label mobility, its accessibility for paramagnetic
molecules and of its dipolar interaction with other nitroxides has developed during the last years as a powerful tool for the determination of protein structures
and conformational changes (for reviews on site-directed spin labeling see, for
example, refs. 23–25). In recent work we have shown that, in addition to the
above studies, the gxx and Azz component shifts as determined for spin labels
located in the protein interior along the BR proton channel provide valuable
information about the polarity of the microenvironment of nitroxide side chains,
R1 [26]. The maximum value of gxx found for position 46 reveals the least polar environment between D96 and the Schiff base. For the BR initial state the
protic character of the extracellular moiety of the proton channel and its more
aprotic counterpart on the cytoplasmic side of the retinal could clearly be identified from the plot of gxx versus Azz. Hence, the enhanced Zeeman splitting in
high-field EPR spectra facilitates a detailed tensor analysis and has considerably
extended the applicability of the method of site-directed spin labeling. In the
a
b
cytoplasmic side
162
B
167
A
C
46
171
53
D
46
167
G
C
E
B
E
53
F
c
171
162
F
G
A
D
171
F
E
extracellular side
Fig. 1. Side (a) and top (b) views of the structural model of bacteriorhodopsin (BR) on the basis of
the data of Subramaniam et al. [12] (PDB-code 1FBB). The seven alpha-helices (A–G) and the interconnecting loops are visible, the Ca atoms of the spin-labeled residues are indicated as spheres. c
Detailed view of helices E and F with position 171 indicated. Here we present an overlay of the
wild type structure (PDB-code 1FBB, dark gray) with the open structure (PDB-code 1FBK, light gray)
of the pseudo M-state found for the unilluminated triple mutant D96G/F171C/F219L. The outward
movement of position 171 in the pseudo M-intermediate becomes clearly visible.
4
C. Wegener et al.
present study our goal is to determine the polarity properties in the proton entrance channel in the microenvironment of position 171 and to investigate hydrogen bonding effects of the introduced nitroxide in the three mutants. In addition, changes of these parameters will be followed after illumination of the protein in order to facilitate a comparison of the polarity properties in the vicinity
of residue F171 in the unilluminated triple mutant with those of the unilluminated
and illuminated double and single mutants.
2 Experimental
Mutagenesis, expression of BR in Halobacterium salinarum and spin labeling with
(1-oxyl-2,2,5,5-tretramethylpyroroline-3-methyl)methanthiosulfonate (MTS) was
performed as described [27]. The triple mutant was provided by J. Tittor and
D. Oesterhelt (Martinsried) [22]. In order to decrease dielectric loss glycerol was
added, to a final concentration of 20 vol%, to the suspension of membranes in
0.1 M phosphate buffer (pH 6.8), 0.1 M NaCl. This concentration of glycerol
does not alter the BR photocycle significantly, e.g., the increase of the decay
time of the M intermediate is less than 25% [28]. The BR samples were transferred into quartz capillaries with an inner diameter of 0.4 or 0.6 mm and then
centrifuged to achieve a final BR concentration of about 0.5 mM in the lower
part of the capillary.
High-field EPR spectra were recorded with a laboratory-built EPR spectrometer operating at 95 GHz (W-band) equipped with a cylindrical TE011 cavity [29,
30]. A superconducting magnet (Cryomagnetics, Bmax = 6 T) provides the desired
magnetic field of 3.4 T. The microwave power is supplied by a 300 mW klystron
(Varian) which can be attenuated to optimal power values at the cavity. Continuous-wave (CW) EPR experiments were performed with a modulation amplitude of 0.1 mT and a modulation frequency of 10 kHz. X-band EPR spectra and
cw saturation data in the presence and absence of molecular oxygen and chromium oxalate were recorded as described by Pfeiffer et al. [27].
Conformational changes of BR were induced by illumination with a quartz
halogen lamp (P = 50 W). Light was filtered with an unsealed combination of filters BG39 and OG550 (Schott, 3 mm thick) in order to limit the spectral range
to 550 < l < 600 nm. The power of the quartz halogen lamp was attenuated to
an energy density at the sample low enough to prevent photobleaching of the BR.
3 Results
A model of the BR initial state structure is shown in Fig. 1. In addition to amino
acid position 171, which is subject to the present study, residue position 162 located in the E-F loop, residue position 167 in the cytoplasmic end of helix F, and
residue positions 53 and 46 in helix B are indicated. With the exception of the
side chain at position 162, which is oriented towards the cytoplasm, the side chains
of all other residues are oriented towards the proton channel. Residue positions 46
Conformational Changes of Spin-Labeled BR
5
and 53 are located in close vicinity to the retinal on the cytoplasmic and extracellular side, respectively. The conformational change observed in BR consists of an
outward tilt of the cytoplasmic end of helix F which results in a considerable displacement of residue F171 [12]. This is illustrated in the comparison of the structure of the BR initial state with that of the triple mutant, D96G/F171C/F219L (Fig.
1c). In the present study the genetically engineered cysteine at position 171 was
reacted with the sulfhydryl specific methanthiosulfonate (MTS) nitroxide spin label to yield the spin label side chain, R1. Inspection of the photocycle of the spinlabeled mutants did not give any evidence for protein unfolding, and it can be
concluded that the overall structure and function of BR is retained [27, 31].
X-band EPR spectra of the single mutant F171R1, the double mutant D96G/
F171R1, and the triple mutant D96G/F171R1/F219L determined at room temperature (T = 293 K) reveal nearly identical spectral shapes which are typical
for an immobilized nitroxide side chain (see, e.g, Fig. 2 of ref. 27). W-band
EPR spectra were measured at 120 K. In the temperature regime below 200 K
the reorientational correlation time of an otherwise unrestricted spin label side
chain exceeds 100 ns [32], i.e., the nitroxide may be considered as immobilized
on the EPR time scale. Librational motion of small amplitude still prevails and
may lead to small deviations of the measured tensor values from their rigid limit
values due to partial motional averaging. However, this effect is small (e.g., less
than 2% for Azz at 200 K [32]) and is further minimized by decreasing the temperature for data collection to 120 K.
The W-band EPR spectra of the MTS spin label attached to position 171 are
shown in Fig. 2. Furthermore, a powder spectrum simulation with a small component line width is depicted to uncover the hyperfine splitting in the gxx and
gyy region (Fig. 2a). The experimental spectra of the single, double, and triple
mutants (Fig. 2b) exhibit the typical nitroxide powder pattern line shape expected
for a dilute distribution of nitroxides. The spectra are clearly resolved into three
separate regions corresponding to the components gxx, gyy and gzz. The variation
of gxx with the type of mutation is clearly visible by the shift of the position of
the low-field absorption line. Generally a polar environment shifts gxx to smaller
values [33]. A corresponding variation of Azz is revealed in the gzz region of the
spectra. In comparing the spectra, the nitroxide in the single mutant shows the
smallest value of gxx (the highest B-field position), whereas it is dramatically
shifted to a higher value for the nitroxide in the triple mutant. The spectrum of
the double mutant exhibits two shoulders on both sides of the maximum absorption in the gxx region. This spectral shape resembles a superposition of the spectra of the single and triple mutant (data not shown).
The gxx and Azz values, which were extracted from the second derivative of
the experimental spectra, are plotted against each other in Fig. 3. In addition,
the tensor component values for the nitroxides attached to positions 46, 53, 162,
167 and of unbound spin label are given for comparison (data taken from ref.
26). These values provide the maximum range of tensor component shifts found
so far for nitroxides located in the BR proton channel. The corresponding values determined now for the nitroxide at position 171 in the triple mutant clearly
extend this range towards a higher shift of both gxx and Azz with respect to the
6
C. Wegener et al.
a
gyy
gzz
EPR signal (first derivative)
gxx
3.400
3.405
3.410
3.415
3.420
B (T)
EPR signal (first derivative)
b
3.400
3.405
3.410
3.415
3.420
B (T)
Fig. 2. Calculated (a) and experimental (b) high-field EPR spectra (first-derivative representation,
T " 120 K, n " 95 GHz) for the spin-labeled BR mutants F171R1 (full line), D96G/F171R1 (dashdotted) and D96G/F171R1/F219L (dotted). For comparison, the simulation of a powder spectrum with
reduced line width is depicted (a). Simulation parameters are gxx " 2.0084, gyy " 2.0061, gzz " 2.0029,
Axx " 0.50 mT, Ayy " 0.50 mT, Azz " 3.41 mT, T2 " 37 ns, additionally convoluted with a Gaussian
of 0.3 mT line width. The variation of gxx and Azz reflects the changes of the polarity of the microenvironment of the nitroxide and/or its hydrogen bonding characteristics. The B-field axis is split
and has different scales to emphasize the shift of Azz. For a detailed discussion, see text.
data for the unbound spin label. This reveals an even less polar environment of
this nitroxide side chain in the triple mutant.
Theoretically, both gxx and Azz are expected to be linearly dependent on the
p spin density at the oxygen atom of the nitroxide group [34–36]. For Azz this
is evident from the relation Azz µ rN and the condition rN ! rO À 1 [37]. For gxx,
however, apart from a direct proportionality to rO, there is an additional dependence on specific electronic properties of the oxygen lone-pair orbitals, such as
their degree of s, px, py-hybridization and their orbital energy [35, 36] (M. Plato
et al., unpubl.). The lone-pair orbital energy En affects gxx via the excitation energy DEnDp* " Ep* # En [33] and is known to be sensitive to a polar environment
and, in particular, to direct H-bonding of the lone pairs. Such specific environ-
Conformational Changes of Spin-Labeled BR
nonpolar
7
polar
2.0089
nonpolar
2.0088
2.0087
gxx
46
aprotic
2.0086
53
2.0085
167
2.0084
protic
2.0083
unbound
polar
162
3.40
3.45
3.50
Azz
3.55
3.60
3.65
3.70
(mT)
Fig. 3. Plot of gxx versus Azz for the single mutant, F171R1 (square), the double mutant, D96G/F171R1
(triangle), and the triple mutant, D96G/F171R1/F219L (circle). In order to facilitate classification of
these data the gxx and Azz values of other spin label side chains of BR were plotted (gray squares,
data taken from ref. 26). Error bars are determined by the signal-to-noise ratio of the second derivative of the EPR spectra. For discussion of the shaded area, see text.
mental effects have been observed experimentally by several groups [26, 37–40]
who found different g vs. A dependences depending on whether the local microenvironment of the nitroxide spin label is protic or aprotic. The data for both protic
and aprotic “solvents” obey linear relations, with a steeper slope for protic solvents (e.g., methanol, water) than for aprotic solvents (e.g., n-hexane, acetonitrile). These two lines intersect at the point of the least protic microenvironment,
e.g., the solvent n-hexane. A similar grouping of the spin label sites located along
the BR proton channel into protic and aprotic sites has been discussed in our
previous paper [26]. The respective values of the nitroxides at positions 46, 53,
162 and 167 (shown in Fig. 3) mark an area the upper and lower boundaries of
which thus represent the more aprotic or more protic properties of the respective sites. Whereas the gxx, Azz values of F171R1 in the single and double mutants fall into this area, in the triple mutant the values of this site show significantly less influence of polar and hydrogen bonding effects on the nitroxide as
compared to all other sites so far investigated. CW saturation measurements (Xband) in the presence and absence of molecular oxygen reveal that the nitroxide
in the triple mutant is not accessible for oxygen. The dimensionless accessible
parameter poxygen, which is proportional to the collision frequency of the nitroxide
with molecular oxygen, amounts to 0.09 for F171R1 [27] and 0.05 for D96G/
F171R1/F219L compared to, e.g., 1.6 for the lipid-exposed position 160 [27].
Thus, a nitroxide reorientation towards the lipid phase as cause for the reduced
polarity of the local microenvironment in the triple mutant can be excluded.
At this point it is very interesting to study changes of gxx and Azz of F171R1
upon illumination of BR. The illumination and freezing protocol was carefully
8
C. Wegener et al.
F171R1
F171R1
EPR signal (first derivative)
a
3.400
3.405
3.410
3.415
3.420
B (T)
D96G/F171R1
D96G/F171R1
EPR signal (first derivative)
b
3.400
3.405
3.410
3.415
3.420
B (T)
Fig. 4. High-field EPR spectra (first derivative representation, T " 120 K, n " 95 GHz) of the illuminated (dotted) and unilluminated (full line) single (F171R1) (a) and double mutants (D96G/F171R1)
(b). A significant g-tensor component shift to higher values is observable in the gxx field region of
the spectrum of F171R1 while, correspondingly, Azz decreases. A superposition of calculated spectra
furnish proof that the spectrum of the illuminated single mutant consists of two components with
the magnetic tensor components of the unilluminated single mutant, gxx " 2.00855, Azz " 3.55 mT,
and of the triple mutant, gxx " 2.00890, Azz " 3.43 mT. The experimental error limits are )0.00005
for the gxx shift and )0.03 mT for the Azz shifts. No such changes are observable for the tensor
components of the illuminated double mutant.
chosen to assure trapping of considerable amounts of the intermediate M of the
photocycle: The samples were cooled down to 230 K under illumination with
the wavelength of the light limited to between 550 and 600 nm. The light was
then switched off and the temperature of the samples was subsequently adjusted
to 120 K. This protocol was shown earlier to lead to a mixture of the intermediate M and the initial state with up to 75% of a trapped M intermediate [21].
The W-band EPR spectra of the illuminated single and double mutants are shown
in Fig. 4 in comparison to those of the initial state in the dark. For the single
mutant, upon illumination considerable changes of the absorption line in the gxx
region are accompanied by a decrease of the hyperfine tensor component Azz (Fig.
Conformational Changes of Spin-Labeled BR
9
4a). The W-band EPR spectrum exhibits a shift of the low-field edge of the gxx
resonance towards higher gxx values and a corresponding shift of the high-field
edge by means of which shoulders are formed on both sides of the absorption
line. A superposition of the spectra of the unilluminated single (55%) and triple
mutants (45%) resembles the observed spectral shape of the illuminated single
mutant (not shown). This is strong evidence that the illuminated single mutant
consists of at least two conformations with the magnetic properties of the single
mutant and of the triple mutant initial states, respectively. The double mutant does
not reveal any changes of the gxx and Azz values upon illumination (Fig. 4b).
4 Discussion
As mentioned above, several EPR studies of solvent effects on nitroxide radicals show that g- and hyperfine tensor components are sensitive to interactions
with surrounding molecules and to the polarity of the local microenvironment [37,
38]. High-field EPR measurements on nitroxide radicals in solvents with different hydrogen bonding capacity have established that the g-tensor component
parallel to the NO bond, gxx, is most sensitive to H-bond interactions [41]. Hydrogen bonding causes a decrease in gxx mainly due to changes in the oxygen
lone-pair orbital which induces a blue shift of the n-p* excitation energy as a
consequence of electron density rearrangement in the NO bond. Recent advanced
quantum chemical g-tensor calculations of hydrogen bond effects on nitroxide spin
labels are in qualitative agreement with this reasoning [42]. In addition to the
hydrogen bonding effect, microenvironments with high dielectric constant polarize the spin density of the NO group of the spin label resulting in a further
decrease of the spin density at the oxygen and increase of the n-p* energy gap.
In summary, major contributions to the gxx component of nitroxide spin labels
originate from H-bond distance and concentration of hydrogen bonding molecules
as well as from polar molecules near the nitroxide group.
Our previous results on the gxx and Azz tensor component shifts of spin label
side chains positioned along the proton channel of BR are in line with these
findings, since gxx and the number of polar residues and internal and external
water in the vicinity of the nitroxide binding site were found to be clearly correlated [26]. We therefore expected changes of gxx and Azz during a conformational change of the protein as a consequence of the change of the microenvironment of the spin label side chain.
Diffraction data reveal considerably different orientations for residue F171 in
wild type BR and the triple mutant D96G/F171C/F219L (Fig. 1). This mutant is
discussed to show the full extent of the conformational change of the illuminated
wild type protein already in the dark. Our results on the gxx and Azz component
shifts of the spin label side chain at position 171 in fact uncover significantly
different hydrogen bonding properties and polarities of the nitroxide microenvironment in the single and triple mutants. The outward movement of helix F is
expected to provide access of water molecules to the amino acid side chains located in the proton entrance channel and, thus, should increase the probability of
10
C. Wegener et al.
hydrogen bonding. In contrast to that expectation, the spin label side chain experiences a decrease of the hydrogen bonding strength and/or polarity of the microenvironment upon opening of the channel. This is clear evidence for a reorientation of the nitroxide towards a hydrophobic environment in the triple mutant.
Molecular modeling and molecular dynamics simulations reveal two energetically
favored distinct orientations of the spin label side chain, one with the nitroxide
ring located between helices E and F close to positions 157 and 172, denoted
“orientation I” [43], the other with the NO group located in the least polar environment of the proton channel between D96 and the retinal, denoted “orientation
II”. In orientation I, the NO group points outward towards the head group region
of the lipid membrane. Spin label side chains attached to F helix surface positions 172 or 176 with their nitroxide rings in close vicinity of orientation I reveal values for Azz (3.58 and 3.54 mT, respectively; results not published) similar
to that of the unilluminated single mutant F171R1 (3.55 mT). Thus, orientation I
would account for the experimental data of the unilluminated single mutant. For
orientation II, gxx and Azz component values are expected according to a nonpolar
environment. This is the case for the triple mutant (cf. Fig. 3). The opening of
the upper cytoplasmic region in the vicinity of D96 which is observed for the
triple mutant thus might change the thermal equilibrium between orientations I
and II in favor of orientation II. This interpretation is in agreement with results
of inter-spin distance EPR measurements which locate the nitroxide at position
171 closer to helix B during the conformational change to the open state [20],
and with our cw saturation measurements which find the nitroxide in the triple
mutant much less accessible for oxygen than in the single mutant. Our results
show that a conformational change with a global increase of the accessibility of
certain protein regions for water molecules not necessarily increases the hydrogen bonding probability of all residues in that area. Instead, the conformational
change may facilitate a reorientation of hydrophilic or hydrophobic side chains
into areas of increased or decreased water accessibility, respectively.
The spectrum of the double mutant D96G/F171R1 shows components of both
the single and the triple mutants and, thus, reveals an initial state structure different from that of the single mutant, at least in the vicinity of position 171. A
thermal equilibrium between two conformations, with the nitroxide orientations
resembling those of the single and triple mutants, may account for the data. The
light-induced conformational change of D96G, which was reported to be different compared to that of the wild type and the triple mutant [12], does not lead
to further changes of the microenvironment of the nitroxide in D96G/F171R1.
Interestingly, the illuminated single mutant F171R1 shows gxx and Azz components again comparable to a mixture of two conformations of the protein with
nitroxide microenvironments resembling those of the unilluminated single mutant
and the triple mutant. The analysis of the intermediate composition of similarly
treated samples by FTIR difference spectroscopy revealed a mixture of the intermediate M and the initial state [21]. Therefore, the light-induced shifts of the
gxx and Azz tensor components must originate from the fraction of the sample that
is trapped in the M intermediate. Hence, the microenvironment and hydrogen
Conformational Changes of Spin-Labeled BR
11
bonding properties of the nitroxide at position 171 in the trapped M state of the
F171R1 single mutant differ significantly from those of the initial state, but are
similar if not identical to those of the triple mutant D96G/F171R1/F219L. We
conclude that the light-induced conformational change of the single mutant results in a redirection of the nitroxide at position 171 towards an orientation found
for this side chain in the triple mutant. This finding is in agreement with the
results of electron diffraction experiments (0.32 nm resolution) of wild type BR
and the triple mutant D96G/F171C/F219L [12]. They show similar electron density difference maps between the illuminated and the unilluminated wild type BR,
and between the triple mutant and the unilluminated wild type BR.
Recent g tensor calculations of the hydrogen bonding effects on the MTS
spin label predict a linear dependence of gxx on the hydrogen bond distance with
a slope of approximately 3$10#4 nm#1 [42]. Only very small changes of gyy are
predicted, whereas gzz is constant in the calculated interval. Hence, the experimentally observed increase of gxx from 2.00855 to 2.00890 during the conformational change, which is well outside the experimental error of )0.00005 for
the relative g shifts, would correspond to an increase of the hydrogen bonding
distance of less than 0.01 nm. This estimate holds if electric field effects on
the g tensor would remain constant during the conformational change. Further
influences of the protein matrix on the g and A tensor values of protein bound
nitroxides are conceivable and are presently being investigated.
The present study shows that the analysis of the gxx and Azz tensor component shifts of spin label side chains hands over a very sensitive tool for determining protein conformational changes provided the tensor component shifts can
be sufficiently resolved. The required precision is achievable by high-field EPR.
Therefore, in addition to measurements of the nitroxide mobility, its accessibility for paramagnetic quenchers and to measurements of inter-spin distances within
doubly spin labeled proteins, the high-field EPR tool considerably extends the
applicability of the site-directed spin labeling technique for better understanding
of the structure-dynamics-function relation in bioenergetics.
Acknowledgements
We are thankful to J. Tittor and D. Oesterhelt (MPI Martinsried) for providing
the triple mutant and to M. Plato (FU Berlin) for helpful discussions. We gratefully acknowledge the support of the Deutsche Forschungsgemeinschaft in the
frame of the Schwerpunkt-Programm SPP1051 “High-field EPR” and the Sonderforschungsbereich SFB 533.
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Authors’ address: Heinz-Jürgen Steinhoff, Max-Planck-Institut für Molekulare Physiologie, Otto-HahnStrasse 11, 44227 Dortmund, Germany