In addition, the Keywords 3D structural modeling; light-harvesting– reaction center core complex LH1–RC; photosynthetic purple bacterium; Raman spectroscopy; Thermochromatium Tch.. For t
Trang 1photosynthetic bacterium Thermochromatium tepidum as revealed by optical spectroscopy and structural modeling Fei Ma1,3, Yukihiro Kimura2, Long-Jiang Yu2, Peng Wang1, Xi-Cheng Ai1, Zheng-Yu Wang2and Jian-Ping Zhang1
1 Department of Chemistry, Renmin University of China, Beijing, China
2 Faculty of Science, Ibaraki University, Mito, Japan
3 Beijing National Laboratory for Molecular Science, State Key Laboratory for Structural Chemistry of Unstable and Stable Species, Institute
of Chemistry, Chinese Academy of Sciences, China
Light-harvesting (LH) complexes are transmembrane
proteins that are involved in the primary steps of
bac-terial photosynthesis: capturing the sun light and
trans-ferring the energy, in the form of electronic excitation,
to the reaction center (RC) Most purple bacteria
con-tain two basic types of LH complexes, i.e the
periph-eral antenna LH2 and the core antenna LH1 [1–3]
X-ray crystallographic structures of LH2 are available
for Rhodopseudomans (Rps.) acidophila strain 10050 [4] and Rhodospirillum (Rs.) molischianum [5] with resolu-tions of 2.0–2.5 A˚ Although the highest available reso-lution for LH1 [6], 4.8 A˚, is not sufficient to display the structural details, it clearly shows that bacterio-chlorophyll (BChl) dimers are sandwiched between a- and b-helices of 15 or 16 subunits arranged in a ring-like manner around the RC In addition, the
Keywords
3D structural modeling; light-harvesting–
reaction center core complex (LH1–RC);
photosynthetic purple bacterium;
Raman spectroscopy; Thermochromatium
(Tch.) tepidum
Correspondence
Z.-Y Wang, Faculty of Science, Ibaraki
University, Mito 310 8512, Japan
Fax: +81 29 2288352
Tel: +81 29 2288352
E-mail: wang@mx.ibaraki.ac.jp
J.-P Zhang, Department of Chemistry,
Renmin University of China, Beijing
1000872, China
Fax: +86 10 62516444
Tel: +86 10 62516604
E-mail: jpzhang@chem.ruc.edu.cn
(Received 25 November 2008, revised 14
January 2009, accepted 14 January 2009)
doi:10.1111/j.1742-4658.2009.06905.x
Native and Ca2+-depleted light-harvesting–reaction center core complexes (LH1–RC) from the photosynthetic bacterium Thermochromatium (Tch.) tepidum exhibit maximal LH1–Qy absorption at 915 and 889 nm, respec-tively To understand the structural origins of the spectral variation, we performed spectroscopic and structure modeling investigations For the
889 nm form of LH1–RC, bacteriochlorophyll a (BChl a) in the native form was found by means of near-infrared Fourier-transform Raman spec-troscopy, a higher degree of macrocycle distortion and a stronger hydrogen bond with the b-Trp)8 residue SWISS-MODEL structure modeling sug-gests the presence of a specific coordination motif of Ca2+at the C-termi-nus of the a-subunit of LH1, while MODELLER reveals the tilt of a- and b-polypeptides with reference to the structural template, as well as a change
in the concentric orientation of BChl a molecules, both of which may be connected to the long-wavelength LH1–Qyabsorption of the 915 nm form The carotenoid spirilloxanthin shows a twisted all-trans configuration in both forms of LH1 as evidenced by the resonance Raman spectroscopic results With regard to the thermal stability, the 915 nm form was shown
by the use of temperature-dependent fluorescence spectroscopy to be approximately 20 K more stable than the 889 nm form, which may be ascribed to the specific Ca2+-binding motif of LH1
Abbreviations
BChl a, bacteriochlorophyll a; Car, carotenoid; fwhm, full width at half maximum; LH1, light-harvesting complex 1; Q y , the absorptive optical transition to the lowest excited state of BChl a; RC, reaction center.
Trang 2structures of a- and b-polypeptides in solution were
determined for Rs rubrum by means of 2D-NMR
spectroscopy [7]
The purple photosynthetic bacterium
Thermochro-matium(Tch.) tepidum was first identified in Mammoth
Hot Springs in the Yellowstone National Park [8] It is
a moderate thermophile with an optimal temperature
range of 48–50C and an upper limit of 55 C, and its
pigment–protein complexes show considerably higher
thermal stability than those from its mesophilic
counterparts such as Allochromatium (Ach.) vinosum,
Rhodobacter (Rb.) sphaeroides and Blastochloris (Bl.)
viridis, which grow at temperatures below
approxi-mately 30C [9] The light-harvesting–reaction center
core complex (LH1–RC) from Tch tepidum is peculiar
with respect to its long-wavelength Qy absorption of
BChl a at 915 nm, which shifts to approximately
885 nm when eluted in presence of NaCl, KCl, KBr,
NaCl or MgCl2 (150 mm) Interestingly, the 885 nm
LH1–RC complex can be fully converted back to the
915 nm form by adding CaCl2[10,11]
Recently, polypeptides of LH1 from Tch tepidum
have been purified and the amino acid sequences
deter-mined [12] In addition, the dimeric feature and the
highly symmetric ring assembly of BChls in LH1, as
well as the interaction between BChl a and carotenoid
molecules, have been confirmed [13] It has been shown
that spirilloxanthin is the major carotenoid
(approxi-mately 92.3%), and that the 889 nm form of LH1–RC
is thermally less stable than the 915 nm form [11,13]
Furthermore, Ca2+has been proven to coordinate in a
ratio of 1 : 1 to an a-, b-subunit when the 889 to
915 nm transformation is induced [11] Our recent study
on the excitation dynamics of the two forms has shown
similar LH1-to-RC excitation trapping kinetics, as well
as similar efficiency of the transfer of excitation energy
from carotenoid to BChl despite some differences in the
BChl-to-carotenoid molecular orientation [14]
Ca2+ plays vital roles in biological activities, e.g as
messengers of signal transduction in the cell, and for
structural stabilization of proteins, etc [15] Ca2+ in
protein usually coordinates seven oxygen atoms from
amino acid residues and water molecules, which
accordingly form a pentagonal bi-pyramid cavity
However, coordination with 6, 8 or even up to 12
atoms is also possible A helix-loop-helix structural
domain constituting the Ca2+ binding motif is found
in a large number of Ca2+-binding proteins, and is
also known as the EF hand [16] Proteins containing
the EF hand are divided into two classes according
to their functions: signaling and buffering⁄ transport
proteins The former undergoing Ca2+-dependent
conformational changes, constitute the largest family,
including well-known members such as the Ca2+ -AT-Pase from skeletal muscle sarcoplasmic reticulum whose transmembrane helices tilt approximately 30 when transformed from the Ca2+-bound form (E1Ca2+) to the Ca2+-free form [E2(TG)] [17,18] The interchangeable 915 and 889 nm forms of LH1–
RC from Tch tepidum provide us with a unique opportunity to investigate the structure–function rela-tionship of these proteins In the present study, we used near-infrared Fourier-transform Raman spectros-copy (FT-Raman) to assess the structural differences
in BChl a molecules between the two forms of LH1–
RC Compared to the 889 nm form, the 915 nm form shows a stronger hydrogen bond (H-bond) interaction between the C10a acetyl carbonyl and the tryptophan (Trp) residue from the b-polypeptide, b-Trp)8, and more severe distortion of the BChl a macrocycle Fur-thermore, the twist of all-trans spirilloxanthin was found to be similar between the two LH1–RC forms
by use of resonance Raman spectroscopy The results
of 3D structural modeling reveal a specific Ca2+ -coor-dination cavity that may induce configurational changes in the polypeptides, and, as a result, in BChl a molecules The results are discussed in terms of the long-wavelength Qy absorption of native LH1–RC Furthermore, the systematic shift of fluorescence spec-tra against temperature shows that the thermal stabil-ity of the intact LH1–RC is approximately 20 K higher than that of Ca2+-depleted LH1–RC
Results and Discussion
Steady-state absorption and fluorescence spectroscopy
The 915 nm form of LH1–RC exhibits much higher thermal stability than the 889 nm form As shown in Fig 1A, the absorption spectra of 915 nm LH1–RC vary slightly from 273 to 323 K, i.e the LH1–Qy absorbance decreases approximately 3% with little change in band width In contrast, for the 889 nm form under similar experimental conditions, dramatic decreases in the LH1–Qy and carotenoid absorption are seen (Fig 1B), together with emergence of a new absorption maximum at 770 nm that is ascribed to monomeric BChl a When the temperature exceeds
303 K, a large spectral change is seen, most likely due
to disassembly of the LH1 complex
Upon increasing temperature, the fluorescence peak wavelengths of both the 915 and 889 nm forms shift to blue, and the emission bands get broader (Fig 2) For the 915 nm form, the peak wavelength shifts from 945.4 to 939.5 nm, and the bandwidth increases from
Trang 3494 to 553 cm)1 [full width at half maximum (fwhm)]
on raising the temperature from 273 to 323 K The
increase in spectral shift or bandwidth in response to
the increase in temperature may indicate the
involve-ment of more thermally populated excitonic states in
the Qy-state manifold of BChl a Using an energy
dif-ference of 120 cm)1between the lowest and the second
lowest excitonic states [19], the population increase in
the second lowest excitonic state in response to a
temperature increase of 50 K was estimated to be
6.4% Given the amount of spectral shift (66 cm)1)
and band broadening (57 cm)1), it is reasonable to
ascribe the fluorescence spectral changes to a new
ther-mal equilibrium in the Qy state As shown in the inset
to Fig 2A, the spectra shift slowly to blue against a
temperature increase below 293 K, and the shift is
faster and shows linear temperature dependence above
293 K In addition, the decrease in fluorescence
inten-sity may be due to the increased rate of internal con-version On the other hand, when the temperature increases from 273 to 303 K, the fluorescence maxi-mum of the 889 nm form shifts from 918.8 to 914.1 nm, while the bandwidth increases from 534 to
569 cm)1 (fwhm) When the temperature exceeds
303 K, the fluorescence intensity decreases consider-ably due to dissociation of the LH1–RC assembly The tendency of spectral shift appears to be signifi-cantly different between the two LH1–RC forms, i.e nonlinear and linear temperature dependence are observed for the 915 and 889 nm forms, respectively, which may reflect their structural differences The
915 nm LH1–RC form exhibits slower (273–293 K) and faster (293–323 K) phases of band shift (Fig 2A); however, the 889 nm complex shows monophasic behavior (273–303 K; Fig 2B) with a slope compara-ble to the faster phase of the 915 nm form Comparing
300 400 500 600 700 800 900 1000
300 400 500 600 700 800 900 1000
0.00
0.05
0.10
0.15
0.20
323 K
293 K
273 K LH1-Q y
Car
Wavelength (nm)
Wavelength (nm)
0.00
0.05
0.10
0.15
323 K
313 K
303 K
293 K
283 K
770 nm
273 K
Car
LH1-Q y
A
B
Fig 1 Steady-state UV-visible spectra of the 915 nm (A) and
889 nm (B) LH1–RC preparations from Tch tepidum at the
indi-cated temperatures Arrows in (B) indicate the direction of
absor-bance change upon temperature increase from 273 to 323 K.
0 1000 2000
3000
10 580
10 600
10 620
10 640
Wavelength (nm)
Wavelength (nm)
0 1000 2000 3000
270 280 290 300
270 280 290 300 310 320
10 880
10 900
10 920
10 940
A
B
T/K
T/K
νm
νm
Fig 2 Fluorescence emission spectra recorded at various temper-atures for the 915 nm (A) and 889 nm (B) LH1–RC preparations from Tch tepidum Arrows show the direction of temperature change from 273 to 323 K in (A) and from 273 to 303 K in (B) Insets show the change of emission maxima (in wave number) against temperature The excitation wavelength was 590 nm.
Trang 4the 915 and 889 nm forms, a difference of 20 K in the
starting temperature of the faster phases was found
(293 versus 273 K), indicating that the
pigment–pro-tein assembly of the 915 nm complex is more stable,
most likely because of the binding of Ca2+ This result
is in agreement with a recent differential scanning
calo-rimetry study on the same core complexes [20], in
which the dissociation temperature of the 915 nm form
was found to be 15 K higher than that of the 889 nm
form, and the enthalpy change for the former was
found to be approximately 28% larger than that for
the latter
The Stokes shifts between absorption and
fluores-cence maxima are 28.9–24.5 nm for the 915 nm form
and 29.8–25.1 nm for the 889 nm form over the
tem-perature ranges 273–323 and 273–303 K, respectively,
and are considerably larger than those of mesophilic
purple bacteria such as Rs rubrum (approximately
15 nm) Therefore, for Tch tepidum, the spectral
over-lap integral between LH1 emission and RC absorption
(maximum at 865 nm) must be much smaller
How-ever, the rates of LH1-to–RC excitation energy
trans-fer are rather similar from the thermophilic to the
mesophilic species [14], implying that the rate is not
strictly proportional to the spectral overlap integral
Resonance Raman spectroscopy
Figure 3A shows the resonance Raman spectrum of a
915 nm form with spirilloxanthin as the major
caro-tenoid component (approximately 92.3%) The key
Raman lines at 1504 cm)1 (m1, C=C stretching) and
1143 cm)1 (m2, C–C stretching) can be assigned to
all-trans spirilloxanthin in LH1 The Raman bands from 15-cis spirilloxanthin in the RC normally seen at
1528, 1239 and 1160 cm)1[21] do not show up because the majority of spirilloxanthin molecules associate with LH1 and only a minor amount in the 15-cis configura-tion binds preferentially to the RC [22] The Raman band at approximately 965.3 cm)1 is characteristic of the out-of-plane movement of C–H (m4), which becomes symmetry-allowed only when the polyene backbone experiences nonplanar distortion [23] As the
m4 mode is localized to and originates from the twists
at C11=C12and C7=C8and their conjugates, C11¢C12¢ and C7¢C8¢, it is concluded that all-trans spirilloxan-thin bound to LH1 takes on a twisted configuration, similarly to the case for LH1 of Rs rubrum [21,23] The Raman spectra do not change appreciably between the 915 and 889 nm LH1–RC forms, indicat-ing that the configuration of spirilloxanthin does not vary despite a large difference in the Qy absorption wavelength of BChl a (26 nm) A similar conclusion was reached in a recent investigation of the same com-plexes by means of circular dichromism spectroscopy [11]
Near-infrared FT-Raman spectroscopy Figure 4 shows the FT-Raman spectra for the 915 and
889 nm LH1–RC forms from Tch tepidum, and Table 1 lists the assignments based on recent work by Frolov et al [24] The key Raman lines labeled with carotenoid correspond to the m1 (1504 cm)1), m2 (1147 cm)1) and m3 (1023 cm)1) modes of spirilloxan-thin (see above), while those labeled R1–R4 originate
Raman shift·cm–1
967.4
997
1145
1187
1278 135213871447
1504
997
965.3
1143
1185
127613521392
1444
1504
A
B
Fig 3 Room-temperature resonance Raman spectra for the
915 nm (A) and 889 nm (B) LH1–RC preparations from Tch
tepi-dum The excitation wavelength was 514 nm.
∗
∗
∗
∗
Raman shift·cm–1
1024 (ν 3 )
1147 (ν 2 )
1147( ν 2 )
1023 ( ν 3 )
1504 (ν 1 )
1504 (ν 1 )
A
B
Fig 4 Room-temperature FT-Raman spectra for the 915 nm (A) and 889 nm (B) LH1–RC core complexes from Tch tepidum The excitation wavelength was 1064 nm.
Trang 5from BChl a, and are sensitive to the core size of
bac-teriochlorin and the molecular conformation of BChl a
These modes are known to be conserved in various LHs
[25,26] It is worthy of noting that the band at
1065 cm)1in the Raman spectrum of 915 nm LH1–RC
is not seen in the 889 nm form (Fig 4A,B), probably
due to variation in the resonance conditions of Raman
excitation [24]
For both forms of LH1–RC, the presence of
meth-ane bridge stretching at approximately 1610 cm)1(R1)
confirms the penta-coordination of BChl a molecules
[27] that is often seen when the a- and b-polypeptides
of LH1 have higher flexibility [28] Raman lines R5 or
R6 overlapped with the intense carotenoid band (m3)
and therefore cannot be resolved For both the 915
and 889 nm forms, the R1 and R4 Raman lines appear
at similar frequencies (Table 1); however, the R2 and the R3 frequencies vary considerably, i.e 1540 versus
1531 cm)1 and 1436 versus 1444 cm)1, respectively The R1–R4 lines of the LH1 complexes from Rb sph-aeroides 2.4.1 and Rhodospirillum (Rsp.) rubrum G9 [26] are conserved in the 889 nm LH1–RC form from Tch tepidum Therefore, the macrocycle configurations
of BChl a are most likely similar among these com-plexes However, the R2 and R3 lines and those with asterisks in the Raman spectrum of the 915 nm form are distinctly different from those of the 889 nm form, both in frequency and intensity, suggesting significant differences in the BChl a conformations between the two LH1–RC forms of Tch tepidum According to recent theoretical studies on the peridinin–chlorophyll– protein complex and the light-harvesting complex II
Table 1 Raman shifts obtained from the near-infrared FT-Raman spectra of BChl a in the 915 and 889 nm LH1–RC forms from Tch tepi-dum (see Fig 4) and the corresponding assignments ‘Carotenoid’ indicates that the Raman lines of BChl a overlap with those originating from carotenoid Intensities are indicated after the Raman shifts.
Raman shift (cm)1)
Key Raman
CH2bend, CH bend
CH bend
carotenoid
carotenoid
a These Raman frequencies are from reference [25] and are given for comparison b R1–R4 are key Raman lines that are sensitive to the core size of BChl a [26] c Assignments based on reference [24] See Scheme 1 for the numbering system of BChl a.
Trang 6(LHCII) chlorophylls [29], distortion of the Chl a
mac-rocycle is the key structural factor governing the Qy
absorptive transition energy
As seen in Fig 4 (and with reference to the
number-ing system of BChl a in Scheme 1), the three Raman
lines above 1600 cm)1 for the 915 nm⁄ 889 nm forms
may be ascribed to the stretching modes of the
methane bridge (1610⁄ 1609 cm)1), the C2 acetyl
(1641⁄ 1641 cm)1) and the C9 keto–C10a acetyl
carbo-nyls (1671⁄ 1676 cm)1) It is known that, for free
BChl a in nonpolar solvent, lines for the two carbonyl
stretching modes appear at 1663 and 1685 cm)1, but
downshift as much as approximately 40 cm)1 for
BChl a bound to protein via an H-bond, and,
impor-tantly, a downshift of the C2acetyl stretching correlates
linearly with the red shift of the Qyabsorption [25] As
the frequency of the particular mode at 1641 cm)1 is
identical between the 915 and 889 nm forms, the
H-bond interaction with the C2acetyl carbonyl cannot
be responsible for the shift of Qyabsorption from 915
to 889 nm Compared with the 889 nm form, the C9 keto⁄ C10a acetyl carbonyl stretching of the 915 nm form shows a downshift of 5 cm)1, indicating a stron-ger H-bond between the Trp)8residue of the b-subunit and the C10aacetyl carbonyl (see below)
3D modeling of Ca2+-binding motifs The amino acid sequences of LH1 from Tch tepidum show the highest homology to the LH1 peptides from
Rs rubrum, i.e the 50.0% and 53.3% (E-value,
2· 10)11⁄ 0) identity for a- and b-polypeptides, respec-tively For comparison, the corresponding identities
to LH2 of Rs molischianum are 35.0% and 40.5% (E-value, 2· 10)4⁄ 6 · 10)9), respectively, and those to the LH2 peptides of Rps acidophila are 28.6% and 34.2% (E-value, 3 · 10)3⁄ 2 · 10)5) However, as the available structures of the a- and b-polypeptides of
H
CH2
H
=
R
Scheme 1 BChl a chemical structure and numbering Right, numbering of carbon atoms according to the Fischer system Left, genetic labeling of meso and pyrrolic carbon atoms.
Trang 7LH1 from Rs rubrum were determined independently
in solution [7], they are not suitable to serve as
tem-plates for the LH1 of Tch tepidum with sufficient
structural details of BChl a molecules and the loop
domain at the C-terminus
Figure 5A shows the BChl a binding sites and the
loop motif of the LH1 polypeptides of Tch tepidum
based on SWISS-MODEL modeling using the LH2
template from Rs molischianum; those obtained using
the LH2 template from Rps acidophila are presented
in Fig 5B With regard to the possible H-bond to
BChl a, the NH2 of b-Trp)8 falls into close proximity
to BChl a, i.e 3.63 A˚ to O10awhen the LH2
crystallo-graphic structure of Rs molischianum is used as a
tem-plate (Fig 5A, upper right) and 3.62 and 5.86 A˚ to
O10band O10a, respectively, when the LH2 of Rps
aci-dophila was used (Fig 5B, upper right) In the LH2s
of Rs molischianum and Rps acidophila, the amino
acid corresponding to b-Trp)8 in LH1 of Tch tepidum
is phenylalanine (Phe), which cannot form an H-bond
with the acetyl carbonyl of BChl a Previous 3D
struc-tural modeling of LH1 of Roseospirillum parvum 930I
proved that the H-bonds between the thiol groups of
cysteine (a-Cys+3, b-Cys)4) and BChl a are responsible
for the long-wavelength LH1–Qy absorption (909 nm)
[30] Similarly, the H-bonds found in the LH1 of Tch
tepidum may be responsible for the extremely red
absorption of BChl a (915 nm), although other factors
such as BChl–BChl excitonic interactions are certainly
also in operation
The possible Ca2+ coordinations optimized by
means of SWISS-MODEL modeling based on the
LH2 templates of Rs molischianum and Rps acidophila,
respectively, are shown in the lower right parts of
Fig 5A,B In both cases, the Ca2+-binding cavities are
localized in the C-termini, which comprise O of Leu)4,
Ser)5, Thr)6, OD1 of Asp)7 and OD1 and OD2 of
Asp)13 in Fig 5A, O of Val)3, Leu)4, Ser)5, Thr)6,
Asp)7and OG of Ser)5in Fig 5B (O, OD1⁄ 2 and OG
are oxygen atoms of the backbone carbonyl, side-chain
acetyl or hydroxyl carbonyl, and side-chain hydroxyl,
respectively) The Ca2+ chelation motifs agree well
with the EF-hand characteristics, i.e they tend to
localize to the helix-loop-helix motifs with a
coordina-tion number of 6 or 7
Figure 6 shows the results of MODELLER
model-ing based on an averaged template (the LH1 from
Rs rubrum and the LH2s from Rs molischianum and
Rps acidophila) The H-bond between b-Trp)8 and
BChl a and the presence of a Ca2+coordination cavity
(consisting of O of Val)3, Leu)4, Ser)5, Thr)6, Asp)7
and OD2 of Ser)13) within a helix-loop-helix motif
are predicted, which is similar to the results of
A
B
αα
α
α β
α β
β
β
Fig 5 Three-dimensional models of a polypeptide subunit of LH1
of Tch tepidum obtained by SWISS-MODEL modeling using the crystallographic structures of LH2 from Rs molischianum (A) and Rps acidophila (B) as templates In each panel, the structures within circles were magnified to show more detail and these are shown at upper and lower right Color codes: deep blue, secondary structure of polypeptide subunit; orange, amino acid; green, BChl a; red, His; pink, Trp)8; yellow, hydrogen atoms in NH2of Trp)8; light grey, oxygen atoms of BChl a; purple, oxygen atoms most probably coordinating to Ca 2+
Trang 8SWISS-MODEL modeling To our surprise, a
consid-erable tilt of the optimized polypeptides with respect
to the template is predicted, especially for the
b-poly-peptide Furthermore, the orientation of the histidine
(His) coordinating to BChl a changes significantly in
a- and b-polypeptides as seen in the top right of
Fig 6 This implies a large difference in the orientation
of BChl a molecules between the LH1 of Tch tepidum
and the template, and, as a result, a large difference in
the BChl–BChl excitonic interactions In addition, the
results show that the locations of a- and b-His residues
are rather different between the LH1 of Tch tepidum
and the template It is therefore expected that
coordi-nation of His residues to BChl a induces considerable
structural heterogeneity in the BChl a molecules bound
to a- and b-polypeptides, and this is supported by a
recent transient spectroscopic study of LH–RC forms
from Tch tepidum [14]
Although the modeling results for Ca2+-induced
conformational changes in the LH1 of Tch tepidum
are preliminary and qualitative, they reveal basic
struc-tural differences between the 915 and 889 nm forms of
LH1–RC from Tch tepidum, e.g the strength of the
H-bond between the b-Trp)8residue and the C10a
ace-tyl carbonyl of BChl a, the excitonic interaction among
the BChl a molecules in LH1, and deformation of the
BChl a macrocycle induced by Ca2+ binding, all of which may account for the low absorptive transition energy of BChl a molecules in native LH1 We pro-pose that the presence of a specific Ca2+-binding motif
in the a-, b-subunit of LH1 is responsible for the long-wavelength LH1–Qy absorption of Tch tepidum, as well as for the high thermal stability of this particular pigment–protein assembly
Conclusion
Based on the spectroscopic and 3D structural modeling results for the 915 and 889 nm forms of LH1–RC from Tch tepidum, this paper proposes a specific
Ca2+-coordination cavity localized to the C-terminus
of the a-subunit of LH1, which agrees with the EF-hand characteristics This Ca2+-binding motif may
be responsible for the reversible conformation change
in the a- and b-polypeptides between the forms, which
in turn lead to changes in the arrangement of BChl a molecules, in the strength of the H-bond between b-Trp)8 and the O10 of BChl a, and in distortion of BChl a macrocycle All of these structural variations are helpful to understand the long-wavelength Qy absorption of the native LH1–RC complex from Tch tepidum Furthermore, thermal equilibrium among the
αα
α α
β
β
β
Fig 6 Three-dimensional model of the LH1 polypeptides of Tch tepidum obtained by MODELLER modeling based on the aver-aged template (LH1 from Rs rubrum and the LH2s from Rs molischianum and Rps acidophila) The model at the top right shows details of the BChl a binding motifs
of the optimized LH1 and the template, while that at the bottom right shows details
of the possible Ca 2+ -binding cavity; both models are re-oriented for clarity with respect to the model on the left Color code: dark green, secondary structure of LH1 polypeptides; blue, secondary structure
of the template; orange, amino acids of LH1 polypeptides; red, His coordinating to BChl a
in LH1 of Tch tepidum; purple, His coordi-nating to BChl a in the template; yellow, hydrogen atoms coordinating to BChl a; light blue: oxygen atoms coordinating to Ca2+.
Trang 9excitonic states of BChls and other structural changes
in the 915 nm form of LH1, as reflected by the
temper-ature-dependent fluorescence spectra, reveal higher
dissociation enthalpy of this complex with respect to
the 889 nm form, which may account for the higher
thermal stability of the native LH1–RC complex from
Tch tepidum
Experimental procedures
Preparation of LH1–RC complexes
Chromatophore was isolated by sonication of the Tch
tepi-dum cells suspended in 20 mm Tris⁄ HCl buffer (pH 8.5)
followed by differential centrifugation at 4C for 15 min
(5000 g) Chromatophores thus obtained were extracted
with 0.35% w⁄ v lauryldimethylamine N-oxide at 25 C for
60 min, followed by centrifugation at 4C for 90 min
(150 000 g) The LH1–RC core complex with an LH1–Qy
absorption maximum at 915 nm (Fig 1A) was prepared as
described previously [24] The final concentration of LH1–
RC was determined to be approximately 10 lm by using a
molar extinction coefficient for BChl a of e915 nm= 4.3·
103mm)1cm)1 [11] As the preparation was eluted using a
linear gradient of CaCl2from 10 to 25 mm, the ionic force
was estimated be approximately 75 mm This LH1–RC
preparation is considered to be intact as the LH1–Qy
absorption maximum, 915 nm, is similar to that of the
chromatophore The 889 nm preparation, i.e the LH1–RC
complex with an LH1–Qyabsorption maximum at
approxi-mately 889 nm (Fig 1B), was prepared by adding 200 mm
EDTA to the intact 915 nm form For spectroscopic
measurements, these preparations were suspended in buffer
containing 20 mm Tris⁄ HCl and 0.8% w ⁄ v
n-octyl-b-d-glucopyranoside (pH 7.5)
Steady-state UV-visible and near-infrared
fluorescence spectroscopy
UV-visible absorption spectroscopy with a spectral
resolu-tion of 0.5 nm was performed using a U-3310
spectropho-tometer (Hitachi, Japan) Near-infrared fluorescence spectra
(spectral resolution of 0.25 nm) were recorded using a liquid
nitrogen-cooled linear photodiode array (OMA V: 10242.2
Princeton Instruments, Trenton, NJ, USA) attached to an
imaging polychromater (SpectraPro 2300i; Acton Research,
Acton, MA, USA), for which excitation pulses at 590 nm
(approximately 2 mJÆpulse)1, approximately 7 ns, 10 Hz)
were supplied by an optical parametric oscillator
(Quanta-Ray MOPO-SL; Spectra Physics, Mountain View, CA, USA)
driven by an Nd3+:YAG laser (Quanta-Ray PRO-230;
Spec-tra Physics) Sample temperatures were controlled exactly in
the range 273–323 K using a water-flow type thermostat
(RTE-110; Neslab Instruments Inc., Newington, NH, USA)
Resonance Raman and near-infrared FT-Raman spectroscopy
Room-temperature resonance Raman spectra (spectral reso-lution of 1.4 cm)1) were recorded with a liquid nitrogen-cooled CCD detector (SPEC-10-400B⁄ LN; Roper Scientific Research, Trenton, NJ, USA) attached to a 0.5 m poly-chromator (grating density 1200 grooves per mm, Spectro-pro 550i; Acton Research Corporation) A continuous-wave
Ar+laser (2060-10S; Spectra Physics) provided the Raman excitation power of 1.8 mW at 514 nm Raman scattering light was collected in a backscattering geometry, and was focused onto the entrance slit of the polychromator after passing through a Raman notch filter (HSNF-514.0-1.5; Kaiser Optical Systems, Ann Arbor, MI, USA) The Raman spectra were obtained using an exposure time of
15 s and a spectral resolution of 1.4 cm)1 The absorbance
of the LH1–RC samples was 5 cm)1at 514 nm
Raman spectra, with pre-resonance to the Qy transition
of BChl a, were recorded on a FT-Raman spectrometer (DIGILAB FTS-3500; Bio-Rad, Krefeld, Germany) with a resolution of 0.5 cm)1; the excitation source was a continu-ous-wave Nd3+:YAG laser operated at 1064 nm The spec-tra were obtained by averaging of 200 scans The optical densities of the two forms of LH1–RC were adjusted to
120 cm)1at the maximal Qyabsorption
3D structural modeling of LH1
The 3D modeling was performed using two methods: SWISS-MODEL, accessed using the Deep View Swiss-PDB Viewer version 4.0 [31–33], and MODELLER [34,35] SWISS-MODEL superimposes a template with the target sequence, and is fully automated by a homology-modeling server (http://www.expasy.ch/spdbv/) The a-, b-polypeptides of LH2 from Rps acidophila (PDB record 1nkz) and Rs molis-chianum(PDB record 1lgh) as well as those of LH1 from Rs rubrum (PDB records 1xrd and 1wrg) were used as the templates MODELLER is used for homology or compara-tive 3D modeling of protein structures It implements com-parative modeling by satisfaction of spatial restraints, and can perform additional tasks such as de novo modeling of loops, etc We used an average of the three templates above (multiple-model) to increase the accuracy Five models were thus obtained, and the one with the lowest discrete optimized protein energy (DOPE potential) was chosen Sequence identity between target LH1 polypeptides from Tch tepidum and each template was calculated using MODELLER
Acknowledgements
This work has been supported by the Natural Science Foundation of China (grant nos NSFC 20703067 and
20673144, and NSFC-JSPS 20711140133) and by the
Trang 10National Basic Research Program of China (grant no.
2009CB220008)
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