However, to date, no molecular mechanism is known for the action of NblA, nor have the gene products been characterized to understand the physical properties of the molecule and thus hel
Trang 1NblA from Anabaena sp PCC 7120 is a mostly a-helical protein
undergoing reversible trimerization in solution
Holger Strauss1, Rolf Misselwitz2, Dirk Labudde1, Sabine Nicklisch3and Kerstin Baier3
1
Forschungsinstitut fu¨r Molekulare Pharmakologie (FMP), Berlin, Germany;2Max-Delbru¨ck-Centre fu¨r Molekulare Medizin (MDC), Berlin, Germany;3Humboldt Universita¨t zu Berlin, Institut fu¨r Biologie/Biochemie der Pflanzen, Germany
The nblA family of genes encodes for small proteins
neces-sary for the ordered degradation of phycobilisomes under
certain stress conditions, a process known as chlorosis
Genes homologous to nblA seem to occur in all
phycobili-some-containing organisms However, to date, no molecular
mechanism is known for the action of NblA, nor have the
gene products been characterized to understand the physical
properties of the molecule and thus help elucidate the
mechanism on a structural basis In this study we report on
the first characterization of an NblA-homologous gene
product The chromosomal gene from the cyanobacterium
Anabaena sp PCC 7120 was cloned, heterologously expressed in Escherichia coli and purified to apparent homogeneity This allowed the protein to be characterized
by analytical ultracentrifugation and CD spectroscopy These experiments show that the NblA protein has a mostly a-helical structure, undergoing an association reaction of folded monomers to form trimers in solution No dimers are detectable
Keywords: phycobilisome; chlorosis; NblA; cyanobacteria; analytical ultracentrifugation
Cyanobacteria are a widespread group of photosynthetic
prokaryotes performing a plant-type oxygenic
photosyn-thesis They are very adaptable organisms that can survive
in a wide variety of environmental conditions [1,2] One
limiting factor for growth is the nitrogen supply and
cyanobacteria have developed various mechanisms to cope
with this nutrient stress
One of the first responses exhibited by cyanobacteria
when they are starved for nitrogen is the degradation of
their major light-harvesting complex, the phycobilisome
Phycobilisomes (PBS), which also represent light-harvesting
antennae of red algae, are large, water-soluble multiprotein
complexes associated with the thylakoid membranes PBS
consist mainly of the pigmented phycobiliproteins that can
constitute up to 50% of the total cellular protein, thus
representing a large nitrogen store [3] Degradation of PBS
is thought to provide substrates for protein synthesis
required for the acclimatization process In addition, PBS
degradation minimizes the absorption of excess excitation
energy under the stress situation
Nondiazotrophic cyanobacteria such as Synechococcus
sp PCC 7942 completely degrade their PBS when starved for combined nitrogen and differentiate into nonpigmented resting cells, able to survive prolonged periods of nutrient stress [4,5] Diazothrophic filamentous cyanobacteria such
as Anabaena sp PCC 7120 adapt to nitrogen limitation (lack of combined nitrogen) by developing differentiated cells, called heterocysts These are specialized for fixation of
N2in an aerobic environment [6] In a filament, approxi-mately 5–10% of vegetative cells undergo this differenti-ation process However, during the first hours of nitrogen starvation all cells start to degrade their PBS [7] When heterocysts mature and nitrogenase is active, vegetative cells resynthesize their light-harvesting complexes, while in heterocysts the PBS content remains very low [8,9] Phycobilisome degradation is an ordered proteolytic process, visible by a colour change of the cyanobacterial cell from blue-green to yellow-green, a process known as chlorosis or bleaching [10] The small polypeptide NblA plays a central role in PBS degradation Its gene, nblA, was first identified in Synechococcus PCC 7942 [11], but nblA homologous genes appear to be present in all PBS-containing organisms, cyanobacteria as well as red algae
In Synechococcus PCC 7942, nblA transcription is induced upon nitrogen and sulfur starvation, and, to a lesser extent, during phosphorus starvation [11] In Synechocystis sp PCC 6803, only nitrogen starvation leads to nblA induction [12] Knock-out mutations of the nblA gene lead to nonbleaching phenotypes under nitrogen-limited conditions [11,13] Several NblA homologous sequences are found in the databases The sizes of the deduced NblA proteins range from 54 to 65 amino acids, corresponding to molar masses
of about 7–7.5 kDa Sequence identity among these NblA proteins amounts to about 30% on average, but no homology has been found to other proteins with known function The molecular mechanism by which NblA triggers
Correspondence to K Baier, Institut fu¨r Biologie/Biochemie der
Pflanzen, Humboldt Universita¨t zu Berlin, Chausseestr 117,
D-10115 Berlin, Germany.
Fax: + 49 30 20938164, Tel.: + 49 30 20938166,
E-mail: kerstin.baier@biologie.hu-berlin.de
or H Strauss, Forschungsinstitut fu¨r Molekulare Pharmakologie,
Robert-Ro¨ssle Str 10, 13125 Berlin, Germany.
Fax: + 49 30 94793 169, Tel.: + 49 30 94793 223,
E-mail: strauss@fmp-berlin.de
Abbreviations: PBS, phycobilisomes; nbl, nonbleaching; AUC,
analytical ultracentrifugation; SV, sedimentation velocity;
SE, sedimentation equilibrium.
(Received 22 May 2002, revised 21 July 2002, accepted 1 August 2002)
Trang 2degradation of PBS is not clear The following hypotheses
have been discussed [11]: NblA may activate a protease
degrading the PBS; alternatively, NblA could tag or disrupt
the PBS, rendering it susceptible to degradation; and finally,
NblA may activate other genes that are involved in the PBS
degradation process Analysis of the structural properties of
NblA could help find out how this polypeptide achieves its
function
The diazotrophic, filamentous cyanobacterium Anabaena
sp PCC 7120 has two nblA genes, one (ORF asr4517) on
the chromosome and another (ORF asr8504) on plasmid
Delta [14] We have cloned and overexpressed nblA from
ORF asr 4517 in Escherichia coli This allowed the NblA
polypeptide to be purified to apparent homogeneity and
thus to study the gene product to determine some of its
structural and physical properties
We used analytical ultracentrifugation (AUC) to study
the hydrodynamic properties of the protein and to
determine its state of association as a function of
concentration The stoichiometry of the association
reac-tion, as well as the extent, in terms of an association
constant at three different temperatures (10, 18 and
26C), were determined CD, together with fluorescent
measurements, was used to probe the gross secondary
structure and any changes observable with temperature
and concentration
M A T E R I A L S A N D M E T H O D S
Construction of the expression plasmid, protein
expression and purification
The chromosomal gene (asr4517) (Table 1) coding for
NblA from Anabaena sp PCC 7120 [14] was amplified, with
total DNA isolated from that strain as template, by
PCR using the following oligonucleotides: 5¢-GTCTTTT
AGGAGTCTCATATGAACC-3¢, complementary to a
DNA region upstream of the N-terminus, with NdeI site
inserted (in italics) and 5¢-GTTGACGCCCCAGGATCCC
CAGCTC-3¢, complementary to a DNA region
down-stream of the C-terminus, with BamHI site inserted (in
italics) The PCR product was digested with NdeI and
BamHI and ligated into plasmid pET11a (Novagen)
resulting in plasmid pBB8 (Fig 1A) For expression of the
NblA protein, plasmid pBB8 was cloned into host strain
E coliBL21 (DE) pLysS (Novagen)
Two liters of Luria–Betrtani medium (Difco Laborator-ies) were inoculated with 100 mL of a bacterial overnight culture (50 lgÆmL)1ampicillin, 34 lgÆmL)1 chlorampheni-col) and grown at 30C to a D600
thio-b-D-galactoside was added to a final concentration of 1 mM and incubation continued for 3 h Cells were harvested by centrifugation (5000 g for 10 min at 4C) and washed with Tris/NaCl/EDTA (50 mMTris/HCl pH 7.5, 150 mMNaCl,
1 mM EDTA) Cells were disrupted by sonication and centrifuged (14 000 g for 10 min at 4C) (NH4)2SO4was added to the supernatant (30% saturation, 10 min centri-fugation at 14 000 g at 4C), the pellet dissolved in 25 mL
of Tris/EDTA (50 mM Tris/HCl pH 7.5, 1 mM EDTA) and applied to a column of Superdex 75 (HiLoad 16/60, Pharmacia Biotech), run in Tris/NaCl/EDTA (1 mLÆmin)1) Fractions of 2 mL were collected and assayed for NblA in a discontinuous tricine/SDS/PAGE system [15] The protein eluted at a volume corresponding
to 19–24 kDa The column was calibrated with BSA, myoglobin and cytochrome c (67, 17.8 and 12.3 kDa, respectively) Fractions containing NblA were pooled and concentrated by precipitation as above After desalting on a PD-10 column (Amersham Biosciences Europe), the preci-pitated protein was applied to a column of Q-Sepharose (1 mL bed volume), equilibrated with Tris/EDTA buffer and eluted with a linear gradient of NaCl (0–300 mM in
30 min) in Tris/EDTA at a flow rate of 1 mLÆmin)1 N blA eluted at 150 mMNaCl (Figs 1B.C)
All purification steps were performed at 0–8C with the exception of chromatography on Q-Sepharose, which was carried out at room temperature
MALDI-TOF mass spectrometry Mass spectrometry measurements were performed on a Voyager-DE STR BioSpectrometry Workstation MALDI-TOF mass spectrometer (Perseptive Biosystems, Inc., Fra-mingham, MA, USA), using a standard protocol as described [16] After analytical ultracentrifugation, sample solutions were taken from the centrifugation cells, pooled, and subjected to the sample preparation procedure without further dilution
Table 1 Results from SV experiments of NblA Parameter estimates were obtained from fits over the whole boundary to the monomer-trimer model The molecular mass parameter was kept constant at the value calculated from the sequence and obtained with MALDI-TOF MS m, molecular mass.
Parameter
Concentration of NblA
S monomer [S)13] (fitted) 0.84 0.83 0.82
S trimer [S)13] (fitted) 2.24 2.23 2.21
D monomer [10 7 cm 2 Æs)1] (calculated) 10.07 10.28 10.09
D trimer [10 7 cm 2 Æs)1] (calculated) 8.96 9.18 9.08 f/f 0 monomer (calculated from m and s) 1.58 1.59 1.62 f/f 0 trimer (calculated from m and s) 1.23 1.24 1.25
Trang 3Protein concentrations were determined
spectrophoto-metrically using the extinction coefficient at 280 nm as
calculated from the sequence [17] Values at all other
wavelengths used were calculated relative to that value from
wavelength spectra recorded with appropriate
concentra-tions of NblA on a JASCO V-550 spectrometer
Absorb-ance measurements at different wavelengths were
transformed to molar concentrations using the law of
Lambert and Beer All optical measurements were carried
out with buffer in dialysis equilibrium with the solution
Circular dichroism and fluorescence measurements
CD studies in the far UV region were performed with a
Jasco J720 spectropolarimeter equipped with a Neslab
temperature control system using 0.01–1.0 cm path length quartz cuvettes and protein concentrations in the range 0.53–53 lM(4–400 mgÆL)1) Measurements were performed
at (10 ± 0.2)C Molar mean residue ellipticities [Q] (degÆcm2Ædmol)1) were calculated using a mean residue molecular mass of 116.0 Da
The content of secondary structure was determined from the far-ultraviolet CD spectra using the variable selection method (program VARSLC1) starting with a set of 33 reference proteins [18]
Fluorescence spectra were measured with a Shimadzu
RF 5001 PC spectrofluorimeter at excitation wave-lengths of 295 nm and 280 nm with bandwidths of
5 nm for both excitation and emission monochro-mator Concentration of protein solutions were adjusted to 5.3 lM (40 mgÆL)1) and were measured in cuvettes of 0.3 cm path length at (10 ± 0.2)C The intensity of the Raman peak of water was used as an internal standard
Thermal-induced unfolding measured by circular dichroism
Thermal unfolding of NblA was carried out in 20 mM sodium phosphate buffer, pH 7.5 monitoring changes in the ellipticity at 222 nm at protein concentrations in the range 0.54–43.7 lM (4.1–330 mgÆL)1) and at a heating rate of
20CÆh)1 The reversibility of unfolding of the protein was checked by slow cooling down to 20C The transition curves were normalized to the fraction of folded protein f, where f ¼ ([Q]) [Qu](T))/([Qn](T)) [Qu](T)) where [Qn] and [Qu] are the mean residue ellipticities of the folded and unfolded protein, respectively, and were corrected for their temperature dependence by linear extrapolation of the pre-and post-melting range [Q] is the observed mean residue ellipticity
Analytical ultracentrifugation Both sedimentation velocity (SV) and sedimentation equi-librium (SE) experiments were performed on a Beckman XL-I analytical ultracentrifuge (Beckman-Coulter, Fuller-ton CA, USA) in a four-hole ANTi60 rotor, using the absorption optics of the instrument The partial specific volume (vv) of NblA was calculated from the sequence [19,20] Values for the density (q) and viscosity (g) of the buffer used were calculated from composition using the options implemented in ULTRASCAN5.0 (B Demeler, University of Texas, Health Science Center at San Antonio,
TX, USA)
2 Values are corrected for the temperatures used [21–23]
SV experiments were performed at 20C and 250 000 g
in double-sector, charcoal filled epon centerpieces capped with quartz windows over a concentration range of 300–
1000 mgÆmL)1(40 lMto 133 lM) Sedimentation patterns were acquired at a single wavelength for a single experiment (286 nm and 278 nm, depending on loading concentration)
in continuous mode every 90 s with a Dr of 0.003 cm Data were analysed by fitting the sedimentation patterns
to the Lamm equation [24–28] and by the method of van Holde-Weischet [29,30] All sedimentation (S)
coefficients (D) as reported here have been corrected for water at 20C [21–23]
Fig 1 Preparation and characterization of NblA from E coli (A)
Restriction map of the T7 lac promoter-nblA region of plasmid pBB8.
For details of construction, see Materials and methods (B)
Purifica-tion of NblA from recombinant E coli cells, harbouring plasmid
pBB8 Discontinuous tricine/SDS/PAGE of different stages of
purifi-cation Lane 1, noninduced cells; lane 2, induced cells; lane 3, soluble
lysate; lane 4, after Superdex 75 and lane 5, after Q-sepharose
chro-matography The positions of standard marker proteins are indicated
on the left (C) MALDI-TOF MS of NblA, after the final purification
step The peak at 3772.05 (m/z) represents the doubly charged
monomer, the peak at 7749.74 (m/z) corresponds to the singly charged
matrix adduct.
Trang 4SE experiments were performed in 12 mm, six-sector
charcoal filled epon
protein solution in each sector at different concentrations
for each of the nine sectors, in the range 20–1000 mgÆL)1
(2.7–133 lM) in repeated experiments Detection
wave-lengths ranging from 225 to 290 nm were chosen so that
Ainitialwas 0.1–0.4
6 for the respective cell and three different
wavelengths were used for detection of each concentration
gradient Data were acquired in step mode with a Dr of
0.001 cm and 20 replicate absorption measurements were
performed at every step point After overspeeding the
solution for 20–30 min at 30 000–38 000 r.p.m
on the rotor speed which was later on used for attainment of
equilibrium [31,32]), the samples were spun in repeated
experiments at various speeds, in the range 18 000–
32 000 r.p.m
8 as indicated Equilibrium was judged to be
reached when a fit to the concentration gradients of a single
molecular species model of the form:
9
cr¼ c0emapp Fþ d ð1Þ where
F¼ ½ð1 qvvÞx2ðr2 r2
didn’t show any
10 systematic deviations in the residuals; cris
the concentration of the solute at position r of the cell, c0the
concentration at an arbitrarily selected reference position,
mappis the apparent molecular mass
volume of the solute, x the angular velocity and d the
baseline offset Temperatures were kept constant during one
experiment In repeated experiments, we chose different
temperatures (10, 18 and 26C) to understand in more
detail the nature of the association reaction
Datasets were globally fitted using the general nonlinear
least-squares procedures as described previously [33] and the
extensions of Eqn (1) for multiple species in reversibly
associating equilibrium [34], taking into account the
associ-ation constants
Data were analyzed using the programs LAMM[25,26],
SEDFIT8.3 [27] andULTRASCAN5.0
R E S U L T S
Analytical ultracentrifugation
The chromosomal nblA gene from Anabaena sp PCC 7120,
ORF asr4517, encodes a polypeptide of 65 amino acids with
a predicted molecular mass of 7542 Da During purification
of recombinant NblA from E coli, the protein eluted from
size exclusion chomatography columns with an apparent
molar mass of 19–24 kDa However, SDS/PAGE and
MALDI-TOF MS confirmed the purity and identity of the
sample (Fig 1B,C), thus suggesting that NblA was
multi-meric or highly elongated in solution We routinely checked
NblA by MALDI-TOF MS to test the stability of the
protein under the experimental conditions used and no
degradation was detected A Van-Holde Weischet analysis
of the sedimenting bundaries at different loading
concen-trations indicated mass-dependent heterogeneitity (not
shown)
Based on these results and the information obtained from
the SE experiments (see below), we used direct boundary
modeling [27] to a monomer-trimer system to gain insights
into the hydrodynamic parameters of the monomer and the trimer Sedimentation patterns of the 300, 400 and
500 mgÆL)1(40, 53 and 66 lM) loading concentrations were fitted over the whole boundary (Fig 2) Apparent values of
S, determined at the lowest and highest loading concentra-tion (for Smonomerand Strimer, respectively), and the value of
Kaat 18C from the SE experiments were used as starting estimates The monomer-trimer model yields random residuals over the whole range of fitted data points, as judged from the conventional presentation and the recently proposed two dimensional bitmap-presentation [28] The hydrodynamic and some statistical parameters obtained from the best-fit values for the three different concentrations are given in Table 1
To understand the stochiometry and the nature of the association reaction in more detail, we have performed SE experiments over a range of concentrations, temperatures and speeds Multiple datasets are best described by a monomer-trimer model, which results in random residuals over the whole region included in the fit (Fig 3) and a monomer molecular mass in good agreement with the theoretical value deduced from the sequence and confirmed with MALDI-TOF MS Using a monomer-dimer-trimer model to deconvolute the data showed that no detectable portion of dimer was present Increasing the temperature increased the fraction of monomer present in solution Higher order associates can be excluded for the concentra-tion range and condiconcentra-tions used in this study, because the average molecular mass level off at 20–22 kDa at the highest loading concentrations when fitted to Eqn (1) Detailed information for the values obtained is given in Table 2 Robustness of the parameter estimates was ascer-tained by Monte-Carlo simulations of the fitted data, using
10 000 iterations for each dataset From this, the 95% confidence intervals were obtained and are reported for the molecular mass and the association constant parameters
CD experiments Under native conditions NblA is well folded with spectral characteristics of proteins with predominantly a-helical
Fig 2 Direct boundary modeling to a monomer-trimer model of the sedimentation patterns obtained with300 mgÆL)1loading concentration (A) Raw data (points) and best fit (solid lines); (B) residuals of the fit; (C) residuals bitmap presentation of the fit.
Trang 5secondary structure elements (Fig 4A) The maximum at
192 nm and double minima at 222 and 208 nm,
respect-ively, point to largely alpha helical structures For reference,
the CD spectrum in the presence of 7M guanidine
hydrochloride, which is typical for unfolded proteins, is
also shown Evaluation of the CD spectrum with the
variable selection method starting with 33 reference proteins
[18] supports this conclusion About 61% a-helix, 10%
b-sheet and 12% turn structures were calculated Secondary
structure prediction with thePHDprogram [35,36] classified
NblA as an all a-helical protein and predicts about 71%
a-helix, 2% b-structure and 28% loop structure The
prediction for residues with a reliability
63% a-helix, no b-structure and 28% loop structure, which
is in good agreement with the secondary structure content
estimated by CD measurements.PHDpredicts two a-helical
regions in the sequence of NblA, a shorter N-terminal helix
from L9 to M25 and a longer C-terminal helix from H27 to
Q55 The N-terminal eight and the C-terminal 10 amino
acids are less structured (Table 3)
The predicted helical character and the position of the
helical elements are corroborated by homologous sequences
in the PDB protein structure database A search
identified for the NblA sequence (from aa 2–28 and 24–65) highly similar sequences with helical structures for gene regulation XRCC4-DNA ligase, PDB entry 1IK9 (Y177– D208) and for synapse-enriched clathrin adaptor protein, PDB entry 1HX8 (D239–P280), respectively No coiled-coil motives in the sequence of NblA were detected by either bioinformatic sequence analysis tools, such as COIL SCAN [WISCONSIN Package Version 10.2, Genetics Computer Group (GCG), Madison, WI, USA], or homology searches
As shown by AUC experiments, the trimeric NblA dissociates at low protein concentrations to monomers To understand the impact of association on secondary struc-ture, we performed CD measurements in the concentration range 0.53–53 lM (4–400 mgÆL)1), which corresponds to 98% to 17% of monomers, respectively The spectra of NblA measured in Tris/NaCl/EDTA are largely identical and the ratio of [Q]222/[Q]208 nmis 1 and does not change considerably in its dependence on protein concentration (Fig 4B) Thus, the folding of NblA in the trimeric and monomeric state is very similar with a high content of a-helical structures
Fluorescence experiments The emission position of tryptophan at an excitation wavelength of 295 nm depends on whether it is localized
in a hydrophobic or hydrophilic surrounding and varies between in the range 320–350 nm For NblA, we found an emission maximum at 346 nm, which correlates with a hydrophilic environment of the single tryptophan residue at position 56 Unfolding of NblA in 7M guanidine hydro-chloride results in a small red shift of the emission maximum
to about 350 nm and a decrease in the fluorescence intensity (not shown)
Thermal-induced unfolding
To measure the thermal stability and to investigate the dissocation/unfolding behavior of NblA, the thermal transition was measured at protein concentrations in the range 0.54–43.7 l (4.1–330 mgÆL)1) in 20 m sodium
Table 2 Results from SE experiments and from global fits to a
mono-mer-trimer model m, molecular mass.
Temperature (C)
Number of datapoints 5390 5044 4362
95% confidence limits
K a [ M )2 ] 5.88 · 10 10
2.17 · 10 10
1.1 · 10 10
95% confidence limits
Upper 7.07 · 10 10 2.31 · 10 10 1.91 · 10 10
2.05 · 10 10
0.64 · 10 10
Fig 3.
15 Global fits of the monomer-trimer model
to the equilibrium gradients obtained at various
loading concentrations, speeds and
tempera-tures Points represent the raw data, solid lines
the best fits On top of the fits are shown the
respective residuals (A) Data obtained at
10 C, 22 000 r.p.m and 28 000 r.p.m.
(B) Data obtained at 18 C, 22 000 r.p.m and
28 000 r.p.m (C) Data obtained at 26 C,
18 000 r.p.m and 27 000 r.p.m.
Trang 6phosphate, pH 7.5 (Fig 4C) Transition curves were
monitored by changes of ellipticity [Q] at 222 nm at a
heating rate of 20CÆh)1 NblA unfolds under these
conditions in a one step manner with an isodichroic point
at about 203.5 nm and the unfolding is largely reversible
(Fig 4D) As expected for the unfolding of noncovalently
associated NblA molecules the melting temperatures
varied with the protein concentration ranging between
about 53C at 0.54 lM (4.1 mgÆL)1) and 66C at
43.7 l (330 mgÆL)1)
D I S C U S S I O N
To our knowledge, this is the first report on properties of an NblA-homologous gene product since the gene was first identified in 1994 in Synechococcus sp strain PCC 7942 [11]
We have cloned the gene from Anabaena sp PCC 7120 and purified the protein without tags We show that NblA from Anabaena sp PCC 7120 is a mostly a-helical protein The results from the thermally induced folding-unfolding experiments indicate the presence of only a single domain
Fig 4 Far-UV-CD spectra of NblA (A), protein concentration dependence of far-UV-CD spectra of NblA (B), thermal denaturation of NblA (C) and
CD spectra at various temperatures and after recooling (D) (A) The spectra were measured in 20 m M sodium phosphate buffer, pH 7.5 (thin line) and in the presence of 7 M guanidine hydrochloride (thick line) Experiments were carried out at (20 ± 0.2) C (B) The spectra were recorded at (10 ± 0.2) C in Tris/NaCl/EDTA buffer and at protein concentrations of 54.0 l M (solid line), 5.4 l M (dashed line), 1.08 l M (dashed-dotted line) and 0.54 l M (dotted line) (407, 40.7, 8.1 and 4.1 mgÆL)1, respectively) The inset shows values of the ratio [Q 222 ]/[Q 208 ] as a function of concen-tration (C) The temperature-induced unfolding was monitored by changes of ellipticity at 222 nm in 20 m M sodium phosphate buffer, pH 7.5 and protein concentrations of 0.54, 5.4, 15.0 and 43.7 l M (4.1, 40.7, 113 and 330 mgÆL)1, respectively) Unfolding curves from left to right (D) CD spectra in the peptide region at a concentration of 43.7 l M and temperatures of 20, 40, 60, 80, and 90 C (solid lines, from bottom to top) and after recooling to 20 C (dotted line) The unfolding was measured with a heating rate of 20 CÆh)1.
Table 3 Sequence and PHD secondary structure prediction of NblA The sequence of NblA is shown in bold figures and directly below is given the secondary structure prediction H, helix; E, extended; L, loop.
Trang 7[37], not unexpected for such a small protein NblA
undergoes trimerization of stable monomers in solution in
a mass-dependent manner In an equilibrium situation, no
dimers are present and no higher associates could be found
Upon association, the gross secondary structure of the
protein doesn’t change in an observable manner The high
frictional ratio f/f0of the monomer could indicate either a
globular structure with a roughed surface or a highly
elongated structure Upon trimerization, f/f0 decreases
moderately Based on purely geometrical considerations
and the hypothesis of elongated monomers, these findings
can be rationalized by a symmetrical arrangement of the
monomers to a trimer with a threefold symmetry The
monomeric building blocks of such a structure would have
two different interaction surfaces along their major axis, one
being identical to the surface of the trimer and one that is
buried within, forming the scaffold for their interaction
At present, it is not clear whether the monomer or the
trimer is the biologically relevant species However, several
arguments favor the trimer to be the species responsible for
NblA action: NblA proteins that we have investigated from
cyanobacterial crude extracts (NblA1 and NblA2 from
Synechocystis sp PCC 6803) or purified in recombinant
form from E coli (NblA1 and NblA2 from Synechocystis
sp PCC 6803 and NblA protein from Anabaena sp
PCC 7120 encoded by plasmid Delta) all behave similarly
on size exclusion chromatography columns, eluting at
positions which correspond to the size of a trimer (data
not shown) This finding suggests that trimerization is
indeed an important prerequisite for NblA action in vivo
Moreover, although the sequence homology among the
known NblA proteins is not very high, PHD predicts a
similar helical arrangement for all of the analysed NblA
sequences from cyanobacteria as well as red algae Thus, we
propose that all NblA-homologous molecules identified so
far share a common overall structure and behave similarly
to Anabaena sp PCC 7120 as reported here
As mentioned in the introduction, one of the proposed
modes of action of NblA is the destabilization of the
structure of PBS, which facilitates attack by proteolytic
enzymes already present in the cell If so, it is tempting to
speculate that for destabilization of PBS, the NblA-trimer
might directly fit into the central channel of the hexamers of
phycobilisome rods (reviewed in [3,38]), thereby displacing
the so-called linker peptides that normally reside there and
thus changing the structure of the phycobilisome rods,
making them amenable to proteolysis However, up to now,
there has been no evidence for a direct interaction between
NblA and PBS
Clearly, experimental evidence for the mechanism of
NblA action is needed Determination of the
three-dimen-sional structure of the protein could yield insights into the
action of this small protein family
A C K N O W L E D G E M E N T S
The authors which to thank E Krause and H Lerch, both FMP, for
performing the MALDI-TOF MS measurements and G Krause
(FMP) for bioinformatics, and Prof J Behlke (MDC) for helpful
suggestions on the AUC Prof H Oschkinat (FMP), Prof H Welfle
(MDC) and Prof W Lockau (HUB) are acknowledged for their
continuing support The authors wish to thank the FMP for financial
support.
R E F E R E N C E S
1 Bhaya, D., Schwarz, R & Grossman, A.R (2000) Molecular responses to environmental stress In The Ecology of Cyano-bacteria (Whitton, B.A & Potts, M., eds), pp 397–442 Kluwer Academic Publishers, Dordrecht.
2 Tandeau de Marsac, N & Houmard, J (1993) Adaptation of cyanobacteria to environmental stimuli: new steps towards mole-cular mechanisms FEMS Microbiol Rev 104, 119–190.
3 Grossman, A.R., Schaefer, M.R., Chang, G.G & Collier, J.L (1993) The phycobilisome, a light-harvesting complex responsive
to environmental conditions Microbiol Rev 57, 725–749.
4 Sauer, J., Schreiber, U., Schmid, R., Vo¨lker, U & Forchhammer,
K (2001) Nitrogen starvation-induced chlorosis in Synechococcus PCC 7942 Low-level photosynthesis as a mechanism of long-term survival Plant Physiol 126, 233–243.
5 Go¨rl, M., Sauer, J., Baier, T & Forchhammer, K (1998) Nitro-gen-starvation-induced chlorosis in Synechococcus PCC 7942: adaptation to long-term survival Microbiology 144, 2449–2458.
6 Wolk, C.P., Ernst, A & Elhai, J (1994) Heterocyst metabolism and development In The Molecular Biology of Cyanobacteria (Bryant, D.A., ed.), pp 769–823 Kluwer Academic Publishers, Dordrecht.
7 Wood, N.B & Haselkorn, R (1980) Control of phycobiliprotein proteolysis and heterocyst differentiation in Anabaena J Bacter-iol 141, 1375–1385.
8 Thomas, J (1970) Absence of the pigments of photosystem II of photosynthesis in heterocysts of a blue-green alga Nature 228, 181–183.
9 Peterson, R.B., Dolan, E., Calvert, H.E & Ke, B (1981) Energy transfer from phycobiliproteins to Photosystem I in vegetative cells and heterocysts of Anabaena variabilis Biochim Biophys Acta 634, 237–248.
10 Allen, M.M & Smith, A.J (1969) Nitrogen chlorosis in blue-green algae Arch Mikrobiol 69, 114–120.
11 Collier, J.L & Grossman, A.R (1994) A small polypeptide trig-gers complete degradation of light-harvesting phycobiliproteins in nutrient-deprived cyanobacteria EMBO J 13, 1039–1047.
12 Richaud, C., Zabulon, G., Joder, A & Thomas, J.C (2001) Nitrogen and sulfur starvation differentially affects phycobilisome degradation and expression of the NblA gene in Synechocystis strain PCC 6803 J Bacteriol 183, 2989–2994.
13 Baier, K., Nicklisch, S., Grundner, C., Reinecke, J & Lockau, W (2001) Expression of two NblA-homologous genes is required for phycobilisome degradation in nitrogen-starved Synechocystis sp PCC 6803 FEMS Microbiol Lett 195, 35–39.
14 Kaneko, T., Nakamura, Y., Wolk, C.P., Kuritz, T., Sasamoto, S., Watanabe, A., Iriguchi, M., Ishikawa, A., Kawashima, K., Kimura, T., Kishida, Y., Kohara, M., Matsumoto, M., Matsuno, A., Muraki, A., Nakazaki, N., Shimpo, S., Sugimoto, M., Takazawa, M., Yamada, M., Yasuda, M & Tabata, S (2001) Complete genomic sequence of the filamentous nitrogen-fixing cyanobacterium Anabaena sp strain PCC 7120 DNA Res 8, 205– 213.
15 Scha¨gger, H & von Jagow, G (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa Anal Biochem 166, 368–379.
16 Andreeva, A.Y., Krause, E., Mu¨ller, E., Blasig, I.E & Utepber-genov, D.I (2001) Protein kinase C regulates the phosphorylation and cellular localization of occludin J Biol Chem 276, 38480– 38486.
17 Gill, S.C & von Hippel, P.H (1989) Calculation of protein extinction coefficients from amino acid sequence data Anal Bio-chem 182, 319–326.
18 Johnson, W.C Jr (1990) Protein secondary structure and circular dichroism: a practical guide Proteins Struct Funct Genet 7, 205– 214.
Trang 819 Zamyatnin, A.A (1972) Protein volume in solution Progr
Bio-phys Mol Biol 24, 109–123.
20 Zamyatnin, A.A (1984) Amino acid, peptide, and protein volume
in solution Annu Rev Biophys Bioeng 13, 145–116.
21 Laue, T.M., Shah, B.D., Ridgeway, T.M & Pelletier, S.L (1992)
Computer-aided interpretation of analytical sedimentation data
for proteins In Analytical Ultracentrifugation in Biochemistry and
Polymer Science (Harding, S.E., Rowe, A.J & Horton, J.C., eds),
pp 90–125 Royal Society of Chemistry, Cambridge.
22 Sober, H (1968) The Handbook of Biochemistry and Molecular
Biology, Chemical Rubber Co., Cleveland, OH.
23 Lide, D.R., ed (1974) Handbook of Chemistry and Physics CRC
Press, Boca Raton, Florida.
24 Lamm, O (1929) Die Differentialgleichung der
Ultrazentrifugie-rung Ark Mat Astr Fys 21B, 1–4.
25 Behlke, J & Ristau, O (1997) Molecular mass determination by
sedimentation velocity experiments and direct fitting of the
con-centration profiles Biophys J 72, 428–434.
26 Behlke, J & Ristau, O (2002) A new approximate whole boundary
solution of the Lamm differential equation for the analysis of
sedimentation velocity experiments Biophys Chem 95, 59–68.
27 Schuck, P (1998) Sedimentation Analysis of noninteracting and
self-associating solutes using numerical solutions to the Lamm
equation Biophys J 75, 1503–1512.
28 Schuck, P., Perugini, M.A., Gonzales, N.R., Howlett, G.J &
Schubert, D (2002) Size-distribution analysis of proteins by
ana-lytical ultracentrifugation: strategies and application to model
systems Biophys J 82, 1096–1111.
29 Van Holde, V.E & Weischet, W.O (1978) Boundary analysis of Sedimentation velocity experiments with monodisperse and pau-cidisperse solutes Biopolymers 17, 1387–1403.
30 Demeler, B., Saber, H & Hansen, J.C (1997) Identification and interpretation of complexity in sedimentation velocity boundaries Biophys J 72, 397–407.
31 Chatelier, R.C (1988) A Parameterized overspeeding method for the rapid attainment of low-speed sedimentation equilibrium Anal Biochem 175, 114–119.
32 Chervenka, C.H (1969) A manual of methods for the analytical ultracentrifuge Beckman Instruments, Inc., Palo Alto, CA.
33 Johnson, M.L., Correia, J.J., Yphantis, D.A & Halvorson, H.R (1981) Analysis of data from the analytical ultracentrifuge by nonlinear least squares techniques Biophys J 36, 575–588.
34 McRorie, D.K & Voelker, P.J (1993) Self-associating systems in the analytical ultracentrifuge Beckman Instruments, Inc., Full-erton, CA.
35 Rost, B & Sander, C (1993) Prediction of protein structure at better than 70% accuracy J Mol Biol 232, 584–599.
36 Rost, B (1996) PHD: predicting one-dimensional structure by profile based neural networks Methods Enzymol 266, 525–539.
37 Jaenicke, R (1999) Stability and folding of domain proteins Prog Biophys Mol Biol 71, 155–241.
38 Sidler, W.A (1994) Phycobilisome and phycobiliprotein struc-tures In The Molecular Biology of Cyanobacteria (Bryant, D.A., ed.), pp 139–216 Kluwer, Dordrecht.