N-Terminal amino acid analyses and mass spectrometry characterized a series of a-PEC peptides which occurred during storage in formic acid.. Mass spectrometry and N-terminal amino acid s
Trang 1Purification, crystallization, NMR spectroscopy and biochemical
analyses of a-phycoerythrocyanin peptides
Georg Wiegand1, Axel Parbel2*, Markus H J Seifert1, Tad A Holak1and Wolfgang Reuter1
1
Max-Planck-Institut fu¨r Biochemie, Martinsried, Germany;2Botanisches Institut der Ludwig-Maximilians-Universita¨t, Mu¨nchen, Germany
The a-phycoerythrocyanin subunits of the different
phy-coerythrocyanin complexes of the phycobilisomes from
the cyanobacterium Mastigocladus laminosus perform a
remarkable photochemistry Similar to phytochromes –
the photoreceptors of higher plants – the spectral
pro-perties of the molecule reversibly change according to the
irradiation wavelength To enable extensive analyses,the
protein has been produced at high yield by improving
purification protocols As a result,several comparative
studies on the Z- and E-configurations of the intact
a-subunit,and also on photoactive peptides originating
from nonspecific degradations of the chromoprotein,were
possible The analyses comprise absorbance,fluorescence
and CD spectroscopy,crystallization,preliminary X-ray
measurements,mass spectrometry,N-terminal amino acid
sequencing and 1D NMR spectroscopy Intact
a-phyco-erythrocyanin aggregates significantly,due to hydrophobic
interactions between the two N-terminal helices Removal
of these helices reduces the aggregation but also desta-bilizes the protein fold The complete subunit could be crystallized in its E-configuration,but the X-ray meas-urement conditions must be improved Nevertheless, NMR spectroscopy on a soluble photoactive peptide presents the first insight into the complex chromophore protein interactions that are dependent on the light induced state The chromophore environment in the Z-configuration is rigid whereas other regions of the protein are more flexible In contrast,the E-configuration has a mobile chromophore,especially the pyrrole ring D, while other regions of the protein rigidified compared to the Z-configuration
Keywords: phycobilisomes; phycoerythrocyanin; protein structure; photochemistry; energy transfer
Phycobiliproteins are a class of chromoproteins bearing
covalently bound linear tetrapyrrole (phycobilins)
chro-mophores They are predominantly involved in the
photo-synthetic light harvesting process of cyanobacteria and
certain algae With respect to this function they are
assembled to supramolecular protein pigment complexes,
i.e phycobilisomes,building up a highly ordered network of
very rigid chromophores which enable an energy transfer
efficiency of almost 100% [1,2]
The phycoerythrocyanin (PEC) complexes located at the
periphery of phycobilisomes are present in only a few
species of cyanobacteria [3] PEC of the thermophilic
cyanobacterium Mastigocladus laminosus is the best
char-acterized complex of this biliprotein class Like other
peripheral phycobiliproteins,e.g phycoerythrin,low light
and high temperature conditions induce a maximum
content of PEC,reaching approximately 30% of total
protein content within the phycobilisomes [4,5] The X-ray
structure of PEC reveals three heterodimeric a,b substruc-tures,so called ‘monomers’,which are associated to a ring shaped disc,designated as ‘trimer’ The b-subunit contains two phycocyanobilin (PCB) chromophores,whereas the a-subunit has a single phycoviolobilin (PVB) chromophore
of which the pyrrole ring A is covalently linked via a thioether bond to Cys84 [6]
Unlike other phycobiliproteins,the PVB chromophores
of the PEC-‘trimers’ present a remarkable reversible photochemistry which has been reported first by Bjo¨rn [7] The observation initiated intense investigations into this unusual spectroscopic behavior,especially regarding the possible function as a sensor pigment [8–10] The sensor function seems to be questionable,however,in particular because of the high content and extremely reduced photochemistry of PEC within the phycobilisomes [4,11,12] Biochemical and spectral data assign the photo-chemistry exclusively to the a-PEC subunit [13,14] Similar
to phytochrome and phytochrome-like photoreceptors of higher plants and cyanobacteria,the PVB chromophore undergoes spectral and molecular changes depending on light quality [15,16] The phototransformation of a-PEC is reflected by the reversible shift in the visible absorption maximum from 505 to 570 nm and the two states were termed E and Z,respectively Isomerization can be performed by irradiation with complementary chromatic light and the two states are quite stable in the dark The molecular mechanism of the photoreaction is similar to that of the phytochromes and is proposed to exists as a chromophore twisting around the D15,16 double bond between the C and D pyrrole rings [9,16] However, the
Correspondence to W Reuter,Max-Planck-Institut fu¨r Biochemie,
Am Klopferspitz 18 A,82152 Martinsried,Germany.
Fax: + 49 (0)89 85783516,Tel.: + 49 (0)89 85782707,
E-mail: reuter@biochem.mpg.de
Abbreviations: FID,free induction decay; HIC,hydrophobic
interac-tion chromatography; MPD,2-methyl-2,4-pentanediol;
PCB,phyco-cyanobilin; PEC,phycoerythrocyanin; PVB,phycoviolobilin.
*Present address: Amersham Biosciences,Munzinger Str 9,79111
Freiburg,Germany.
(Received 4 June 2002,revised 28 August 2002,
accepted 29 August 2002)
Trang 2situation may be more complicated in a-PEC,as at least
type I and type II Z/E-isomerizations have been
spectrosc-opically discerned The ratio of the two types has been
suggested to be controlled by sulfhydryls of Cys98 and
Cys99 in the protein [8,10] Whereas, the structures of the
PVB isomers are well defined by spectroscopical
charac-terizations,the participitation of the protein in the
phototransformation process of the a-subunit is almost
unknown Some reasons for this may be the considerable
difficulties in the preparative purification of a-PEC and the
problems arising during analyses of the protein at high
concentrations
The present study describes a very effective method of
isolation and purification of PEC and its photoactive
a-subunit Crystallization and preliminary X-ray
experi-ments suggest pronounced conformational alterations of
both the protein and the PVB chromophore,depending on
light quality N-Terminal amino acid analyses and mass
spectrometry characterized a series of a-PEC peptides
which occurred during storage in formic acid The Z- and
E-configurations of one chromopeptide with an excellent
solubility and stability were investigated by 1D NMR
spectroscopy The information presented here are mainly
focused on the preparations,handling and characterizations
of a-PEC peptides which will be a prerequisite for successful
studies on the detailed molecular events during
phototrans-formation
M A T E R I A L S A N D M E T H O D S
Strain, culturing conditions and isolation
of phycobilisomes
The thermophilic cyanobacterium M laminosus (syn
Fischerellasp PCC 7603) was photoautotropically grown
at 48C,10 lEÆm)2Æs)1 and gassed with 2% (v/v) CO2
enriched air The cells were harvested when an optical
density of 0.7 at 740 nm was reached At these conditions
the phycobilisomes are attributed by the maximum content
of PEC [4]
The phycobilisomes were isolated by step-gradient
cen-trifugation principally following the buffer conditions
described by Reuter and Wehrmeyer [17] Scaling up of
the phycobilisome preparation was necessary for the
development of the effective purification procedure of the
a-subunit Cells of M laminosus with a wet weight of 40 g
were disrupted at 17C in a self-constructed cell mill The
glass beads (1 mm) were filtered off and the filtrate of about
180 mL was incubated with 4% (w/v)
N,N-dimethyldode-cylamine N-oxide (Fluka,Buchs,Switzerland) for 1 h at
17C This homogenate was clarified by centrifugation at
48 000 g for 30 min at 17C The resulting supernatant of
approximately 180 mL was directly applied to
step-gradi-ents comprising 10 mL 40% (w/v),15 mL 30% (w/v),
15 mL 20% (w/v) and 15 mL 10% (w/v)
sucrose,respect-ively Centrifugation was performed in a Ti45 rotor
(Beckman Coulter,USA) at 40 000 g for 16 h at 17C
The separated phycobilisomes banding between 40% (w/v)
and 30% (w/v) sucrose were eluted and subsequently
precipitated by a final concentration of 2.0 M potassium
phosphate,pH 7.0 These products are stable at 4C for at
least 2 years without any changes in their protein
compo-sition
Purification of PEC complexes About 1000 mg of precipitated phycobilisomes were sedi-mented by centrifugation at 74 000 g for 30 min at 17C The sediment was resolved in distilled water and dissoci-ation was performed by gel filtrdissoci-ation on a Sephadex G25 column (Amersham Biosciences,40 mm· 250 mm) and equilibrated with 5 mMpotassium phosphate,pH 7.0 The eluted phycobilisomes were applied directly to an anion exchange column (40 mm· 150 mm) packed with DEAE-Trisacryl M (Serva,Heidelberg,Germany) and equilibrated with the dissociation buffer At this pH and ionic strength the PEC complexes with and without linker polypeptides eluted completely,whereas all other biliprotein complexes remained on the column The resulting PEC fraction of about 250 mg was precipitated with a final concentration of 2.0Mpotassium phosphate,pH 7.0
Purification of the a-PEC subunit Sedimented PEC was resolved in distilled water and the biliprotein complexes were dissociated by gel filtration on a PD-10 column (Amersham Biosciences) in 0.3% (v/v) formic acid The dissociation into a- and b-subunits is accompanied by the loss of the excitonic coupling between the corresponding chromophores Therefore,the complete-ness of dissociation can be followed by the color change of PEC from pink to blue Separation of the subunits from each other was obtained by isocratic hydrophobic interac-tion chromatography in 0.3% (v/v) formic acid on a column (10 mm· 30 mm) packed with isopropyl-substi-tuted Sephacryl-S300 (Amersham Biosciences) The proce-dure of the isopropyl substitution of the gel will be published elsewhere During the chromatography,the a-subunits show negligible interactions with the gel, whereas the elution of b-subunits and linker polypeptides
is strongly retarded The eluted homogeneous a-PEC fraction of at least 80 mg was concentrated by ultrafiltra-tion up to 20 mgÆmL)1
Figure 1 summarizes the purification steps which are necessary for the isolation of the intact,photoactive a-subunit Some of the different steps are based on previous methods,e.g induction of the maximal PEC content [4,5], anion exchange chromatography on DEAE [18],or use of formic acid as isolation medium [19]
Nevertheless,some advantages of the preparation meth-ods should be described Based on a maximal phycobilisome concentration of 30 mgÆmL)1 within the cells [20],the isolation yield of about 80% of intact phycobilisomes is very high The effectiveness of the cell breakage (¼ 95%) and the complete precipitation of the phycobilisomes without dis-sociation are responsible for the extraordinary high yield (results not shown) A previous study described an unusual fractionation of PEC on DEAE–cellulose [18] This non-specific separation was not observed on DEAE-Trisacryl M,therefore the elution time and dilution of the sample was considerably reduced In addition,short-time dissociation
by gel filtration with formic acid and the fast separation by hydrophobic interaction chromatography (HIC) reduce the time consumption and the denaturation effects How-ever,the most important step was the chromatography on the isopropyl-substituted Sephacryl-S300 minimizing non-specific interactions of a-PEC with the gel matrix
Trang 3Optical spectroscopy
The spectra of a-PEC were recorded after saturated
irradiation with 577 nm light transforming the E-isomer
and 500 nm light transforming the Z-isomer,respectively
Absorbance spectra were measured with a Lambda 2 UV/
visible spectrophotometer (Perkin-Elmer) and circular
dichroism (CD) was recorded on a Dichrograph VI (ISA)
The spectral band width was 0.25 nm,the scan speed
5 nmÆs)1 Fluorescence spectra were recorded with 2 nm
resolution on a Spex model 221 fluorimeter Details of the
measurements are described by Parbel et al [21]
Crystallization of a-PEC
Unless otherwise stated,all preparations were carried out
under red light with an emittance maximum of 650 nm
(Phillips,the Netherlands; TLD 15) Crystallization growth
was controlled in a modified microscope at a wavelength of
620 nm Parallel crystallization experiments were conducted
after transforming the a-PEC in the E- and Z-state,
respectively The state of the two isomers was monitored
by absorbance spectrometry in the range of 250–650 nm
Using the vapor diffusion hanging-drop method,the
proteins were crystallized in a pH range of 4.0–8.5 because
at these pH values both protein and chromophore show a
high stability Crystallization of the E-form could only be observed in the presence of different salts,e.g ammonium sulfate,ammonium phosphate,sodium-potassium phos-phate or Tris phosphos-phate,as precipitants Other precipitants like poly(ethylene glycol) or 2-methyl-2,4-pentanediol (MPD) have not,as yet,been found to be successful Crystals of the Z-form have never been obtained,although the crystallization conditions of both protein states have been identical Thin plates with dimensions of approxi-mately 0.2· 0.6 · 0.3 mm of the E-isomer grown in the presence of 1.0Mdibasic Tris phosphate,pH 8.0,at 18C were analyzed by diffraction studies and mass spectrometry For cryo-measurements the crystals were transferred into
3M Tris phosphate,pH 8.0,which serves as cryo-protec-tant The crystals were frozen directly in liquid nitrogen and the X-ray diffractions were recorded under white room-light
at temperatures between)140 and )160 C
‘Native’ PAGE PAGE was performed in 3-mm thick 10% (w/v) polyacryl-amide slab gels with Tris/boric acid (42 mM/100 mM,
pH 7.9) Gels were polymerized with 0.1% (v/v) tetrameth-ylethylenediamine and 0.03% (w/v) ammonium peroxodi-sulfate [17] Samples of 1.5 mL containing 10–15 mgÆmL)1 protein were electrophoresed in the Tris/boric acid buffer system for 2400 VÆh)1 at a constant power of 18 W,at
10C and continuous buffer circulation in a DESAGA VA-200 apparatus (DESAGA,Germany) After separ-ation,the protein bands were cut out and the gel slices were squeezed between two glass plates The homogeneous gel pastes were eluted for 3 h under continuous stirring with a 10-fold volume of water After elution the homogenates were centrifuged for 1 h at 75 000 g and the supernatants were filtered through a 0.22-lm poly(vinylidene difluoride) membrane (Millipore,USA) [22] The peptides were con-centrated by ultrafiltration and the photoactivity was tested
by absorbance spectra after alternative irradiation with the two light qualities
Mass spectrometry and N-terminal amino acid sequencing Mass spectrometry of the a-PEC peptides originating from the preparation of the hydrophobic interaction column,the crystals and the ‘native’ PAGE was performed by the electrospray method in a single quadrupol mass spectro-meter API165 (Applied Biosystems,Langen,Germany) The spectra were deconvoluted with theBIOTOOLsoftware
of the manufacturer Removal of salts within the samples was performed by hydrophobic interaction chromato-graphy on ReproSil-Pur C18-AQ,3l, 1· 150 mm (Dr
A Maisch,Ammerbuch,Germany) The peptides were eluted with a gradient from 10% (v/v) trifluoroacetic acid in
H2O to 0.08% (v/v) trifluoroacetic acid in acetonitrile All sequences were determined with an Applied Biosys-tems sequencer model 473 A following the manufacturer’s instructions
Structural comparison of the intact a-PEC and the degraded a-PEC peptide 2 The picture comparing the secondary structures of the intact subunit and peptide 2 was produced with the 3D
Fig 1 Overview of the isolation and purification steps of a-PEC from
Mastigocladus laminosus Approximately 250 mg of PEC-linker
com-plexes could be separated from 1000 mg of phycobilisomes The
pre-cipitated PEC-fraction can be stored without alteration at least for a
period of 2 years Starting with 250 mg of the linker-PEC complexes,
the purification at dissociating conditions in 0.3% (v/v) formic acid
results in a final preparation of approximately 80 mg of homogeneous
a-PEC.
Trang 4visualization program ‘WebLab ViewerPro,Version 3.20’
(Molecular Simulations Inc.) The coordinates derived from
the structure analyses of phycoerythrocyanin [6]
Light-dependent 1D NMR spectroscopy
of a-PEC peptide 2
Prior to NMR measurements,peptide 2 in 20 mM
sodium-potassium phosphate,pH 7.2 with a protein concentration
of 15–20 mgÆmL)1 was irradiated with light of 571 and
503 nm inducing the E- and Z-configuration,respectively
The complete transfer into both configurations was
obtained by an illumination time of 1 h Continuous
spinning of the NMR tube minimized the self-shadowing
of the highly concentrated sample.1H-NMR measurements
were carried out in the dark without spinning on a Bruker
DRX600 spectrometer equipped with a1H-13C-15N
triple-resonance probehead including triple-axis gradients All
spectra were recorded at a temperature of 27C To
suppress the water resonance a jump-return pulse sequence
was used [23] For each spectrum 512 free induction decays
(FIDs) with 32 k time domain points comprising a sweep
width of 9 kHz were recorded The interscan delay was set
to 1 s The 90 pulse was determined to be 8.4 ls The
spectra were processed by fast Fourier transformation
including a Gaussian window function and digital filtering
of low frequencies in the range of 1.5 p.p.m to enhance
water suppression Only 12 k of the recorded 32 k time
domain points were used for transformation to increase
signal-to-noise ratio The final spectra were processed to
32 k frequency domain points
R E S U L T S
Spectral behavior of a-PEC in 0.3% (v/v) formic acid
The steady state absorption,fluorescence and CD spectra of
a-PEC depending on pre-illumination are represented in
Fig 2 The a-subunit in the E-configuration is characterized
by a long wavelength absorbance maximum at 503 nm with
a pronounced shoulder near 566 nm,an extremely low
fluorescence and a CD minimum near 505 nm The sharp
peak (arrowhead) in the fluorescence spectrum originates
from the excitation light The absorbance shoulder near
566 nm,the broad fluorescence maximum at 588 (arrow) and
the minimum in the CD spectrum near 325 nm are typical for
signals of a-PEC in the Z-configuration Therefore,the
presence of these signals in the spectra of the E-isomer
indicates an incomplete transformation of the molecule or at
least of the chromophore In contrast,the Z-configuration of
a-PEC reveals uniform maxima at 566 nm (absorbance),
588 nm (fluorescence),566 nm (CD) and a minimum at
329 nm (CD) The spectral data of the proteins in the E- as
well as the Z-state in 0.3% (v/v) formic acid are nearly equal
to those in conventional buffers near pH 7.0 [9,10,12] Thus,
the ‘native’ state of the chromoprotein has been assumed
Crystallization of a-PEC
In order to obtain information about changes of the
polypeptide properties in the Z- and E-state,respectively,
crystallization was performed with the protein in both
configurations One major problem was the aggregation
Fig 2 Optical spectroscopyof the E- and Z-configurations of the a-subunit in 0.3% (v/v) formic acid The spectral behavior of the chromoprotein in is nearly identical to that at neutral pH which con-firms the suitability of the isolation and purification method It must
be noted that the chromophore cannot completely be transferred into the E-configuration This is shown by the arrows in the absorbance, fluorescence and CD spectra The fluorescence of a-PEC in the E-configuration is extremely low,therefore the excitation light,marked
by an arrowhead,is seen in the spectrum.
Trang 5behavior of the protein at pH values near 7.0,especially in
the Z-configuration Therefore,variations in the protein
concentrations (5–7.5 mgÆmL)1) were strongly limited The
crystallization behavior of the E-isomer is identical in the
dark and in green light (results not shown) This was tested
by parallel crystallization attempts in the dark and under
weak monochromatic green light All common precipitants
have been used but only different salts at varying ionic
strength and pH values have been successful Two typical
crystallization conditions comparing the E- and the
Z-configurations are demonstrated in Fig 3 Despite the
identical crystallization conditions,only the E-configuration
crystallized (Fig 3a,b), whereas the Z-configuration always
showed a type of phase separation (Fig 3c,d) The
branch-ing of the crystals occurred under nearly all conditions,
however,the size of single,homogeneous crystal plates was
sufficient for further analyses
Unfortunately,X-ray analyses of such plates were
unsuccessful because the diffraction of the crystals decreased
very rapidly during the measurements This phenomenon
has been observed for different crystals,even at low
temperatures between)140 and )160 C Because of the
extreme changes in the diffraction patterns,a unique
determination of the space group and the unit cell was not
possible Nevertheless,within the limits of the
measure-ments,we tentatively determined an orthorhombic space
group with two molecules in the asymmetric unit What is
the reason of the strongly decreasing diffractions? The
frozen crystals have been mounted and measured under
white room-light At this condition,light-induced
conform-ational changes which destroy the well ordered crystal
packing might be possible The molecular flexibility of
different crystallized phycobiliproteins at temperatures in
the range from )100 to )160 C has frequently been
observed during the freezing and measuring procedures (Reuter,unpublished results) In addition,different inter-mediate chromophore states of PEC were recorded depend-ing on the measurdepend-ing temperatures [24,25] The results clearly demonstrate the molecular mobility of phycobili-proteins,even at low temperatures,but the influence of light
on the crystal packing of a-PEC during the measurements remains uncertain
Purification and analyses of a-PEC peptides The storage time of a-PEC in 0.3% (v/v) formic acid at 4C was approximately 6 months At the end of this time,the crystals shown in Fig 3 could not be reproduced This fact initiated an analysis of the sample by mass spectrometry revealing at least seven peptides with molecular weights between 16 000 and 14 000 Da (results not shown) At present,the reasons for the degradation are uncertain A proteolytic splitting of the a-subunit by proteases may be possible,although the pH of 2.2 of the formic acid probably inhibits the activity of most peptidases Another postulation
is the acid-induced degradation of the a-PEC during long-term storage Specific acid-catalyzed degradation reactions have previously been reported for other proteins [26] The most probable explanation would be a nonspecific acid-catalyzed hydrolysis of a-PEC which is facilitated by a partial unfolding of the two N-terminal helices (Fig 4) The resulting high flexibility of this peptide region may be responsible for destabilization of favored peptide bonds within the protein This view is in line with the variability of the amino acid sequences for which the degradation occurs However,cooperation between the three mechanisms cannot be excluded Further studies on the instability of the isolated a-subunit are in progress and some aspects will
be stressed in the discussion section
Fig 3 Crystallization of a-PEC has been successful onlywith the
molecule in the E-configuration (a,b) In principle,all crystals have been
grown at 17 C by the hanging-drop method with vapor diffusion
concentration Only salt precipitation resulted in crystals as shown in
the figure (a) Potassium phosphate,pH 7.5; (b) Tris phosphate,
pH 8.0; (c) potassium phosphate,pH 7.5; (d) Tris phosphate,pH 8.0.
Crystals of (b) have been tested by X-ray analysis They diffracted up
to 2.8 A˚ but structure analysis could not been performed because the
lifetime of the crystals during the measurements was extremely short
even at temperatures between )140 and )160 C.
Fig 4 Comparison of the secondarystructure of the intact a-PEC and the peptide 2 obtained bynonspecific degradation The alignment was performed with the structure viewer program WEBLAB VIEWER PRO and could be generated concerning the results of mass spectrometry and N-terminal amino acid sequencing (Table 1) The two N-terminal helices are responsible for the aggregation of a-PEC in solution The mobile D pyrrole ring is marked by an arrow.
Trang 6The preparative separation of the peptides was achieved
by the high performance ‘native’ PAGE,resolving five
colored bands which have been analyzed by UV/visible
spectroscopy,N-terminal sequencing and mass
spectro-metry (Fig 5,Table 1) The similarity of absorbance and
fluorescence as well as the complementary
phototransfor-mation of the peptides is indicative for the unchanged
chromophore environment of the peptides This observation
could be confirmed by the comparative amino acid analyses
and mass spectrometry The chromopeptides 1–3 showed
different N-terminal degradations,resulting in partially
different charges of the peptides Nevertheless,the
elec-trophoretic separation cannot be explained solely by the
peptide charges because bands with nearly the same charge (bands 1B,1C and 2) migrated quite differently in the gel It can be speculated that either structural factors or distinct aggregations of the peptides are responsible for the individ-ual migration behavior The aggregation of the peptides 1A, 1B and 1C is shown from the behavior of these peptides during concentration by ultrafiltration As shown in Table 1,they aggregate at pH 7.0,similar to the intact a-PEC subunit
Mass spectrometry of the PEC complexes and purified a-PEC was performed directly after isolation The deter-mined molecular mass of the corresponding a-subunits agrees exactly with the calculated mass,including amino acids and the PVB chromophore In contrast,within the crystals,two peptide masses differing by 16 Da have been detected This fine but significant distinction reproducibly occurred in the crystal analyses and points to a modifi-cation of the chromoprotein during crystallization Within the error limits,the difference of 16 Da corresponds well
to an addition of oxygen Although,the site of the oxidation could not be determined,it is probable that Cys98 and/or Cys99 of the a-subunit are partially oxygenated The reaction mechanisms and conditions have not been investigated thoroughly,but it is an interesting result,especially regarding the photochemistry
of the types I and II [8, 10]
Structure of peptide 2 The results summarized in the Table 1 clearly show that the two N-terminal helices are not necessary for the photo-chromism Therefore,the molecular events accompanying the isomerization of the chromophore should be equivalent within the intact a-PEC and the derived peptides The excellent solubility of peptides 2 and 3 at pH 7.0 recom-mended their employment for further studies such as crystallization and NMR spectroscopy Unfortunately, depending on light,ionic strength and pH,the peptides are much more sensitive to degradation than the intact subunit The reactions and their physical reasons have not been investigated systematically,however,all crystallization experiments failed and the peptides often lost their color
Table 1 Comparison of the N-terminal sequences and molecular masses of the a-PEC peptides separated by‘native’ polyacrylamide gel electrophoresis Numbers in parentheses are minor components of the samples.
Peptides Molecular mass N-terminus
Molecular properties Photoactivity a-PEC 18 151.6 MKTPLTEAIAÆÆAADLRGSYLSÆÆNTELQAVFGRÆÆFNRARAGLEA Aggregating at pH 7.0
+ Crystals of a-PEC 18 151.6
18 167.8
MKTPLTEAIAÆÆAADLRGSYLSÆÆNTELQAVFGRÆÆFNRARAGLEA Original molecule
Modified molecule Peptide 1A 15 803.2
(15 473.8)
ÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆLQAVFGRÆÆFNRARAGLEA Aggregating at pH 7.0
+ Peptide 1B 15 585.0 ÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆAVFGRÆÆFNRARAGLEA Aggregating at pH 7.0
+ Peptide 1C 15 157.0 ÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆGRÆÆFNRARAGLEA Aggregating at pH 7.0
+ Peptide 2 14 945.2
(14 797.2)
ÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆFNRARAGLEA ÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆNRARAGLEA
Soluble at pH 7.0 +
Peptide 3 14 525.6 ÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆÆARAGLEA Soluble at pH 7.0
+
Fig 5 High performance ‘native’ polyacrylamide electrophoresis of the
a-PEC peptides Cathode (–) is at the top and anode (+) at the bottom
of the picture All peptides show the ‘normal’ photoactivity suggesting
a nearly unchanged chromophore environment The peptides were
analyzed by mass spectrometry and N-terminal amino acid
sequen-cing In both its E- and Z-configurations,peptide 2 was characterized
further by NMR spectroscopy.
Trang 7(results not shown) An explanation for this behavior can be
derived from the structural comparison of intact a-PEC and
peptide 2 (Fig 4) Within ‘monomeric’ PEC,hydrophobic
interactions between the N-terminal helices of both the
a- and b-subunits stabilize the complex [6] At pH 7.0,
similar interactions take place in the solutions of isolated
a-PEC and the diffraction data suggest a ‘dimeric’
arrange-ment of the subunits within the crystals Consequently,the
association to ‘homodimers’ is proposed to be responsible
for the enhanced stability of the intact a-subunit in contrast
to that of the peptides The low pH of 2.2 in 0.3% (v/v)
formic acid,or at least the partial degradation of the two
helices,prevents the interactions and reduces the
aggrega-tion However,complete loss of the helices or even more of
the N-terminus significantly decreases the physical stability
of the chromopeptides Peptide 2 is characterized by a small
stabilizing section of the second N-terminal helix and a
sufficient solubility Therefore,providing a good
compro-mise between the two opposite molecular properties,this
chromopeptide enabled light-dependent analysis by 1D
NMR spectroscopy
Molecular alterations of the a-PEC peptide 2
demonstrated by NMR spectroscopy
The purpose of the NMR study was not the detailed
structural analysis of the two chromopeptide
configura-tions Moreover,the study should answer some important
questions concerning the methodological knowledge and
the molecular events depending on photochemistry: (a) Is
peptide 2 suitable for further NMR studies? (b) Is the
photochemistry of the chromophores accompanied by
significant changes in the protein structure? (c) Is it possible
to discern between chromophore and protein signals? (d) Is
the photoconversion between the two states of the
chromopeptide complete or incomplete,as indicated by
the spectral data of the E-configuration (Fig 2)
Initial NMR spectroscopy was performed using the
intact a-PEC,but protein aggregations caused extreme
broadening of the signals In contrast,the 1D NMR
spectra of peptide 2 in its E- and Z-configuration,
respectively,show the well separated peaks of a monomeric
protein (Fig 6) A reliable comparison between the spectra
of one sample is possible as the light equipment enabled
complementary irradiation within the NMR tube without
changing the protein environment For clarity,only the
two important regions (NH and aliphatic) of the spectra
are presented The main differences between the spectral
data of the E- and Z-configurations are emphasized by the
E/Z-difference spectrum Multiple spectral deviations in
the height as well as the chemical shifts of the peaks can be
seen The various differences between nearly all regions of
the spectra are indicative of parallel light-dependent
structural changes of the peptide and the chromophore
The interpretation of the NMR spectra is rather difficult
because protein and chromophore signals overlap
Obvi-ously,the presence of two peaks between 10 and 11 p.p.m
which do not change and their positions within the spectra
suggests that they represent the two tryptophanes,Trp51
and Trp128,in the peptide [13,27],although an unusually
shifted signal from another amino acid residue cannot be
excluded At least three peaks from the E-configuration
and their slightly shifted negative counterparts from the
Z-configuration are resolved in the aliphatic region of the difference spectrum Because of the height and the sharpness of the peaks,they are assumed to be derived exclusively from the aliphatic residues of the distinct chromophore states These signals probably reflect the isomerization and mobility of the D pyrrole ring The dominant peaks of the peptide in the E-configuration show the enhanced mobility depending on reduced chromophore protein interactions in this state The integration of well resolved peaks should enable an estimation of the state populations obtained by complementary illuminations The protein peaks at)0.033 p.p.m and )0.099 p.p.m.,as well
as the protein peaks at 9.44 p.p.m and 9.38 p.p.m.,can be attributed to the Z- and E-states,respectively Integration
of both pairs of peaks yields the ratios of state populations
of Z/E¼ 12%/88% for the E-state and approximately Z/E¼ 65%/35% for the Z-state These estimations are consistent within the various peaks of the NMR spectra but are contrary to the optical spectra,where only the E-form of a-PEC shows a significant amount of the complementary spectral state [9,10,21]
D I S C U S S I O N This study presents the purification and molecular analyses
of photoactive a-PEC peptides from phycobilisomes of
M laminosus Preliminary results of crystallization and NMR spectroscopy offer reliable information on the relations between the protein backbone and the photo-chemically active chromophore of the peptides
Fig 6 1D NMR spectroscopyof a-PEC peptide 2 in 20 m M sodium-potassium phosphate, pH 7.2 The spectra were recorded after irradiation with light of 571 nm (E-configuration) and 503 nm (Z-configuration),respectively To emphasize the spectral deviations the difference spectrum E-configuration–Z-configuration is presented The spectra of the single sample have been recorded three times within
a period of 3 months Only the last spectrum,recorded after 3 months, showed significant deviations which could be attributed to a nonspe-cific degradation of the chromopeptide (results not shown) The chromophore peaks are marked by arrows and the integrated protein peaks are labeled by arrowheads.
Trang 8Methodological aspects
In order to obtain high amounts of a-PEC,the purification
methods have been scaled up without adversely changing
the physical and chemical conditions of previous studies
[18,19,21] This means that the isolation media are almost
identical,whereas the dissociation conditions for the
purification of PEC complexes and a-PEC subunits,as well
as the time consumption of all steps,have been optimized
In the ‘native’ PAGE of the isolated PEC fraction (Fig 1),
only the two naturally occurring PEC-linker complexes are
present,confirming the brief dissociation and separation
conditions [4] The second important preparation step was
that of hydrophobic interaction chromatography
Dissoci-ation of PEC and separDissoci-ation of the subunits take place
within 2–3 h,which is extremely shortened in comparison
with established separation methods [12,18,19]
a-PEC from M laminosus was recently crystallized under
white light,but the photoactive state of the proteins within
the crystals has not been determined [19] Therefore,it
remains uncertain whether those crystals were composed of
E-, Z-,or possibly both,states of the protein However,the
X-ray measurements of these crystals,as well as those of
the crystals in this study,failed Despite cryo-conditions,the
molecular order of the crystals decreased rapidly during
measurements The reason for this is unknown,although
the occurrence in both studies,as well as the markedly
distinct crystallization behaviors of the E- and Z-states,
point to the influence of light on the protein structure,even
at low temperatures It may be of interest that no cracks
developed in the crystals during measurement
The considerable problem of the light sensitivity of
a-PEC in all preparation,crystallization and measuring
steps demands special light equipment In crystallization,
microscopic control and irradiation for NMR spectrometry,
the light conditions have been optimized Unequivocally,
the X-ray measurements also need a protection light,and a
long wavelength (650 nm) red light source is favored
Rapid degradation of a-PEC during all preparations has
often been observed [19] Certainly,one reason is the
enhanced accessibility of isolated subunits to proteolytic
enzymes Nevertheless,other factors exist which are
responsible for the degradation (see Results) The analyses
of the peptides revealed various splitting positions of the
amino acid chain This variability cannot be explained by
specific acid- or protease-catalyzed hydrolyses of the
protein Additionally,the stability of the chromopeptides
decreases rapidly,depending on the presence and length of
the two N-terminal helices,which has been proven by gel
filtration after the last NMR measurements (3 months) of
peptide 2 at pH 7.2 This sample showed a significant
amount of degraded peptides (results not shown) With
respect to all results,an ‘autolytical’ degradation at
prefer-ential regions of the peptides can be suggested
Molecular features of a-PEC peptides
The aggregation behavior and the tendency to degrade of
isolated a-PEC strongly limited the investigation methods
elucidating the molecular mechanisms of the
photoconver-sion [10] The isolation in formic acid enables working with
high protein concentrations,although the influence of low
pH between 2.0 and 2.5 on the molecular structure is not
completely clear Optical properties as well as photoactivity are almost equal in the pH range of 2.2–8.5 [9,10,12,19,21],
so that a nearly unchanged protein structure around the chromophore must be assumed Aggregation of a-PEC is assigned exclusively to the two N-terminal helices of the molecule that bind to each other via hydrophobic patches deviating from the association of the a- and b-subunits [6] Subsequently,the dimers unspecifically associate to supra-molecular particles Although,the excellent solubility of peptides 2 and 3 confirms this view,the explanation is not complete and the influence of low pH values also has to be considered Low pH induces a partial and possibly a temporary unfolding of the N-terminal helices,depressing dimerization A rapid degradation of these helices in formic acid which may be caused by their destabilization support this hypothesis Thus,the physical stability of a-PEC is strongly correlated to the interactions of the N-terminal helices or at least parts of these helices (Table 1)
The photochemistry of a-PEC peptides The photoactivity of the a-subunit strongly depends on its multiple protein interactions within the different association states of the PEC complexes [11,12,21] The assembly of
‘monomeric’ and ‘trimeric’ complexes is accompanied by a decrease of the photochemistry from 100% of the isolated a-PEC to 8% for the ‘trimers’ Naturally,linker-free phycoerythrocyanin does not exist Therefore,the slightly enhanced photoactivity of 11% of the linker-PEC com-plexes is of special interest Structural and spectral results clearly show that some linker polypeptides are responsible for an increased flexibility of allophycocyanin and phyco-cyanin complexes [28; Reuter,unpublished results] A similar behavior in the PEC-linker complexes would explain their relatively high photoactivity
Optical spectroscopy,as well as theoretical considera-tions,characterized the changes of the chromophore configuration on a substructural level [9,10,21,24,29–31] Strong coupling of excited states within the chromophore and charge transfer states from the surrounding polar amino acid residues are assigned either to stabilize the E- and Z-configurations or to enable the fast photoinduced struc-tural changes [30] The chromophore of the protein in the E-configuration also shows pronounced Z-characteristics spectrally (Fig 2),suggesting either a higher mobility or the existence of different intermediate states of the D pyrrole ring [24,31] The role of the apoprotein conformation in the spectral behavior of the chromophore is unknown because almost all applications focus on the chromophore and its neighboring amino acids
A first indication for considerable structural deviations of a-PEC in the E- and Z-states can be derived from their crystallization behavior The E-state crystallizes under various conditions whereas crystals,or at least microcrys-tallization,of the Z-state have never been observed This result correlates well with the NMR data,where the protein peaks of the molecule in the E-conformation are much more homogenous than that of the Z-conformation On the other hand,the mobility of some aliphatic groups of the E-chromophore are clearly increased compared with those
of the Z-chromophore (Fig 6) The NMR data can be interpreted as stabilization of the Z-chromophore configur-ation by an enhanced protein flexibility This situconfigur-ation has
Trang 9actually been simulated by molecular dynamics and was
described as oscillation of the chromophore and its
environment [30] In contrast,the protein in the E-state is
more rigid,although the D pyrrole ring of the chromophore
moves between its E and the Z positions
At present,the function of the photochemistry in PEC is
uncertain because the analysis in the environment of the
phycobilisomes is not currently possible According to
evolution studies on the phycobiliproteins of cyanobacteria
and rhodophyceae,PEC is the youngest member of this
protein family [32] Unequivocally, a-PEC is not a
sponta-neous mutation of a phycocyanin gene because two special
lyases are involved in the synthesis and attachment of the
chromophore [33,34] Concerning the light harvesting, the
advantage of PEC complexes compared with phycocyanin
complexes is the broadening of the phycobilisome
absorb-ance in the green light gap,whereas the photochemistry of
a-PEC may function in a radiationless energy dissipation
However,the missing fluorescence of PEC in intact
phycobilisomes and different adapted cells of M laminosus
support this suggestion [4]
A C K N O W L E D G E M E N T S
This work was financially supported by the Deutsche
Forschungsg-emeinschaft,Sonderforschungsbereich 533 (projects A1,A2,A3) The
authors wish to thank K.-H Mann for N-terminal amino acid analyses
and F Siedler and S Ko¨rner for mass spectrometry.
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