Springer 2005
Photosynthesis Research (2005) 85: 15–32
Review
Elucidation of the molecular structures of components
of the phycobilisome: reconstructing a giant
Noam Adir
Department of Chemistry and Institute of Catalysis, Science and Technology, Technion – Israel
Institute of Technology, Technion City, Haifa 32000, Israel (e-mail: [email protected]; fax:+972-48295703)
Received 11 April 2004; accepted in revised form 13 August 2004
Key words: antenna proteins, cyanobacteria, energy transfer, photosynthesis, X-ray crystallography
Abstract
The molecular architectures of photosynthetic complexes are rapidly becoming available through the power
of X-ray crystallography. These complexes are comprised of antenna complexes, which absorb and transfer
energy into photochemical reaction centers. Most reaction centers, found in both oxygenic and nonoxygenic species, are connected to transmembrane chlorophyll containing antennas, and the crystal
structures of these antennas contain information on the structure of the entire complex as well as clear
indications on their modes of functional association. In cyanobacteria and red alga, most of the Photosystem II associated light harvesting is performed by an enormous (3–7 MDa) membrane attached complex
called the phycobilisome (PBS). While the crystal structures of many isolated components of different PBSs
have been determined, the structure of the entire complex as well as its manner of association with
Photosystem II can only be suggested. In this review, the structural information obtained on the isolated
components will be described. The structural information obtained from the components provides the basis
for the modeled reconstruction of this giant complex.
Abbreviations: APC – allophycocyanin; Cc-PC – Cyanidium caldarium phycocyanin; Fd-PC – Fremyella
diplosiphon phycocyanin; Gm-PE – Griffithisia monilis phycoerythrin; LHC – light harvesting complex;
Ml-APC – Mastigoclaudus laminosus allophycocyanin; Ml-PC – Mastigoclaudus laminosus phycocyanin;
NMA – c-N-methyl asparagines; PBPs – phycobiliproteins; PBSs – phycobilisomes; PC – phycocyanin;
PCB – phycocyanobilin cofactor; PDB – the Protein Data Bank; PE – phycoerythrobilin; PEB – phycoerythrin; PEC-phycoerythrocyanin; PEG – polyethylene glycol; PS I – Photosystem I; PS II – Photosystem
II; rms – root mean square; Ps-PE – Porphyridium sordidum phycoerythrin; Pu-PC – Polysiphonia urceolata
phycocyanin; Py-APC – Porphyra yezoensis allophycocyanin; S7-PC – Synechococcus sp. PCC7002 phycocyanin; Sp-APC – Spirulina platensis allophycocyanin; Sp-PC – Spirulina platensis phycocyanin; TEM –
transmission electron microscopy; Te-PC – Thermosynechcoccus elongatus phycocyanin; Tv-PC – Thermosynechcoccus vulcanus phycocyanin
Introduction
The emergence of the phycobilisome
There is evidence that organisms exhibiting
rudimentary photosynthetic capabilities appeared
on earth about 3.5 GYr (3.5 · 10)9 years) ago.
Within about 0.5–1 GYr, the first oxygenic
prokaryotic organisms appear to have arisen, as
seen in biomarkers and microfossils from that
period (Awramik 1992; Buick 1992; Knoll et al.
1996; Brocks et al. 1999; De Marais 2000; Xiong
16
and Bauer 2002). The records of these early
organisms appear to have originated from
organisms rather similar to those of modern-day
cyanobacteria, and thus, the photosynthetic
apparatus found in cyanobacteria may represent
a close approximation to those ancient oxygenevolving systems. It is these same organisms that
altered the chemistry of the earth, by evolving
vast quantities of oxygen into the atmosphere
and thereby inhibited the further domination by
anoxygenic species. In time, these primitive species evolved and endosymbiotically merged with
non-photosynthetic
unicellular
organisms
(Hedges et al. 2001) into the more advanced
eukaryotic photosynthetic species. The major
difference in the photosynthetic apparatus of
cyanobacteria and red-algae [which appeared at
about 1.4 GYr ago (Hedges et al. 2004)], in
comparison to the more ancient non-oxygenic
bacteria, the off-shoot prochlorophytes (Ting
et al. 2002), and the more advanced green algae
and higher plants, is the presence of one of the
largest antenna proteins found in all of the
photosynthetic organisms – the phycobilisome
(PBS). The term phycobilisome was first coined
by Gantt and Conti (Gantt and Conti 1966a), on
the basis of their size and shape as visualized by
electron microscopy. Electron micrographs
showed a series of large granules aligned regularly on the thylakoid membranes of different
cyanobacteria and red alga, which were about
twice the size and of similar shape to that of
ribosomes. However, even earlier the components of the PBS attracted the attention of early
biologists, due to their brilliant colors. Indeed
the first crystals of an apparent phycobiliprotein
(PBP) were already reported in the late 19th
century (Cramer 1862; Molisch 1894, 1895). A
comprehensive review on the early study of PBS
components was recently compiled by Tandeau
de Marsac (Tandeau de Marsac 2003). The PBS
serves as a harvester of light energy in the
spectral gap between the major chlorophyll
absorbing bands (500–660 nm), allowing the
species bearing these antennas to utilize the
entire visible range of sunlight. All PBS complexes
have been shown to contain two substructures: a
core structure found closest to the membrane
surface, and a series of rods emanating out from
the core (Glazer 1985; Glazer 1989; Huber 1989;
MacColl 1998). Both core and rod substructures
contain subunits which covalently bind the lightharvesting linear tetrapyrolle bilin pigments, and
additional subunits lacking pigments called linker
proteins. Within the substantial range of cyanobacterial and red algal species, the number of
components which comprise the core and rods is
variable; however it is believed that common
structural features control the formation of the
PBS (Anderson and Toole 1998). Unlike photosynthetic reaction centers, no present day existing
biochemical or structural evidence has been identified for precursors of the PBS. Phylogenetic
analysis (Apt et al. 1995), has traced the evolution
of the different subunits back to a proposed primary ancestor, which may have been a precursor
for all globin-type proteins (Schirmer et al. 1985;
Pastore and Lesk 1990). These observations suggest that the evolution of the PBS may have
occurred relatively rapidly following the appearance of the first cyanobacterial species, and that
their ratio of functional efficiency to physiological
expenditure is large enough to have survived the
enormous range of ecological niches where
cyanobacteria can be found (Samsonoff and
MacColl 2001). Through a process of gene
duplication and evolutionary optimization, four
major subgroups of phybiliproteins (PBPs) can
be found: allophycocyanin in the cores (APC,
kmax ¼ 652 nm), phycocyanin in rods, closest to
the core (PC, kmax ¼ 620 nm), phycorerythrin
(PE, kmax ¼ 560 nm) and phycoerythrocyanin
(PEC, kmax ¼ 575 nm) in the rods, distal to the
cores. The physical arrangement of the different
PBPs creates a clear energy gradient from PE (or
PEC) through PC to APC and down into the
chlorophyll bed of the reaction center
(kmax ¼ 670–680 nm).
The maximal absorption wavelengths noted
above are due to the overlap of the absorption
characteristics of the 2–6 bilin pigments bound
to each minimal PBP unit, a heterodimeric
complex called the (ab) monomer, as modified
by the level of PBP aggregation (see below). Two
bilins are invariably linked to conserved cysteine
residues at position 84 on each residue (using the
PC sequence numbering), with the additional
pigments bound at additional cysteines. The
different types of bilins (MacColl 1998) include
phycocyanobilin (PCB, found in PC, PEC and
APC), phycoerythrobilin (PEB, found in PEs in
either singly or doubly linked forms), phycovi-
17
olobilin (found only in PEC) and phycourobilin
(found in some PEs).
Functional characteristics of the PBS
Attached primarily to reaction centers of Photosystem II (PS II), the PBS can functionally
link more than 600 energy absorbing pigments to
a single Photosystem II dimer (Glazer 1989). In
addition to the chlorophyll a molecules attached
to the PS II internal antenna subunits (CP43 and
CP47), this brings the total antenna cross section
to more than 350 cofactors per reaction center.
This can be compared to about 100 chlorophylls
per cyanobacterial Photosystem I (PS I) monomer (Jordan et al. 2001). In plant and green
algae systems, the antenna beds are somewhat
larger (Danielsson et al. 2004), but still smaller
than that provided by the PBS. This enlarged PS
II antenna base allows many cyanobacterial
species to increase the ratio of PS I/ PS II to
between 3 and 6 (Shen et al. 1993). In green
algae and plants, which contain only membranebound light harvesting complexes, the ratio of
PS I to PS II is close to 1 (Danielsson et al.
2004). Lowering the amount of PS II may be
physiologically beneficial due to the inherent
instability in PS II activity, which under extreme
conditions can lead to photoinhibition and cell
death (Prasil et al. 1992; Adir et al. 2003). Prevention of photoinhibition is achieved by rapid
replacement of the D1 subunit of the PS II
reaction center, which can be achieved more
efficiently by lowering the total amount of PS II.
Many reports exist showing the ability of the
PBS to transfer energy to PS I in vitro (Kirilovsky
and Ohad 1986) and in vivo (Rakhimberdieva
et al. 2001); however, whether binding to PS I is
mediated by specific interactions or by transient
association is unclear (Aspinwall et al. 2004).
Indeed, direct measurements of fluorescence
recovery using confocal microscopy has indicated
that the PBS is quite mobile in vivo, much more
than the photosystems (Mullineaux et al. 1997;
Sarcina et al. 2001), indicating that the association between antenna and photosystems in
cyanobacteria may be much looser than in other
systems.
Other than light absorption, the only
additional function of the PBS so far identified is
as an emergency source of nutrients to be used in
the case of nitrogen, sulfur or carbon starvation
(Collier and Grossman 1994; Richaud et al. 2001;
Li and Sherman 2002). This ordered disassembly
of the PBS complex requires the presence of the
gene products of a number of nbl genes (Grossman
et al. 2001), although different disassembly
pathways may occur in different organisms.
However, the specific mechanism of disassembly is
still unclear. Since the PBS can under certain
conditions account for up to 60% of the total
protein mass in the cyanobacterial cell, this is
+indeed a significant reservoir.
The ability of some cyanobacterial species to
adapt chromatically to their environment by
changing the composition of their PBS (Bennett and
Bogorad 1973; Bryant and Cohen-Bazire 1981;
Grossman et al. 1993; Kehoe and Grossman 1994)
has been shown to occur due to changes in the
expression of PBP proteins (PC and PE), and does
not occur due to PBS disassembly or by degradation
of PBS subunits (Bennett and Bogorad 1973).
Energy transfer within the PBS
Energy transfer within PBS subunits and subcomplexes have been measured by a variety of
spectroscopic techniques (Sauer and Scheers 1988).
Species-specific
differences
not-withstanding,
energy transfer rates are extremely fast (Knox
1999) and overall quantum yields are very high –
about 95% (Searle et al. 1978; Glazer 1989;
MacColl 1998). This has been found to be the case
for all photosynthetic antenna complexes, and has
been proposed to be due to optimization of both
relative orientations and distances between
cofactors. However, the PBS is distinct in having
rather large distances between cofactors, unlike the
chlorophyll-based antenna systems that have been
described at molecular resolution such as LH1 and
LH2 from purple bacteria (Koepke et al. 1996;
Prince et al. 1997; Roszak et al. 2003), LHC II
from plants (Kuhlbrandt and Wang 1991; Liu et al.
2004) and the internal and external antennas of
Photosystems I and II in both cyanobacteria and
plants (Jordan et al. 2001; Zouni et al. 2001; BenShem et al. 2003; Kamiya and Shen 2003). In the
chlorophyll-based antennas, distances between
adjacent cofactors are typically close enough to
envision closely interacting absorption rings or
aggregates. In the bacterial LH2 complex, the two
rings of bacteriochlorophylls have spacing of 9 Å
18
and 20 Å (center to center) respectively, and the
distance between rings is also 20 Å. The addition
of carotenoid molecules makes this antenna quite
cofactor dense. In the antenna beds of both PS I
and PS II, there are many chlorophyll molecules,
positioned with very complex arrangements, which
can roughly be separated into two groups, on either
face of the membrane – however typical distance
are on the order of 10 Å. The densest of all chlorophyll based systems is the chlorosome found in
green sulfur (Chlorobiaceae) and green filamentous
bacteria (Chloroflexaceae), in which an entire
organelle encloses thousands of pigment molecules
stacked in close proximity, without intervening
protein subunits (Frigaard et al. 2001; Vassilieva
et al. 2002; Montano et al. 2003). While very different in its light-harvesting strategy, some similarities exist between the chlorosome and PBS. The
bulk pigment of the chlorosome is bacterial chlorophyll c, and can be found in 10–30 long aggregated rods, without intervening protein. This large
array efficiently transfers the absorbed energy to an
intermediate bacterial chlorophyll a/protein complex called the base-plate, which transmits the
energy into the reaction center (Blankenship et al.
1995; Olson 1998; Montano et al. 2003). No
homology exists between the chlorosome and PBS
on either the level of pigment aggregation or protein sequence, and thus it can be assumed that these
two complexes evolved separately, to utilize the
available volume outside the thylakoid membrane
for light-harvesting.
Within each minimal PC unit, the distances
between the a84 and both the b84 and b155 bilins
is 50 Å, while the b84 and b155 bilins are separated by 40 Å. Association of the PC monomers
into trimers and hexamers allows a slightly shorter
distance to occur between select bilins, but none
are closer than 20 Å (Schirmer et al. 1986; Nield
et al. 2003). In assembled PE hexamers, which
contain five bilins per monomer, the density is
slightly higher, with the nearest approach between
two bilins on the order of 20Å (Ficner et al. 1992).
Thus it can be assumed that efficient energy
transfer does not particularly require the dense
packing of cofactors – rather that dense packing
maximizes the potential for light absorption with
the lowest expenditure on protein synthesis. Since
this is not the case for the PBS, it could be
assumed that the secondary role of the PBS,
serving as a reservoir of nutrients for starved cells,
must be of high enough importance to have preserved this form of antenna system.
The central mechanism for energy transfer proposed by Forster (1948), requires the existence of
weak coupling between nearby chromophores
electronic energy levels. Due to the relatively large
distances between cofactors in the PBS, this coupling is indeed weak, and structure-based theoretical energy transfer rate calculations utilizing the
Förster mechanism match well with the experimentally obtained values. Debreczeny and
coworkers measured energy transfer rates in isolated PC and obtained values between 50 and 500 ps
for different pathways within the (ab) monomer
(Debreczeny et al. 1993). Formation of (ab)3
trimers allowed for much faster energy transfer in
the 0.5–1 ps range (Beck and Sauer 1992), showing
the importance of the level of complex formation
when dealing with the functional characteristics of
the PBS. Ultrafast two-color pump-probe
spectroscopic measurements on different organizational states of APC have revealed energy-transfer
at less than 100 fs, which have been suggested to
occur due the formation of dimer-exciton states
(Edington et al. 1995, 1996). These states can only
exist between the PCBs on adjacent monomers, and
the very fast decay component is probably outside
the time-frame accessible to Förster resonance
energy transfer. It is still unclear what the effect of
further aggregation (to full cylinders, cores or PBSs)
will have on the energy transfer rates, especially due
to the presence of linker proteins. However use of
isolated subunit also allows for extremely high
quality descriptions of the cofactor/protein/solvent
environments during absorption and energy transfer (Homoelle and Beck 1997; Homoelle et al.
1998). Detailed discussions on energy transfer
within isolated PBP can be found in a number of
excellent reviews (Glazer 1989; Huber 1989; MacColl 1998; Knox 1999).
Determination of the three dimensional structure
of the PBS and isolated components
Early structural studies by electron microscopy
From the 1960s on, the power of the electron
microscope was used to first identify, and then
analyze the ultrastructural characteristics of PBS
from a variety of cyanobacteria and red-algae. In
19
these studies, it became clear that the large granules
identified by Gantt as adhering to the photosynthetic membranes (Gantt and Conti 1966a, b), were
made up of recurring structural motifs, namely a
multi-unit core coupled to a varying number of
extended rods. Samples were typically fixed with
glutaraldehyde and then negatively stained. Magnification of the specimens using transmission
electron microscopy (TEM) was typically on the
order of 250,000. In this fashion a number of PBS
types were analyzed from both cyanobacteria and
red alga. The major structural aspects of the core
and rod organization were thus visualized. The
structure of the core subcomplex was found to be
variable according to organism, and contained 2–5
cylinders. Each cylinder had a diameter of greater
than 100 Å and had a typical thickness of about
120 Å. High-resolution TEM indicated that the
thickness of each cylinder was due to multiple rings
of a thickness of about 30 Å. These thinner rings
were recognized to be comprised of (ab)3 trimers of
APC. The arrangement of the cylinders within the
core is hexagonal close packed, and thus the total
height of the core is less than the sum of the
dimensions of each individual disk (see below). The
dimensions of the components which make up
the rods were comparable to those of the core, but
the basic unit was a disk of 60 Å in width, indicating that in the rods the basic structural unit is an
(ab)6 hexamer. However, due to inconsistencies in
sample preparation, and perhaps due to damage to
the PBS during sample preparation, each uniquely
resolved PBS showed variation in the angle of
radiation of the rods out from the core. The results
of these studies resulted in a number of models
showing proposals for the structure of the entire
PBS, with variation in the arrangements and associations between rods, and between rods and cores.
One of the most popular models, often used in the
literature, is of rods radiating out at approximately
even spacing surrounding the core. This form of
PBS is typically termed the hemi-discoidal model
(Anderson and Toole 1998; MacColl 1998).
Structural determination of PBP by X-ray
crystallography
Crystallization of PBPs
To date, 19 crystal structures of different components of PBS from cyanobacteria and red algae
have been determined (Table 1), including eleven
PC structures (Fisher et al. 1980; Schirmer et al.
1985, 1986, 1987; Duerring et al. 1991; Stec et al.
1999; Adir et al. 2001; Jiang et al. 2001; Padyana
et al. 2001; Wang et al. 2001; Adir et al. 2002;
Adir and Lerner 2003; Adir et al. 2003; Nield et al.
2003), four PE structures (Ficner et al. 1992;
Chang et al. 1996; Jiang et al. 1999; Ritter et al.
1999), 3 APC structures (Brejc et al. 1995; Liu
et al. 1998, 1999; Reuter et al. 1999) and one
structure each of PEC (Duerring et al. 1990) and a
cryptophyte PE found in the thylakoid lumen that
does not form PBS (Wilk et al. 1999) and will not
be discussed in this review. While it is not always
specifically mentioned in the related publications,
in most cases the isolated protein that was crystallized was in its (ab)3 trimeric form [or (ab)6c
form in the case of PE] . This form has a high
degree of hydrophobic interactions in the (ab)
monomer interface, which apparently adds a high
degree of stability in low ionic-strength solutions.
This detail is important, since it indicates that one
can assume with a high degree of certainty that the
interactions on the level of the (ab) monomers and
(ab)3 trimers visualized crystallographically are
indeed as close as possible to those found in vivo.
While the level of sequence homology is quite
high among the different PBPs of different species
(Apt et al. 1995), the method of obtaining well-diffracting crystals of PBPs from different sources was
not consistent with respect to the conditions of
crystallization growth. This includes the type of
precipitating reagents (salts, PEG and isoproponol),
the ionic strength, the pH of the crystallization
liquor (between 5.0 and 8.5) etc. When approximate
conditions for crystallization are identified, the
proteins crystallize readily, typically within hours to
days. More than one group, including our own, has
found PBPs crystals in solutions containing high
concentrations of contaminating protein, showing
the high propensity for self-assembly by PBPs. The
crystallization process must indeed be controlled to
avoid overly rapid crystallization. The structure of
PC from T. elongatus (Te-PC) was recently determined at high-resolution (1.45 Å), by utilization of
the back-soaking method which allows for
improved crystal growth (Nield et al. 2003; Saridakis and Chayen 2003).
The first PBP structures determined were PC
from M. laminosus (Ml-PC) (Schirmer et al. 1985,
1987) and Synechochoccus sp. PCC7002 (S7-PC,
previously called A. quadruplicatum) (Schirmer
20
Table 1. Structures of PBSs determined by X-ray crystallography
Species (abbreviation)
PBP
type
PDB
code
Resolution
(Å)
Asymmetric
unit
Reference
M. laminosus (Ml-PC)
Synechococcus PCC7002 (S7-PC)
F. diplosiphon (Fd-PC)
C. caldarum (Cc-PC)
S. platensis (Sp-PC)
PC
PC
PC
PC
PC
PC
PC620
PC620
PC612
PC
PC
PEC
APC
APC
APC
PE
PE
PE
PE
PE
–
–
1CPC
1PHN
1GHO
1HA7
1I7Y
1KTP
1ON7
1JBO
1F99
–
1ALL
1KN1
1B33
–
1LIA
1B8D
1EYK
1QGW
2.1
2.5
1.66
1.65
2.2
2.2
2.5
1.60
2.7
1.45
2.4
2.7
2.3
2.2
2.2
2.2
2.8
1.9
2.25
1.63
(ab)
(ab)3
2·(ab)
2·(ab)
2·(ab)6
2·(ab)6
(ab)
(ab)
(ab)
(ab)
(ab)3
(ab)
(ab)
(ab)
2·(ab)3
2·(ab)
(ab)3
2·(ab)
2·(ab)
(a1a2bb)
Schirmer et al. (1985,1987)
Schirmer et al. (1986,1987)
Duerring et al. (1991)
Stec et al. (1999)
Wang et al. (2001)
Padyana et al. (2001)
Adir et al. (2001)
Adir et al. (2002)
Adir and Lerner et al. (2003)
Nield et al. (2003)
Jiang et al. (2001)
Duerring et al. (1990)
Brejc et al. (1995)
Liu et al. (1999)
Reuter et al. (1999)
Ficner et al. (1992)
Chang et al. (1996)
Ritter et al. (1999)
Contreras-Martel et al. (2001)
Wilk. et al. (1999)
T. vulcanus (Tv-PC)
T. elongates (Te-PC)
P. urceolata (Pu-PC)
M. laminosus (Ml-PEC)
S. platensis (Sp-APC)
P. yezoensis (Py-APC)
M. laminosus (Ml-APC)
P. sordidum (Ps-PE)
P. urceolata (Pu-PE)
G. monilis (Gm-PE)
G. chilensis (Gc-PE)
Rhodomonas CS24
et al. 1987 ). The Ml-PC structure was determined
by the multiple isomorphous replacement method
(Davis et al. 2003), and has been the source of
phasing of many of the other crystal structures
determined by molecular replacement (either
directly or indirectly, in many cases through use of
the molecular structure of PC from F. diplosiphon
(Fd-PC) (Duerring et al. 1991)). In addition, the
structure of PEC from M. laminosus (Ml-PEC) was
also determined (Duerring et al. 1990). All three of
these structures have unfortunately not been
deposited in the PDB; however they serve as
examples of the great efforts that research groups
made to obtain intermediate resolution structures
(2.5–2.7 Å) in the 1980s. Structure determination of
the Ml-PC at 2.1 Å (Schirmer et al. 1987) required
the use of synchrotron radiation, and due to the lack
of the cryo-crystallographic techniques developed
in the mid 1990s, multiple crystals were required for
complete data collection. The expansive level of the
description of the roles of a majority of the residues
in these early papers is rarely found in modern
crystallographic publications, mainly due to the
existence of the PDB found at http://www.rcsb.org/
pdb (which allows the downloading of the atomic
coordinates) and to widely available computer-
based analysis and visualization tools that enables
the sharing of the structural data with others of the
scientific community.
Along with the detailed three-dimensional
description of the PBS structure, the primary
objective of these studies was to describe the possible pathways of energy transfer between adjacent
cofactors. Since in many cases the crystallized
protein was either in the (ab)3 or (ab)6 forms, the
elucidation of more extensive energy transfer
pathways requires the assumption that the further
assembly of the trimeric rings during the crystallization process resembles the organization of the
PBS in vivo. This assumption has been a mainstay
of all further structural studies, even though all
structures except one lack the linker PBS components. This point will be expanded below.
General structural characteristics of the PBPs
The PBS is built up step-wise from basic building
blocks in an orderly and controlled process of self
assembly (Glazer et al. 1983; Glazer 1989;
Anderson and Toole 1998; MacColl 1998). Very
early in the study of PBPs it appeared that these
proteins were of high molecular weight (Svedberg
and Lewis 1928), and it was not until later that it
21
was recognized that some substructures of the PBS
are stable enough to withstand isolation, thus
allowing the isolation of complex forms of PBPs.
In the early work by Svedberg, PC and PE were
thought to have molecular weights of 106 and
208 kDa, respectively, which we can relate today
as being an (ab)3 form of PC and a (ab)6 form of
PE. Each of the basic a and b monomers contain
eight a helices (Figure 1), six of which (helices A
through H) fold into a globin-like structure with
the covalently linked bilin cofactors in a position
analogous to that of the porphorin moiety in
globin proteins. The two additional helices (X and
Y) form the association domain (with a buried
surface area of about 1000 Å2) between the two
subunits in the formation of the (ab) monomer.
On the level of the tertiary structures, most of the
differences between the different PBPs lie within
loops bridging between the helical regions, and
contain most of the cofactor binding niches. As the
number of cofactors increases (from APC to PC
and PE), the polypeptides contain extra loops
needed to accommodate the additional cofactors.
The geometry of the polypeptide backbone is very
regular, except for the almost invariant threonine
b77 residue, which interacts with the a84 cofactor
on the adjacent (ab) monomer in the trimer
structure, and whose constrained backbone is in a
disallowed region of the Ramachandran plot
(Schirmer et al. 1987).
Figure 1. Molecular structure of the phycocyanin (ab) monomer. Atomic coordinates of the Tv-PC620 1.6 Å structure (1KTP)
are depicted in stick representation. The overall fold is represented in ribbons: yellow, the a subunit; blue, the b subunit. The
three PCB cofactors are in CPK representation and colored
according to atom type and are labeled according to the consensus PC sequence. Solvent molecules are shown as red crosses.
Figures 1 and 2 were prepared using InsightII (Accelrys).
The crystal packing of the first two PC structures was different, with the Ml-PC crystal
(Schirmer et al. 1985, 1987) comprised of (ab)3
trimeric rings with only limited crystal contacts,
and the S7-PC crystal showing the formation of
(ab)6 hexameric disks [made up of two trimeric
rings (Schirmer et al. 1986; 1987). The trimeric
ring is formed by mostly hydrophobic contacts
with a buried surface area of about 500 Å2. The
S7-PC structure had a greater degree of order on
the level of both the backbone and side chain
atoms (as indicated by quality of the electron
density and the B-factors), and this was suggested
to be a result of the addition of trimer–trimer
interactions. The question of the source of the
dissimilar organization of PC in the different
crystals was not addressed by these authors.
Cofactors
In addition to the polypeptide fold, the positions
and environments of the cofactors were analyzed
in great detail. The major structural features
which lead to the extended cofactor structures
were found to be due to specific interactions with
mostly conserved charged and polar residues.
Superposition of the three cofactors in the PC
structures showed a high degree of similarity,
however some differences were apparent, especially in ring IV (the pyrrole furthest from the
thioether linkage). This is due to the level of
direct protein–cofactor interaction (rings I and II
are sequestered more deeply within the protein),
as well as by the level of assembly. A detailed
description of the cofactors can be found in a
number of reviews (Glazer 1985, 1989; Huber
1989; Anderson and Toole 1998). Each cofactor
contains two propionic acids jutting out from
rings II and III. These potentially negatively
charged groups are usually found in electrostatic
contact with positively charges residues such as
arginine. Thus, the extension of the cofactor must
be a closely controlled balance between the natural tendency of the cofactor to close in to a ringlike or helical structure, and the linearizing power
of the protein. Slight changes within this balance
are utilized to tune each cofactor to the appropriate absorption wavelength, thus maximizing
the spectral cross-section, and allowing for efficient energy transfer. The absorption of the different cofactors in different environments has
been measured in vitro (Debreczeny et al. 1993;
22
Anderson and Toole 1998; Pizarro and Sauer
2001).
Higher levels of assembly
Of the PBP crystal structures determined thus to
date, 7 PC structures, all three PE structures, and
two of the APC structures show the presence of
hexameric discs in crystal. Only two PC structures
were the result of crystallization of trimers that did
not associate further into hexamers: Ml-PC and a
unique PC subpopulation isolated from T. vulcanus, Tv612-PC. The exceptional Ml-APC structure (Reuter et al. 1999), which is the only crystal
structure containing a linker protein, is trimeric
with unique perpendicular interactions between
the two trimers in the asymmetric unit.
Unlike the trimeric ring, the interactions
between two trimers that form a hexameric disk
were primarily attributed to charged and polar
interactions (Schirmer et al. 1986). The total buried surface is about 3000 Å2. In the S7-PC structure (Schirmer et al. 1986) as well as many others,
the two trimer rings were positioned face to face,
with little rotational displacement. Thus, each (ab)
monomer is fully contacting an upside-down (ab)
monomer and contacts are thus between a and b
subunits from different rings. While the hexamer
interface for each monomer couple is on the same
order as that of the buried surface in the (ab)
monomer, and both interfaces are charged/polar
by nature, the (ab) monomer is very stable (Anderson and Toole 1998), while the hexamer
typically is easily disassembled in solution. This
may indicate that just a limited number of saltbridges and/or hydrogen bonds may be the source
of this added stability in the monomers. Indeed,
the addition of only a few intermediate strength
polar interactions have been proposed to be the
source of added stability in PBPs isolated from
thermophilic cyanobacteria (Adir et al. 2001).
The hexameric discs assemble further in crystal
into rods. It is certainly logical to assume that
there is some connection between the rods seen in
electron micrographs, and those that appear during crystallization. However in all of the PC and
PE structures to date, no linker proteins have been
obtained. In fact, it has been noted that the presence of linkers can inhibit crystal growth (Stec
et al. 1999). The interactions between adjacent
hexamers within the rod structures are rather
limited (with a buried surface area of less than
500 Å2), and are mostly polar. This indeed agrees
with the in vitro instability of the rods. Interestingly, under some conditions, isolated PBPs can
form extremely long rod like assemblies (Yu et al.
1981). Linker proteins are probably involved in the
termination of rod elongation. In Synechococcus
sp. PCC7002, a 9 kDa linker unit was shown to
have an effect on the homogeneity of rod lengths
(de Lorimier et al. 1990a), however additional
components are probably required for both
structural and functional reasons (de Lorimier
et al. 1990b; Gomez-Lojero et al. 2003).
In addition to the rod formation, hexamers also
interact laterally in crystal. This is of course a prerequisite of crystal lattice formation. In all hexameric forming PBPs analyzed crystallographically
so far, the hexamers interact such that the rods all
run directly parallel to each other (Figure 2). This
too has a similarity to the apparent association of
rods seen in electron micrographs (Glazer 1989). In
these pictures, rods typically form doublets running
parallel to each other, with up to three doublets
surrounding each PBS core. However in some case
Figure 2. Levels of PBS association as seen in the crystallographic unit cell of Tv-PC620. The asymmetric unit in this
crystal form is an (ab) monomer (Figure 1). The a and b
subunits are shown in Ca traces for simplicity in yellow and
blue, respectively. Application of crystallographic symmetry
reconstructs a number of physiologically important aggregation
states: the (ab)3 trimer (the top of each ring), the (ab)6 hexamer
disk (the bottom trimer is directly below the top trimer and is
thus superimposed), the ((ab)6)n rod (not shown, and results in
additional hexamers layered one on top of the other) and the
(ab)6 hexamer–(ab)6 hexamer interaction between adjacent
rods. Notice that the three PCB cofactors are separated
spatially: a84 PCBs (red) are buried in the (ab) interface of the
trimer, b84 PCBs (green) are all located within the trimer and
hexamer disk and probably associate with linker proteins and
b155 PCBs (pink) are all located on the outside circumference
of the disks and may help in energy transfer between disks and
between rods.
23
the rods appear to diverge, leading to models
showing the rods surrounding the core in a nonparallel fashion (Glazer 1989; Huber 1989; MacColl
1998; Adir et al. 2002; Adir and Lerner 2003).
Indeed, the reasons behind the rod interactions may
be due to the method of sample preparation prior to
microscopy (such as the fixation and drying steps).
If this is the case, then the situation found in crystal
may not represent the true rod association mode.
This level of aggregation shows a significant degree
of dissimilarity in the crystallized PBPs from
different organisms, which could be an additional
indication that this is not the same as the rod
interactions in vivo. This subject is addressed further
below.
Additional structural aspects
Structural features of APC subunits. While only
about 25–30% sequence identity exists between
APC, PC and PE from the same species (Apt et al.
1995), the three-dimensional structures are highly
similar (pair wise rmsd of 1.3 A between equivalent a-carbons). There are only two PCB cofactors
on each APC monomer, and while the molecular
structure and environment of the b84 bilin is
similar to that found in PC, the environment of the
a84 cofactor is modified, leading to the large redshift in the total (ab)3 APC absorption spectra
(Brejc et al. 1995).
One of the major differences in the APC subunit is the formation in crystal of a more loosely
associated hexamer, due to slight sequence variations in the APC versus PC or PE (Brejc et al.
1995; Liu et al. 1999). It is not clear what the
source of this looseness might be; however, the
somewhat lower rigidity could facilitate the perpendicular association between the APC core and
the rods. Evidence that the native form of APC
(within the context of the complete PBS) is a trimer has been published (Bryant et al. 1979), and
so whether a ‘hexameric’ nature is actually necessary for APC function is unclear. Concentrated
APC can however form long rod-like crystalline
needles (Bryant et al. 1976) showing that APC too
has the ability to form long stacks. In the Ml-APC
structure (Reuter et al. 1999), APC is found as
trimers, with the two trimers of the asymmetric
unit associated in an almost perpendicular
arrangement. This mode of association does not
appear to be similar to rod–core association as
seen in EM studies, and so is probably a pure
crystallization effect, perhaps due to the presence
of linker (see below).
The most prominent APC trimers in the core
are of the type (aAPCbAPC)3. However additional
APC trimer rings, with red shifted absorption
spectra exist, encoded by the ApcD, ApcE and
ApcF genes (Ducret et al. 1998). The ApcE gene
product is known as the LCM, and is considered
to be the core-membrane linker component
(Capuano et al. 1991). This component has a
molecular weight of greater than 75 kDa,
depending on the species, and it may have an
additional role in core assembly (Isono and Katoh
1987). No crystal structures of these minor, but
extremely important subunits have been determined to date.
Structural attributes of PE subunits. PE exists as an
additional PBP only in some species. The basic PE
structure is a (ab)6 hexamer in cyanobacteria, and
a (ab)6 c, with the chromophore bearing 30 kDa c
subunit inserted within the hexameric disk in some
red algae. The first PE structure was that of B-PE
(Ficner et al. 1992) in which the (ab) monomer
contains five PEBs, with 2 PEBs and 2 PUBs
attached to the c subunit. PE contains doubly
linked cofactors, which has a noticeable affect on
its conformation and thus on its absorption. The
c subunit could not be visualized in the four PE
crystal structures. In the Gm-PE structure, three
residues of the c subunit were built into electron
density within the (ab)6 disk (Ritter et al. 1999),
thus providing evidence for the general position of
this subunit. The identified residues are located in
the vicinity of the bilins. The reason for the ‘loss’
of the c subunit in the electron density is probably
due to the threefold crystallographic averaging
applied during structure determination (Ficner
et al. 1992; Ritter et al. 1999), although heterogeneity in the position of this subunit cannot be
discounted.
Linker proteins. Only a single crystal structure has
an identifiable linker protein – the Ml-APC
structure (Reuter et al. 1999). In this structure, in
which the APC trimers associate in a perpendicular fashion, the linker is seen completely within the
trimer ring. The linker contains a three strand
b-sheet with two short a-helices, and makes contact with only 2 of the three APC monomers. Most
of the contact surface is with residues and rings II
24
and IV of the bilin on the APC b-subunits, with
concomitant structural changes to both. The
presence of the linker has a significant effect on
the entire trimer, making it somewhat more flat.
The asymmetric unit of this APC-linker form
included two trimers, and the position of the linker
with in each trimer was not identical, suggesting
that the linker binding might be somewhat flexible.
This flexibility may be compounded by the additional heterogeneity in the APC cylinders due to
the presence of the minor forms of APC as
described above.
Solvent molecules embedded within the PBP
structure. In the process of crystallographic
refinements, solvent molecules contributing to the
diffraction power of the crystals are modeled
(typically as oxygen atoms of water molecules)
into the electron density map. The number of
water molecules added to the protein molecular
structure is also dependent on additional crystallographic parameters such as the temperature of
data collection (which affects the overall rigidity of
the molecule) and the resolution. The crystal
structures of the different PBPs are quite compact
with few cavities. It is difficult to make straightforward comparisons between the solvent contents
of the final molecular structures of the various
PBPs due to the great variety in experimental
conditions. Of the various structures with resolutions below 2.2 Å, there are between 70 and 470
water molecules per (ab) monomer. Most of the
structural water surrounds the exterior of the
protein surface, which is largely polar, with many
potentially charged surface residues. However a
significant fraction of water molecules can be
found within the proteins folds, and associated
with the cofactors. At high resolution, these waters
can be found at especially important interaction
interfaces. In the 1.6 Å Tv-PC structure, water
molecules were found at the interaction interfaces
of both the monomer and hexamer, interacting
with both protein and the PCB (Adir et al. 2002).
One of the most critical positions is in between
ring D of the b155 PCB, and residues Aspa28 and
Asnb35, which together with the solvent molecules
form a strongly interacting unit. In most mesophilic strains the a28 residue is either a non-polar
phenylalanine or a non-charged asparagine. The
PC from this thermophilic organism was shown to
be thermostabile in its isolated form (Inoue et al.
2000), and the waters bound to the interface could
potentially strengthen the stability of association
at the elevated temperatures of native surroundings. Additional water molecules can be found in
the high resolution structures in hydrogen bonding
distance from both propionic acid groups of the
cofactor (Adir et al. 2002). The negatively charged
propionic acids associate with positively charged
residues (typically arginine), and taken together
might put stress on the PCB molecule leading to
increased curvature. The bound waters may help
balance these forces by partially negating the
repulsive effect the propionic acids have one on the
other, thus helping the PCB assume its proper
structure.
Methylation of asparagine residues. In 1986, Glazer
and co-workers identified a unique post-translational modification of the APC b subunit. They
identified an asparagine residue at position 72
(using the PC consensus sequence numbering)
which undergoes the addition of a methyl group to
the c-nitrogen of the side chain (Klotz et al. 1986).
The resulting modified residue, N-methyl asparagine (NMA) was later found to occur as a result of
the presence of a methylating enzyme that could
by biochemically isolated and genetically analyzed
(Swanson and Glazer 1990). The isolated activity
was able to identify and methylate the b72 residue
in vitro, indicating that methylation in vivo may
occur after trimer assembly. Methylated bAsn72
residues were identified in PC, PE and PEC as
well, and the NMA was identified crystallographically by reinterpretation of the electron density
maps of Ml-PC and S7-PC (Duerring et al. 1988)
and Ps-PE (Ficner et al. 1992). The position of the
methyl group on NMA is close to ring II and its
propionic acid group, and thus may modify the
polar environment of this cofactor enough to alter
its absorption maximum, and indeed methylation
induces a small red-shift in isolated PC. When
compared to PC isolated from methylase-lacking
mutant strains, methylated PC is also more efficient in energy transfer from PC to APC. No
problems in the assembly of the PBS in the
methylase-lacking strains could be seen, and thus
methylation is not required for proper assembly.
However most of these experiments, as well as the
crystallographic studies were performed in PBPs
lacking linker proteins. Thus, the combination of
NMA with linker interactions may have alterna-
25
tive or additional consequences. Consistent with
this idea, the linker visualized with APC (Reuter
et al. 1999) does interact with the NMA residue.
The existence of asparagines at this position is
highly conserved, but there are species in which
not all of the PBPs contain this residue (Apt et al.
1995). This indicates that for certain cases, perhaps
due to changes in the linker protein, methylation
is not required, or indeed is deleterious. It is certainly possible to envision that isolation of the b84
bilin from the solvent could be achieved by a
combination of natural amino-acid substitutions
coupled with the linker protein. Indeed, one could
envision that the evolutionary development of a
methylation system might only be required, if it
serves to control some aspect of function, thus
suggesting that under other conditions nonmethylated b72 might be required.
During the process of isolation of APC from T.
vulcanus, we recently isolated a PC fraction that
had an absorption maximum at 612 nm, as compared to the typical Tv-PC which absorbs at
620 nm (Adir and Lerner 2003). The Tv-PC612 was
isolated in its trimeric form, however, it crystallized in a different space group than Tv-PC620, and
did not form hexamers in crystal. Careful analysis
of the region of b72 showed no indication of the
extra methyl carbon in Fo–Fc simulated annealing
omit electron density maps, explaining the blueshifted spectra.
What are the possible roles of this form of PC?
It is certainly possible that this fraction could
merely be a population of non-methylated PC,
prior to assembly into newly formed PBS. In the
very careful analysis of PBPs performed in the past
of a variety of cyanobacterial species, no previous
mention of such a PC was made (Bryant et al.
1976; Yu et al. 1981; Lundell and Glazer 1983).
The PBS is however a dynamic complex, which
can change composition, association and position
according to the needs of the organism. Thus it is
also possible that the PC612 is a minor constituent
of a minor type of PBS, which would be difficult to
identify in the presence of the major PBS forms. A
blue-shifted fraction of PC was reported to have
been isolated from the thermophilic cyanobacteria
Synechococcus lividus (Edwards et al. 1996). This
PC was shown to preserve its altered absorption
characteristics and have greater thermal stability
at different aggregation levels. The fluorescence
emission of this PC form was red shifted compared
to typical PC, however the molecular explanation
for the blue-shift was not identified, nor was the
presence or absence of NMA assessed. This additional observation indicates that additional PC
forms may exist and that they may play specific
roles in PBS function.
In our lab, the PC612 form was never isolated
in the presence of bulk PC620, rather it only
co-purified with APC, which was removed from the
thylakoid membrane. We have thus suggested that
this form of PC might serve as a functional bridge
between some of the bulk PC rods and the APC in
the tricylindrical core (Figure 3) and that in the
absence of NMA, and in the presence of linker
proteins, there might be the possibility of modifying
the absorption of the PC612 further to the red, so
that it would be in between PC620 and APC (which
absorbs at 652 nm). It has been difficult to obtain a
satisfactory quantitative ratio of PC612, to PC620,
and in any case it is important to identify its position
with respect to the intact PBS. Preliminary measurements of an isolated PBS subfraction containing core and rod components (with a mass of greater
than 1 MDa) showed by fluorescence measurements the presence of an entity absorbing at about
Figure 3. Model of the PBS consistent with Tv-PC620 and
Tv-PC612 crystal structures. The three blue rings represent the
APC core. The four yellow rectangles represent four PC rods
running parallel to both the membrane (pink – 40 Å in width) and
themselves. The two orange rectangles represent the additional
two PC rods, perpendicular to the membrane. The two green
boxes are the approximate dimensions of two PSII monomers,
which bind a single PSB. The two red crescents represent the
putative positions of the non-methylated Tv-PC612, which may
serve as a linker between PC rods and the core. There may be
additional linker PCs connecting the other rods.
26
632 nm (unpublished data). We are now in the
process of refining the isolation of this partial PBS
particle in our hope to obtain a careful quantitative
determination of its components, as well as to
determine its crystal structure.
Models of the PBS
The ultimate goal of all of the structural studies
performed on PBS components has been to obtain
a physical picture of energy absorption and transfer through the complex to the reaction center. In
the absence of a high resolution structure of the
PBS, this visualization of the PBS has been
attempted by structure-assisted modeling, with an
attempt to arrive at a model which will satisfy the
information obtained by the low-resolution EM
studies. Indeed, almost every research group dealing with the determination of crystal structures of
PBS components has suggested models of this sort,
based on the aggregation state of the PBPs in
crystal. The similarity between the rods that form
during crystallization of most PCs, APCs and PEs,
certainly suggest that the some of same interactions
come into play during rod formation in vivo, but
without the contribution of the linker proteins.
What parts of the different models are consistent?
In all of the models proposed to date, two structural elements are mostly agreed upon: the PBPs
(ab)3 trimer and the APC core (Figure 3). Since the
trimer is often the organizational unit isolated from
the PBS and crystallized, we can say with a high
degree of confidence that the dimensions of the
trimers in vivo are indeed 110 Å in diameter and
30 Å in thickness. The absorption spectra of the
PBS in vivo and in vitro are similar to that of isolated trimers indicating that the distances
between the cofactors are very similar to those
crystallographically determined. The APC cores
consist of 2–5 cylinders (depending on the species)
in 1–2 hexagonal close-packed layers. The thickness of the core in most models is 4 trimeric rings,
equivalent in width to two loosely packed hexamer
disks, thus creating a 120 Å cylinder. APC cores
of 3 or 5 cylinders are organized in two rows, with a
height of about 190 Å, (30 Å less than the sum of
the diameters of two cylinders). Since the rods also
have diameters of 110 Å, this height difference may
affect the positioning of the rods surrounding the
cores. While the rod associated with the bottom
APC row fit exactly (since they have the same
diameter), the second rod cannot fit in exactly the
same fashion, since the second APC layer is packed
into the ‘saddle’ formed by the first row (Figure 3).
Models of the next level of arrangement, the
(ab)6 hexamers are generally consistent. Most of
the structures of PC show the presence of crystallization-induced hexamer formation by face to
face association of two trimers, and PE exists both
in solution and in crystal as hexamers of similar
nature. In these hexamers the thickness is slightly
less than that of two trimers – about 55 Å. Two
PCs have been crystallized in forms that do not
contain hexamers (Ml-PC and Tv-PC612). In the
case of Tv-PC612, the connecting region of the B to
E helices of the a subunit is less ordered than in
hexamer forming PCs. It is difficult to determine
what is the cause/effect of the lack of order/lack of
hexamer formation, and additional studies on
larger complexes are necessary (see below).
In the three existing APC structures there are
three variations on trimer packing: in the Sp-APC,
two trimers associate ‘back-to-back’ through
b-subunit interactions; in the Py-APC the association is ‘face-to-face’ through mostly a-subunit
interactions. In the Ml-APC crystals, which contain a linker protein, two trimers associate in an
almost perpendicular fashion, and hexamers are
not present. From these studies it is difficult to
ascertain whether APC forms ‘real’ hexamers (as
do PC and PE) or are loosely associated trimers.
Rod formation
In most PC and PE crystals, the hexamers pack
one on top of the other without rotational variance (Figure 2). Thus each cofactor is aligned
directly above or below the same type of cofactor
in adjacent hexamers. In the Cc-PC structure,
there is both a rotation (30) and translation of
the one hexamer with respect to the adjacent
hexamer. This allows a slightly (2.5 Å) tighter
packing between hexamers. The Cc-PC hexamer
pairs appear to fit more snuggly, and the
interaction faces may align better with respect to
the electrostatic surface potential. This arrangement also opens up an alternative energy transfer
27
pathway between b155 cofactors on adjacent
hexamers, however this is the only structure with
this arrangement. In the Pu-PC structure (Jiang
et al. 2001), hexamers do not form rods at all,
rather they associate somewhat like the APC in the
Ml-APC structure (Reuter et al. 1999). These last
structures not withstanding, there is general
agreement between the crystal structures and EM
studies that the rods should be at the least close in
structure to those visualized crystallographically.
The lack of the linker proteins within the rod
structures is a source of uncertainty in our
understanding of this level of association.
Rod association
In most PC and PE crystals, adjacent rods associate
in a parallel fashion (Figure 2). Each rod is typically surrounded by six adjacent rods. This
arrangement is obviously not equivalent to the rod
associations seen in electron micrographs. In most
of these micrographs, six rods are seen emanating
from the core, and cases of additional rods have
been identified (Glauser et al. 1992a). Usually,
these six rods are organized into three rod-pairs.
Most pairs indeed appear to be aligned in a parallel
fashion, thus indicating that there may be some
relationship between the crystals and the PBS.
However what limits the rods from forming additional interactions is not known. If, as it is presumed, that the linker proteins are all sequestered
within the rods, they cannot be the reason for this
state. In the Tv-PC and other crystal structures, rod
association does not include direct interactions
between b155 PCB on the circumference of the
hexamer disk (Adir et al. 2002), while in the Sp-PC
they do (Padyana et al. 2001). If such association
between some, but not all b155 PCBs occurred, one
might expect the appearance of an additional band
in the absorption spectra of undissociated PBS,
however this band has not been reported.
Assembly of rods onto the core
No crystal structure contains experimental evidence that can be associated directly with the
mechanism of connection of the rods to the core.
Early models showed the possibility of arranging
6–8 rods around a hemidiscoidal core. On the basis
of the early EM studies, and the rod associations
in some crystals, it seems possible that at least
some of the rods are assembled in a parallel fashion. The Pu-PC structure (Jiang et al. 2001) led
these authors to propose that four rods form two
pairs, and the remaining two rods jut out at angles
almost perpendicular to each other and at about
45 to the rod pairs. This proposal is a modification of an earlier proposal by MacColl (MacColl
1998) which has single rods along the membrane
and two pairs at right angles. Anderson and Toole
depicted a 6-rod radial view of the PBS in their
review on PBS assembly events (Anderson and
Toole 1998). If the full radial model is correct, it
would indicate that the rod–rod association pattern seen in almost all PBP crystal structures is
only due to the crystallization process and crystal
packing constraints, and has no bearing on this
level of functional assembly. There are however
architectural problems with the radial model, due
to the association point between PC and APC. If a
two-level, 3 cylinder core is 120 Å in thickness
(four APC trimers), and the height of the core is
190 Å (Figure 3). The circumference around the
hemidiscoidal core is not sufficient to accommodate six rods at equal angles, without interpenetration between adjacent rods. Since the crystal
structures show the PC hexamers to be quite solid,
it is not apparent how interpenetration could
occur. This problem would also arise in the Pu-PC
model. A model showing 6 rods surrounding a
140 Å tricylindrical core was built based on EM
studies (Bryant et al. 1979). In order to avoid
interpenetration, the rods are staggered in a zig–
zag pattern. This would not necessarily negate the
associations seen in the crystal structures (since
there are still rod–rod contact points in the vicinity
of the rod–core attachment point), however this
model is dependent on the core being thicker than
120 Å.
An additional possibility is that the six rods
form three pairs, and that they are arranged as
pairs surrounding the core at right angles
(Figure 3). This model has been proposed in the
past by Glazer (1989), Huber (1989), and ourselves
(Adir and Lerner 2003). This arrangement is consistent with many of the crystal structures, however two problems arise. The joining of the rod
pairs to the core is different between the two pairs
along the membrane, as opposed to the pair perpendicular to the membrane. This might be
achieved by different linker proteins, but no solid
evidence exists. The second problem is that the top
28
APC cylinder is offset by 50 Å (on both sides)
from the APC layer below. If the rods are parallel
as in the crystal structures, and also in the same
register (offset by about a trimer ring diameter),
there will be a gap between the terminal PC and
the core of about a trimer. This could be fixed by
having an extra trimeric ring or hexameric disk on
the top rod of each pair, thus closing the gap. A
second possibility is that the ends of the rods may
have a special trimeric form of PC as isolated from
T. vulcanus (Adir and Lerner 2003). This trimer
might help assembly by (i) gripping on to the
outside circumference of the APC core, as result of
the flexibility of trimers hexamers; (ii), adapt the
rod termini to the 30 Å difference in height
between the rod pair (220 Å) and the core (190 Å)
and (iii) fix the gap between rod and core caused
by having only one APC cylinder on the top of the
core.
Of course, the total lack of structural data on
the positions of the different linker proteins within
the PBS, makes all of the models speculative, since
it is clear that rod–core attachment is linker protein dependent (Glauser et al. 1992b).
growth. However modern day crystallography has
been able to accomplish seemingly impossible
tasks with structural determinations of ribosomes
(Schluenzen et al. 2000; Yusupov et al. 2001),
photosynthetic reaction centers (Jordan et al.
2001; Zouni et al. 2001; Ferreira et al. 2004), etc.
The second method is to obtain high-resolution
EM images (7–10 Å) using modern cryo-techniques to obtain electron density maps, into which
the smaller isolated component structures at
molecular resolution are fitted. This strategy has
been very successful for very large complexes such
as the T4 viral baseplate (Kostyuchenko et al.
2003). The mode of attachment of the PBS to PS II
may be even more difficult to determine experimentally, however high quality models can be built
using the strategies mentioned above.
Acknowledgements
This work was supported by the Israel Science
Foundation founded by the Israel Academy of
Sciences and Humanities (438/02) and the Technion Fund for the Promotion of Research.
Future prospects
As described in this review, our molecular understanding of the light-harvesting process by PBPs is
very detailed up to the level of the (ab)6 hexamer.
Past this point, the rest of the levels of assembly
are educated conjecture. On the level of isolated
components, it is clear that there is a great need in
additional high-resolution structures of the linker
proteins themselves, and/or of PBPs in the presence of linkers. Beyond that, what the future
requires is the merging of the molecular details we
have already obtained, with supramolecular details
of the entire PBS. These details are absolutely
required to understand the directionality of energy
transfer, the high quantum yield, along with the
mechanism of assembly and disassembly. Obtaining this experimental information can be accomplished in two ways. The best and most direct
method will be the crystallization and structure
determination of the entire or partial PBS. This
will be of course no trivial task, due to its enormous size, and the special conditions in which the
PBS is stable (high concentration of phosphate
salts) which may not be conducive for crystal
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