Linker polypeptide

The use of sequence information to frame structural, functional, and evolutionary hypotheses represents a major challenge for the postgeno-mic era. Central to an understanding of the evolution of sequence families is the concept of the domain a structurally conserved, genetically mobile unit. When viewed at the three-dimensional level of protein structure, a domain is a compact arrangement of secondary structures connected by linker polypeptides. It usually folds independently and possesses a relatively hydrophobic core (Janin and Chothia, 1985). The importance of domains is that they cannot be divided into smaller units— they represent a fundamental building block that can be used to understand the evolution of proteins. 

Fig. 11. Phycobilisome of Masligocladus laminosus (Fischerella PCC 7603) represented in a schematic view. The structure is adapted from the PBS of Synechocystis 6701 [77] using structural and spectral data from M. laminosus biliproteins and linker polypeptides [22,31,82,97,105,135-137]. For the designation of the rod and core substructures, the nomenclature of Glazer [86] is applied.

Phycocyanin hexamers (a /3 )(, (A ax 620-635 nm), together with one of the linker polypeptides from the 30 kDa family, form the discs in the PBS rods attached to the APC core in the PBS [124,125], In several cyanobacteria the type of C-PC and the number of C-PC discs in the PBS rods is regulated by light intensity and/or light quality (chromatic adaptation, reviewed in Refs. 126 and 127). C-PC from M. laminosus (Fig. 12) has two PCB chromophores bound to the subunit, the second one being bound to Cys within an insertion of 10 amino acid residues at position 151-160 [113]. The PCB chromophores are singly bound to Cys and Cys - by ring A (Fig. 10). 

PS II [134], Primary structure information is available for linker polypeptides of Synechococcus 6301 [77] and M. laminosus [105]. The complete amino acid sequences of the and the have been established and large N-terminal... 

Several methods for the isolation of PBS have been established [81,104,105,138]. Principally they are based on the observation that PBS are only stable in solutions of high ionic strength, e.g. 0.75-0.9 M potassium phosphate. The PBS are detached from the membranes with 2% Triton X-100. The function of the intact PBS is tested by fluorescence emission spectra at 680 nm upon excitation at 550-650 nm. Most of the structural data describing the PBS originate from the hemidis-coidal type, as reviewed in Refs. 1,77 and 79-86. The complex architecture of the PBS rods and core is best described for the PBS of the cyanobacterium Synechococcus 6301 (a cyanobacterium which contains C-PC but neither PEC nor C-PE in the rods) with a bicylindrical core and for the PBS of the cyanobacterium Syne-chocystis 6701 (which contains C-PC and C-PE in the rods) with a tricylindrical core (reviewed in Refs. 1, 139 and 140). Each cylinder in the core is formed by four complexes of APC trimers with linker polypeptides cylinder A,... 

B, (a P/3 P)3 lOK C, (af a P/3 P)10K D, ( a /3 )218.5K-99K. The six rods contain four biliprotein hexamers, each hexamer associated with a different linker polypeptide of the 30 kDa family. A rod-core-PS II connecting 18 S subassembly particle was isolated from Synechococcus 6301 with the polypeptide composition... 

Further energy transfer within the rods may be performed by the /3f-chromophores of the PBS rods, modulated by the various 30 kDa-linker polypeptides, and directed along the central rod channel towards the APC complexes of the PBS core. Excitation energy transfer in the PBS was found to occur in up to five steps with different energy transfer rates [134] within the discs (hexamers) from (s)- to (f)-chromophores and from (f)- to (f)-chromophores, from disc to disc, from the rods to the APC complexes in the core, to the APB-Lcm complex and to Chi a in the PS II complex. The disc-to-disc excitation energy transfer (20 ps) was supposed to be the rate-limiting step. 

In a medium of high ionic strength, in vitro hexamers can build up into short rods. However, such aggregation appears to require the presence of a so-called linker polypeptide (symbol L ), which apparently serves as a kind of glue. Since it has been shown that phycobiliproteins purified to strict homogeneity cannot form these phycobilisome subassemblies. The presence ofthese linker polypeptides in... 

Fig. 8. Representations for the phycobilisome rod components and linker polypeptides (A) and mechanism of assembly (B). Figure adapted from Mdrschel and Rhiel (1987) Phycobilisomes and thylakoids The light-harvesting system of cyanobacteria and red algae. In JR Harris and RW Horne (eds) Membranous Structure, p 238. Acad Press drawn after results of Glazer (1982) Phycobilisomes structure and dynamics. Annu Rev Microbiol 36 183...

Fig. 9. Schematic representation of the phycobiiisome from Anabaena variabilis showing the location of the billproteins and linker polypeptides. See text for details. Adapted from Glazer (1984) Phycobilisomes A macmmolecular complex optimized for light energy transfer. Biochim Biophys Acta 768 42.

Phycobiiisome rods of Anabaena variabilis contain mostly phycocyanin (PC), a minor amount of phy-coerythrin (PE), and four linker polypeptides with molecular weights of 27, 29, 30.5 and 32.5 kDa. The phycocyanin and phycoerythrin content depends on culture conditions, such as light quality, temperature, etc. The order ofphycobiliprotein complexes along the rod is indicated for Anabaena variabilis in Fig. 9 the experimental confirmation of this sequence will be described below. 

Extensive fractionation of the dissociation products from A. variabilis phycobilisomes led to the isolation of three PC complexes, (a(3)3-LRc , (aP)3-LR - and (aP)3 LR , and one phycoerythrocyanin complex (aP)3 L °. With suchtrimer-linker complexes available, in vitro reconstitution experiments could be performed to help clarify the sequence of the components in the rods and how the linker polypeptides mediate rod assembly. As already shown in Pig. 8, a large portion of the linker polypeptide is buried inside the trimer, and only the small segment ofthe linker that is projecting outside the trimer is apparently able to link with the next trimer or hexamer. 

Limited tryptic degradation ofthe linker polypeptides Lr - and Lrc , still associated with their respective biliprotein complexes, resulted in and L, respectively, i.e., 4.5- and a 6-kDaportions, presumably belonging to the exposed domains, were removed by tryptic degradation. These results are shown in Fig. 10(A). 

As shown in Fig. 10 (B, 1), after the 4.5-kDa portion ofthe linker polypeptide had been removed from the original trimer complex, leaving (ap)3-L, its non-linker side (left face in the figure) is apparently unaffected as it can still attach another trimer to form a hexamer. However, without the projected domain this hexamer cannot attach (aP)3-LRc to its right, as illustrated earlier in Fig. 8 (B, 3). It was also shown that(aP)3-L with the projection still intact, apparently cannot attach (aPje L to its right either. These results suggest that (ocP)3-Lrc demands a specific and restricted location in the assembly, namely, the position nearest the core. 

Fig. 10. Aggregation behavior of Anabaena variabilis phycocyanin compiexes with the 32.5- and 27-kDa linker poiypeptide after tryptic degradation. Original data from Yu and Glazer (1982) Cyanobactehal phycobilisomes. Roles of the linker polypeptides in the assembly of phycocyanin. J Biol Chem 257 3430 and Glazer (1982) Phycobilisomes structure and dynamics. Annu Rev Microbiol 36 189. Figure drawn in a form similar to that of Fig. 8, using the graphic representation of MSrschel and Rhiel.

A similar finding also confirmed (aP)3-L to be at this location in a rod subassembly in Synechoccocus 6301. Lundell, Williams and Glazer reported earlier that in a solution containing the 27- and 33-kDa linker polypeptides and phycocyanin, the length ofrods formed is inversely proportional to the ratio ofthe 27-kDa to the 33-kDa polypeptides, indicating that (aPjg-L most likely occupies the terminal position where it inhibits any further lengthening ofthe rod. 

It is obvious that for radiant energy absorbed by any one of the chromophores to be transferred efficiently along the rod toward the core and eventually to the chlorophyll in the thylakoid complex, it is necessary that the hexamer-linker complexes along the rod have appropriate spectral properties. Since all three hexamer disks in some rods may contain only PC, its spectral properties must be appropriately modulated to fulfill the condition for directional energy transfer. In Synechococcus 6301, for example, the spectral properties of the three PC hexamer disks along the rod are indeed modified by their linker polypeptides from the outer end of the rod to the core, as shown here from left to right ... 

This trend in spectral properties promotes energy transfer in the proper direction. Therefore, linker polypeptides not only have the function of linking biliprotein complexes together and controlling the position of the hexamer units along the rod, but also of modifying the spectral properties of the linked hexamers in order to achieve efficient energy transfer toward the core. 

Fig. 12. (A) Time-resolved fluorescence spectra of A. nidulans phycobilisomes measured at 77 K. Excitation by 6-ps, 580-nm argon laser pulse. Three small ticks in the topmost spectrum (at 932 ps) indicate locations of maximum fluorescence at 0 ps. (B) Rise and decay of various fluorescent components derived from deconvolution of the fluorescence spectra. Assignment of individual fluorescent components are shown in the right margin. (C) Energy flow among individual chromophores in the phycobilisomes. The asterisk in (B) and (C) indicates a linker polypeptide is attached to the trimer. See text for discussion. Figure source Mimuro (1989) Studies on excitation energy How in the photosynthetic pigment system structure and energy transfer mechanisms. Bot Mag Tokyo 103 244.

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