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Saschenbrecker, Sandra (2007): Folding and assembly of RuBisCO: Structural and functional characterization of the RuBisCO assembly chaperone RbcX. Dissertation, LMU München: Fakultät für Chemie und Pharmazie



To become biologically active, proteins have to acquire their correct three-dimensional structure by folding, which is frequently followed by assembly into oligomeric complexes. Although all structure relevant information is contained in the amino acid sequence of a polypeptide, numerous proteins require the assistance of molecular chaperones which prevent the aggregation and promote the efficient folding and/or assembly of newly-synthesized proteins. The enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), which catalyzes carbon fixation in the Calvin-Benson-Bassham cycle, requires chaperones in order to acquire its active structure. In plants and cyanobacteria, RuBisCO (type I) is a complex of approximately 550 kDa composed of eight large (RbcL) and eight small (RbcS) subunits. Remarkably, despite the high abundance and importance of this enzyme, the characteristics and requirements for its folding and assembly pathway are only partly understood. It is known that folding of RbcL is accomplished by chaperonin and most likely supported by the Hsp70 system, whereas recent findings indicate the additional need of specific chaperones for assembly. Nevertheless, this knowledge is incomplete, reflected by the fact that in vitro reconstitution of hexadecameric RuBisCO or synthesis of functional plant RuBisCO in E. coli has not been accomplished thus far. In this thesis, attempts to reconstitute type I RuBisCO in vitro did not result in production of active enzyme although a variety of reaction conditions and additives as well as chaperones of different kind, origin and combination were applied. The major obstacle for reconstitution was found to be the incapability to produce RbcL8 cores competent to form RbcL8S8 holoenzyme. It could be shown that the RbcL subunits interact properly with the chaperonin GroEL in terms of binding, encapsulation and cycling. However, they are not released from GroEL in an assembly-competent state, leading to the conclusion that a yet undefined condition or (assembly) factor is required to shift the reaction equilibrium from GroEL-bound RbcL to properly folded and released RbcL assembling to RbcL8 and RbcL8S8, respectively. Cyanobacterial RbcX was found to promote the production of cynanobacterial RbcL8 core complexes downstream of chaperonin-assisted RbcL folding, both in E. coli and in an in vitro translation system. Structural and functional analysis defined RbcX as a homodimeric, arc-shaped complex of approximately 30 kDa, which interacts with RbcL via two distinct but cooperating binding regions. A central hydrophobic groove recognizes and binds a specific motif in the exposed C-terminus of unassembled RbcL, thereby preventing the latter from uncontrolled misassembly and establishing further contacts with the polar peripheral surface of RbcX. These interactions allow optimal positioning and interconnection of the RbcL subunits, resulting in efficient assembly of RbcL8 core complexes. As a result of the highly dynamic RbcL-RbcX interaction, RbcS can displace RbcX from the core-complexes to produce active RbcL8S8 holoenzyme. Species-specific co-evolution of RbcX with RbcL and RbcS accounts for limited interspecies exchangeability of RbcX and for RbcX-supported or -dependent assembly modes, respectively. In summary, this study helped to specify the problem causing prevention of proper in vitro reconstitution of type I RuBisCO. Moreover, the structural and mechanistic properties of RbcX were analyzed, demonstrating its function as specific assembly chaperone for cyanobacterial RuBisCO. Since the latter is very similar to RuBisCO of higher plants, this work may not only augment the general understanding of type I RuBisCO synthesis, but it might also contribute to advancing the engineering of catalytically more efficient crop plant RuBisCO both in heterologous systems and in planta.