Cooperative Subunit Refolding of a Light‐Harvesting Protein through a Self‐Chaperone Mechanism

Abstract

The fold of ap rotein is encoded by its amino acid sequence,b ut how complex multimeric proteins fold and assemble into functional quaternary structures remains unclear.H ere we show that two structurally different phycobiliproteins refold and reassemble in ac ooperative manner from their unfolded polypeptide subunits,w ithout biological chaperones.R efolding was confirmed by ultrafast broadband transient absorption and two-dimensional electronic spectroscopyt op robe internal chromophores as am arker of quaternary structure.O ur results demonstrate ac ooperative,s elfchaperone refolding mechanism, whereby the b-subunits independently refold, therebyt emplating the folding of the asubunits,w hich then chaperone the assembly of the native complex, quantitatively returning all coherences.O ur results indicate that subunit self-chaperoning is ar obust mechanism for heteromeric protein folding and assembly that could also be applied in self-assembled synthetic hierarchical systems.The folding of proteins was shown to be encoded by amino acids in 1960s.[1] Cooperativity [2][3][4] and the realization that multiple folding pathways are possible [5] are the keys to resolving Levinthalsf amous paradox, [6] which states that if aprotein were to explore all possible conformations,itwould take longer than the age of the universe to fold-yet most proteins fold within seconds.M any proteins in nature are found as multimeric or quaternary complexes, [7] with database searches suggesting that more than 80 %o fp roteins are multimeric,w ith between 15-50 %b eing hetero-oligomeric. [8,9] Thep revailing view on how these complexes form starts with the assumption that the individual subunits are first folded, or sometimes disordered (fuzzy), prior to the oligomerization step. [8,10] Thef olded monomer of the homooligmeric GroEL chaperonin catalyzes its own oligomerization [11] in aprocess known as self-chaperoning.Such behavior has also been observed in the folding of the bI5 RNAi ntron with its CBP2 protein cofactor.[12] How,o ri f, quaternary structures can be unfolded and then refolded in vitro,a nd whether the subunits need each other to fold and form aquaternary structure,r emains an open question.There are two different quaternary types of phycobiliproteins in the cryptophytes,the closed-form and the open-form protein. Both quaternary structures share the same types of subunits,t hat of two identical approximately 20 kDa bsubunits,a nd two identical or non-identical approximately 7-9 kDa a-subunits,w hich combine to make the approximately 60 kDa abab-heterodimer ( Figure S1). The Rhodomonas sp.p hycobiliprotein, phycoerythrin 545 (PE545 [13] ), is ad imer of two non-identical ab-monomers (a 1 ba 2 b,F igure 1a,P DB:1 XF6 [14] ). Each globular b-subunit covalently binds three linear tetrapyrroles (bilins), while the two nonidentical a-subunits (a 1 and a 2 )a re short extended polypeptides,each with asingle covalently bound bilin chromophore. Thed imer adopts the closed-form quaternary structure that brings two chrom...

Determination of the protonation preferences of bilin pigments in cryptophyte antenna complexes

“…Note that at these values protonation/deprotonation of protein residues and partial unfolding of the complexes may affect the absorption spectra, making it more difficult to correlate absorption changes to variations in the protonation pattern of the pigments. 56 Indeed, the absorption spectra of all the proteins varies at 4, matching the approximate ! of Asp and Glu side chains, but after the eventual deprotonation of these aminoacids all spectra remain mostly unchanged as is increased from 4.6 to 6.5.…”

Molecular dissection of the soluble photosynthetic antenna from a cryptophyte alga

“…f. Shows the interactions between the N-terminus of 10 the αL subunit and the PEB β82 chromophore in HaPE645, which are similar to those seen in g. for the PCB β82 chromophore in HvPC612. References (41)(42)(43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53)(54)(55)(56)(57)(58)(59) 10…”

Conformational Regulation of Multivalent Terpyridine Ligands for Self-Assembly of Heteroleptic Metallo-Supramolecules

“…Cooperative self-assembly prevails ubiquitously in construction and modulation of functional biomacromolecules in nature . The cooperativity typically involves conformational regulation of receptors or configurational preorganization of two or more building blocks to either increase or decrease binding affinity in subsequent binding events. The bio-inspired molecular design with cooperativity has been demonstrated in creation of artificial systems for applications in molecular recognition, catalysis, ion sensing, and so on.…”