Kinetics control preferential heterodimer formation of platelet-derived growth factor from unfolded A- and B-chains.
The folding and assembly of platelet-derived growth factor (PDGF), a potent mitogen involved in wound-healing processes and member of the cystine knot growth factor family, was studied. The kinetics of the formation of disulfide-bonded dimers were investigated under redox reshuffling conditions starting either from unfolded and reduced PDGF-A- or B-chains or an equimolar mixture of both chains. It is shown that in all cases the formation of disulfide-bonded dimers is a very slow process occurring in the time scale of hours with a first-order rate-determining step. The formation of disulfide-bonded PDGF-AA or PDGF-BB homodimers displayed identical kinetics, indicating that both monomeric forms as well as the dimerized homodimer have similar folding and assembly pathways. In contrast, the formation of the heterodimer occurred three times more rapidly compared with the formation of the homodimers. As both monomeric forms revealed similar renaturation kinetics, it can be concluded that the first-order rate-determining folding step does not occur during monomer folding but must be attributed to conformational rearrangements of the dimerized, not yet disulfide-bonded protein. These structural rearrangements allow a more rapid formation of intermolecular disulfide bonds between the two different monomers of a heterodimer compared with the formation of the disulfide bonds between two identical monomers. The preferential formation of disulfide-bonded heterodimers from an equimolar mixture of unfolded A- and B-chains is thus a kinetically controlled process. Moreover, similar activation enthalpies for the formation of all different isoforms suggest that faster heterodimerization is controlled by entropic factors.
Müller
Carsten
C
GBF National Research Center for Biotechnology, Biochemical Engineering Division, Mascheroder Weg 1, 38124 Braunschweig, Germany.
Richter
Susanne
S
Rinas
Ursula
U
D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D004789:Enzyme Activation; D004926:Escherichia coli / Q000378:metabolism; D019791:Guanidine / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008956:Models, Chemical; D010982:Platelet-Derived Growth Factor / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D020033:Protein Isoforms; D017433:Protein Structure, Secondary; D013696:Temperature; D013816:Thermodynamics; D013997:Time Factors
D004220:Disulfides; D010982:Platelet-Derived Growth Factor; D020033:Protein Isoforms; D019791:Guanidine