Spontaneous dissociation-association of monomers of the human-stem-cell-factor dimer

Abstract

In its native state, recombinant human-stem-cell-factor (SCF) dimer can spontaneously and rapidly undergo hybridization when two different SCF dimer species are incubated together. SCF species differing in molecular charge, e.g., a wild-type SCF form and a variant with Asp at position 10 instead of Asn, were used in the hybridization studies; the original species and newly formed dimer hybrid can be separated and quantified by cationic-exchange h.p.l.c. The hybridization reaches an equilibrium where the ratio of hybrid dimer to each of the original species is 2. Kinetic studies of the initial rate of hybridization enable a rate constant for monomer dissociation to be determined. This rate constant is influenced by pH, temperature and salt concentration. The pH and salt effects suggest that salt bridges between charged amino acids at the monomer-monomer interface may be present. From the temperature effects, the activation energy for monomer dissociation was determined to be 85.6 kJ/mol, which is typical for oligomeric proteins. Heavily glycosylated recombinant SCF from Chinese-hamster ovary cells exchanged equally well with the bacterially derived non-glycosylated SCF, indicating that the attached carbohydrate moieties had no effect on monomer exchange.

Crystal structure of human stem cell factor: Implication for stem cell factor receptor dimerization and activation

“…Dimerization of SCF is sensitive to pH and salt concentration changes (25). This property is likely due to the fact that the interface is formed in part by polar interactions via salt bridges at the periphery and by water molecule-mediated hydrogen bonds among buried polar residues at the core of the interface.…”

Human Stem Cell Factor Dimer Forms a Complex with Two Molecules of the Extracellular Domain of Its Receptor, Kit

“…5 shows the results of a simultaneous fit of Model 1 to data for sKit plus E. coli-derived SCF mixtures made up at both the two sKit per SCF dimer and one sKit per SCF dimer molar ratios, and at three different rotor speeds. This fit is excellent and returns an intrinsic dissociation constant for each site of 17 [12][13][14][15][16][17][18][19][20][21] nM. As implied by Fig.…”

“…Soluble SCF exists as noncovalently associated homodimer at concentrations where it has previously been possible to study its quaternary structure (19), although monomer dissociation/reassociation is rapid (20). Whether or not membranebound SCF is dimeric has not been determined.…”

Interleukin-6 dimers produced by endothelial cells inhibit apoptosis of B-chronic lymphocytic leukemia cells

“…Differences in the mode of dimerization that affects the stability of the IL-6 D can be predicted to decrease the mediation of survival activity, as has been described for the noncovalently associated hrSCF dimer, which can undergo spontaneous rapid monomer dissociation. 42 Accordingly, it is also possible that IL-6 D derived from ECV-304 cells exhibit more stability than hrIL-6 D derived from E coli. Indeed, IL-6 D contained in the ECCM were heat stable.…”