Molten globule monomer to condensed dimer: role of disulfide bonds in platelet factor-4 folding and subunit association

Abstract

Platelet factor 4 (PF4) exhibits high affinity for heparin and exists as a tetramer in solution under physiologic conditions. Reduction of the two disulfide bridges in PF4 increases the protein's dissociation constant for heparin approximately 20-fold and shifts the highest apparent aggregation state from tetramer to dimer as evidenced by gel filtration, chemical cross-linking, and 1H-NMR studies. 1H-NMR spectra of reduced PF4 monomers generally show narrower, less dispersed, upfield-shifted NH and alpha H resonances, suggesting the presence of an unfolded monomer state. Reduced PF4 monomer folding, however, is evidenced by the presence of about 12 relatively long-lived backbone NHs and by CD spectra that indicate conservation of overall secondary structure. These data suggest the presence of a molten globule-type state. Urea denaturation shifts this apparent molten globule to a fully unfolded state characterized by more random coil-like resonance shifts. The reduced PF4 dimer state yields NMR and CD data consistent with preservation of tertiary structural folds found for the native species. In this regard, the reduced PF4 folding transition is thermodynamically linked with dimer formation which stabilizes tertiary structure. Monomer-dimer association equilibria for reduced PF4 essentially follow the same pH and salt titration trends as reported previously for native PF4 dimers [Mayo, K. H., & Chen, M. J. (1989) Biochemistry 28, 9469-9478], indicating that that dimer interface is generally conserved in the absence of disulfide constraints. Reduced PF4 tetramers are not apparent under any conditions investigated, suggesting that disulfides are necessary for efficient antiparallel beta-sheet alignment between dimer pairs.

Molecular Features of the Collagen V Heparin Binding Site

“…The clustering of positive charges through the formation of dimers and tetramers can increase the affinity for heparin as has been described for the platelet factor-4 protein (31). Therefore, we propose that the positively charged ring observed for the homotrimer can reinforce the interaction with heparin and thus explain the higher affinity obtained.…”

Dynamics and thermodynamic properties of CXCL7 chemokine

“…S2), suggesting that disulfide bonds do not play a critical role in maintaining structural integrity in either the monomer or dimer. This result agrees with reported experimental data on a closely related chemokine, CXCL4 …”

“…This result agrees with reported experimental data on a closely related chemokine, CXCL4. 64 To quantify changes in thermodynamic stability for each disulfide bond configuration, the change in Gibbs free energy, enthalpy, entropy, total energy of the Hbond network, and the number of interfacial H-bonds formed upon dimerization, is compared at 300K. Table I shows that dimerization of CXCL7 is energetically favorable, and enthalpically driven for all disulfide bond configurations.…”

Anthrax vaccine powder formulations for nasal mucosal delivery

“…Similar characteristics are also observed for rPA at pH 6–8. This suggests the appearance of molten globule‐like states33,34 between these temperatures. Such states are often prone to protein aggregation 35,36.…”