Molten globule monomer to condensed dimer: role of disulfide bonds in platelet factor-4 folding and subunit association.
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.
Mayo
K H
KH
Jefferson Cancer Institute, Department of Pharmacology, Thomas Jefferson University, PHiladelphia, Pennsylvania 19107.
Barker
S
S
Kuranda
M J
MJ
Hunt
A J
AJ
Myers
J A
JA
Maione
T E
TE
D002850:Chromatography, Gel; D002942:Circular Dichroism; D003432:Cross-Linking Reagents; D004220:Disulfides / Q000737:chemistry*; D006493:Heparin / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D010084:Oxidation-Reduction; D010978:Platelet Factor 4 / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D012965:Sodium Chloride / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature
D003432:Cross-Linking Reagents; D004220:Disulfides; D010978:Platelet Factor 4; D012965:Sodium Chloride; D006493:Heparin