Chemokines are a family of small signaling proteins that are responsible for the chemotaxis of leukocytes. Chemokine oligomerization may modify the biological activity of the chemokine monomer. We use a combination of computational (Anisotropic Network Model (ANM), molecular dynamics (MD) simulations, minimal Distance Constraint Model (mDCM) and experimental (Circular Dichroism (CD) spectroscopy) methods to characterize the dynamics and stability of the monomer and dimer of CXCL7, one of the most abundant platelet chemokines. The effects of dimerization and disulfide bond formation on the stability/flexibility within secondary structure elements are investigated. Taken together, a consistent picture emerges. From ANM, the Cys5-Cys31 and Cys7-Cys47 disulfide bonds show respectively large and small fluctuations in protein motion due to the location of Cys47 within the beta sheet. The mDCM predicts the heat capacity for the dimer to be more than twice that of the monomer, suggesting a higher degree of cooperativity of folded-unfolding transition in the dimer. A predicted higher free energy barrier suggests that dimerization enhances kinetic stability, but surprisingly, the dynamics and mechanical couplings remain virtually unchanged. Combining CD measurements with a modified Gibbs-Helmhotz equation provides estimates for changes in enthalpy and entropy for folding/unfolding and dimer dissociation. These estimates are used in conjunction with the mDCM to account for solvation free energies in a semi-empirical fashion, which allows us to discern solvation and conformational entropy contributions. Changes in free energy due to dimerization with respect to different disulfide bond configurations are compared. The disulfide bond Cys5-Cys31, which connects two highly flexible regions, has a larger global effect on rigidity and flexibility than Cys7-Cys47. These mechanical and thermodynamic characteristics of CXCL7 help explain the role dimerization plays in modifying the function of chemokines that may be also applicable in vivo.

Stabilization effects of disulfide bonds and dimerization on CXCL7

249th National Meeting (2015)

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