Biophysical characterization of a soluble CD40 ligand (CD154) coiled-coil trimer: Evidence of a reversible acid-denatured molten globule

The CD40 ligand molecule is unique, consisting of a receptor-binding domain anchored by an isoleucine zipper moiety. Exact determination of the multim eric state and its tendency to form molten globules has not been elucidated . Corroborating evidence of a trimerized molecule in aqueous solution was o btained from size-exclusion chromatography, laser light scattering, and ana lytical ultracentrifugation. A reversible acid-denatured molten globule sta te was observed from circular dichroism. and fluorescence spectroscopy data . The molten globule state was characterized by a loss of tertiary structur e with associated retention of secondary structure near pH 3. Once returned to pH 7, the acid-denatured state refolded over the course of 7 days resul ting in approximately 90% recovery of the native structure. The molten glob ule state was characterized by a broadening of structural features in the s econd-derivative spectra of Fourier transform infrared spectroscopy. A comp onent band at 1650 cm(-1) was shown to be a-helix and originate from amide carbonyl vibrations of the isoleucine zipper. Differential scanning calorim etry measurements characterized the pH-sensitive molten globule state at pH 3.3 as one lacking a well-defined unfolding transition with an accompanyin g baseline shift at 58 degreesC (a consequence of increased heat capacity). The tendency to for. molten globules during acid denaturation stress permi ts an opportunity to study the process of partial protein unfolding with im plications concerning stability. Although reversible molten globules can be formed, it is important to recognize the unusual nature since the molten g lobule state is formed exclusively within the beta -sheet receptor-binding region. (C) 2001 Academic Press.