Folding/Unfolding of Glycolipid Transfer Protein: Molten Globule-Like Intermediates?

Abstract

Glycolipid transfer proteins (GLTPs) are small, soluble, single-chain proteins (∼24 kDa) that selectively accelerate the intermembrane transfer of glycolipids in vitro. The GLTP-fold is unique among lipid-binding proteins. However, little is known about GLTP stability and folding/-unfolding. During isolation of heterologously expressed GLTP, FPLC size exclusion chromatography showed peaks corresponding to monomer, multimer, and a third peak of intermediate elution volume. Unexpectedly, native gel electrophoresis showed that the intermediate protein peak migrated as monomer rather than dimer, raising the possibility of a molten globule-like state. Intrinsic GLTP tryptophan fluorescence showed a blue-shifted (∼2nm) emission wavelength maximum (λmax), indicating an altered tryptophan environment compared to monomer. ANS binding resulted in a large blue shift (∼20nm) in λmax and dramatically enhanced emission intensity (∼120%). Far-UV-CD showed retention of ordered secondary structure (>95%), but substantially reduced cooperativity during thermally-induced melting. Near-UV-CD analysis of induced optical activity of GLTP Trp/Tyr residues was insufficient to establish tertiary folding changes. To further evaluate GLTP unfolding intermediates, the effect of urea was studied. Trp emission changes suggested a two-step unfolding pathway involving intermediate formation at 4M urea and characterized by blue-shifted Trp emission. Additional urea induced further unfolding marked by red-shifted Trp emission. Far-UV-CD analyses of the 4M urea-induced intermediate indicated reduced ordered secondary structure and cooperative melting at lower temperature compared to native GLTP, but the near-UV-CD signal did not provide definitive insights into tertiary folding status. ANS binding showed 1nm blue shift and 60% increase in fluorescence intensity compared to untreated GLTP. Conditions are identified under which GLTP may exist in molten globule-like and other partially unfolded states. Studies on the significance of these intermediates with respect to function are underway. [Support: NIH/NIGMS GM45928 & GM34847, NIH/NCI CA121493, The Hormel & Mayo Foundations]