Spectral Characterization Of Het-C2, A Glycolipid-transfer Protein

Abstract

Het-C2 is a small 23 kDa protein, isolated from the fungi Podospora anserina, homologous to mammalian GLTP, and capable of transferring glycospingolipids in vitro. The crystal structure of Het-C2 is unknown, but molecular models suggest conservation of the GLTP-fold. Here, the locations of the Trp residues in Het-C2 have been investigated to gain further insights into their function. Sequence homology shows one of Het-C2’s two Trp residues aligned with GLTP Trp96 which resides in the sugar headgroup liganding site. The other Het-C2 Trp did not align with either of GLTP's other two Trp residues. The Trp fluorescence spectrum of native Het-C2 exhibited an emission maximum at 355nm which red shifted 2nm upon denaturation with 8M urea, indicating Trp localization to a more polar environment. Acrylamide and KI quenched >90% of the average Trp fluorescence confirming that Het-C2 Trp residues are not buried in hydrophobic environments but reside in exposed polar regions. The linearity of Stern-Volmer plots for native Het-C2 and urea-denatured (8M) Het-C2 suggested dynamic quenching at physiological pH and ionic strength. The Stern-Volmer constants were higher for native protein than denatured protein. Upon interaction with probe-sonicated POPC vesicles, the Trp emission maximum blue shifted (∼2nm) and decreased in intensity (∼13.5%). Including glycolipid in the vesicles slightly enhanced the blue shift (∼3nm) and significantly decreased Trp intensity (∼21%). Far-UV-CD of Het-C2 showed secondary structure dominated by alpha-helices and with a highly cooperative, thermally induced melting transition near 43°C. Near-UV-CD indicated the induced optical activity of Trp/Tyr residues was unaffected by interaction with vesicles lacking or containing glycolipid. The results are analyzed and discussed within the context of the known locations and functions of human GLTP's three Trp residues. [Support: NIH/NIGMS GM45928 & GM34847, NIH/NCI CA121493, The Hormel & Mayo Foundations]