title: Conformational folding and stability of the HET-C2 glycolipid transfer protein fold: does a molten globule-like state regulate activity?
abstract: The glycolipid transfer protein (GLTP) superfamily is defined by the human GLTP fold that represents a novel motif for lipid binding and transfer and for reversible interaction with membranes, i.e., peripheral amphitropic proteins. Despite limited sequence homology with human GLTP, we recently showed that HET-C2 GLTP of Podospora anserina is organized conformationally as a GLTP fold. Currently, insights into the folding stability and conformational states that regulate GLTP fold activity are almost nonexistent. To gain such insights into the disulfide-less GLTP fold, we investigated the effect of a change in pH on the fungal HET-C2 GLTP fold by taking advantage of its two tryptophans and four tyrosines (compared to three tryptophans and 10 tyrosines in human GLTP). pH-induced conformational alterations were determined by changes in (i) intrinsic tryptophan fluorescence (intensity, emission wavelength maximum, and anisotropy), (ii) circular dichroism over the near-UV and far-UV ranges, including thermal stability profiles of the derivatized molar ellipticity at 222 nm, (iii) fluorescence properties of 1-anilinonaphthalene-8-sulfonic acid, and (iv) glycolipid intermembrane transfer activity monitored by Förster resonance energy transfer. Analyses of our recently determined crystallographic structure of HET-C2 (1.9 Å) allowed identification of side chain electrostatic interactions that contribute to HET-C2 GLTP fold stability and can be altered by a change in pH. Side chain interactions include numerous salt bridges and interchain cation-π interactions, but not intramolecular disulfide bridges. Histidine residues are especially important for stabilizing the local positioning of the two tryptophan residues and the conformation of adjacent chains. Induction of a low-pH-induced, molten globule-like state inhibited glycolipid intermembrane transfer by the HET-C2 GLTP fold.
authors.affiliation: The Hormel Institute, University of Minnesota, Austin, MN 55912, USA.
authors.forename: Roopa
authors.initials: R
authors.lastname: Kenoth
authors.forename: Ravi Kanth
authors.initials: RK
authors.lastname: Kamlekar
authors.forename: Dhirendra K
authors.initials: DK
authors.lastname: Simanshu
authors.forename: Yongguang
authors.initials: Y
authors.lastname: Gao
authors.forename: Lucy
authors.initials: L
authors.lastname: Malinina
authors.forename: Franklyn G
authors.initials: FG
authors.lastname: Prendergast
authors.forename: Julian G
authors.initials: JG
authors.lastname: Molotkovsky
authors.forename: Dinshaw J
authors.initials: DJ
authors.lastname: Patel
authors.forename: Sergei Y
authors.initials: SY
authors.lastname: Venyaminov
authors.forename: Rhoderick E
authors.initials: RE
authors.lastname: Brown
mesh_terms: D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000378:metabolism; D006017:Glycolipids / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D017510:Protein Folding; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D005656:Fungal Proteins; C511922:GLTP protein, human; D006017:Glycolipids; C087973:HET-C2 protein, Podospora anserina; C030363:lipid transfer protein; D014364:Tryptophan