Glycolipid acquisition by human glycolipid transfer protein dramatically alters intrinsic tryptophan fluorescence: insights into glycolipid binding affinity.
Glycolipid transfer proteins (GLTPs) are small, soluble proteins that selectively accelerate the intermembrane transfer of glycolipids. The GLTP fold is conformationally unique among lipid binding/transfer proteins and serves as the prototype and founding member of the new GLTP superfamily. In the present study, changes in human GLTP tryptophan fluorescence, induced by membrane vesicles containing glycolipid, are shown to reflect glycolipid binding when vesicle concentrations are low. Characterization of the glycolipid-induced "signature response," i.e. approximately 40% decrease in Trp intensity and approximately 12-nm blue shift in emission wavelength maximum, involved various modes of glycolipid presentation, i.e. microinjection/dilution of lipid-ethanol solutions or phosphatidylcholine vesicles, prepared by sonication or extrusion and containing embedded glycolipids. High resolution x-ray structures of apo- and holo-GLTP indicate that major conformational alterations are not responsible for the glycolipid-induced GLTP signature response. Instead, glycolipid binding alters the local environment of Trp-96, which accounts for approximately 70% of total emission intensity of three Trp residues in GLTP and provides a stacking platform that aids formation of a hydrogen bond network with the ceramide-linked sugar of the glycolipid headgroup. The changes in Trp signal were used to quantitatively assess human GLTP binding affinity for various lipids including glycolipids containing different sugar headgroups and homogenous acyl chains. The presence of the glycolipid acyl chain and at least one sugar were essential for achieving a low-to-submicromolar dissociation constant that was only slightly altered by increased sugar headgroup complexity.
Zhai
Xiuhong
X
The Hormel Institute, University of Minnesota, Austin, Minnesota 55912.
Malakhova
Margarita L
ML
The Hormel Institute, University of Minnesota, Austin, Minnesota 55912.
Pike
Helen M
HM
The Hormel Institute, University of Minnesota, Austin, Minnesota 55912.
Benson
Linda M
LM
Mayo Proteomics Research Center, Mayo Clinic College of Medicine, Rochester, Minnesota 55905.
Bergen
H Robert
HR
Mayo Proteomics Research Center, Mayo Clinic College of Medicine, Rochester, Minnesota 55905.
Sugár
István P
IP
Department of Neurology, Mount Sinai School of Medicine, New York, New York 10029.
Malinina
Lucy
L
Structural Biology, CIC bioGUNE, Parque Tecnológico de Vizcaya, Ed. 800, Derio 48160, Spain. Electronic address: lucy@cicbiogune.es.
Patel
Dinshaw J
DJ
Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10065. Electronic address: pateld@mskcc.org.
Brown
Rhoderick E
RE
The Hormel Institute, University of Minnesota, Austin, Minnesota 55912. Electronic address: reb@umn.edu.
D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D005453:Fluorescence; D006017:Glycolipids / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D006860:Hydrogen Bonding; D011485:Protein Binding / Q000502:physiology; D014364:Tryptophan / Q000737:chemistry* / Q000378:metabolism
D002352:Carrier Proteins; C511922:GLTP protein, human; D006017:Glycolipids; D014364:Tryptophan