Glycolipid acquisition by human glycolipid transfer protein dramatically alters intrinsic tryptophan fluorescence: Insights into glycolipid binding affinity

Xiuhong Zhai, Margarita L. Malakhova, Helen M. Pike, Linda M. Benson, Harold Robert (Bob) III Bergen, István P. Sugár, Lucy Malinina, Dinshaw J. Patel, Rhoderick E. Brown

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

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. ∼40% decrease in Trp intensity and ∼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 ∼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.

Original languageEnglish (US)
Pages (from-to)13620-13628
Number of pages9
JournalJournal of Biological Chemistry
Volume284
Issue number20
DOIs
StatePublished - May 15 2009

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Glycolipids
Tryptophan
Fluorescence
Sugars
Lipids
human GLTP protein
Sonication
Ceramides
Microinjections
lipid transfer protein
Phosphatidylcholines
Protein Binding
Dilution
Extrusion
Hydrogen
Carrier Proteins
Hydrogen bonds
Proteins
Ethanol
X-Rays

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

Glycolipid acquisition by human glycolipid transfer protein dramatically alters intrinsic tryptophan fluorescence : Insights into glycolipid binding affinity. / Zhai, Xiuhong; Malakhova, Margarita L.; Pike, Helen M.; Benson, Linda M.; Bergen, Harold Robert (Bob) III; Sugár, István P.; Malinina, Lucy; Patel, Dinshaw J.; Brown, Rhoderick E.

In: Journal of Biological Chemistry, Vol. 284, No. 20, 15.05.2009, p. 13620-13628.

Research output: Contribution to journalArticle

Zhai, Xiuhong ; Malakhova, Margarita L. ; Pike, Helen M. ; Benson, Linda M. ; Bergen, Harold Robert (Bob) III ; Sugár, István P. ; Malinina, Lucy ; Patel, Dinshaw J. ; Brown, Rhoderick E. / Glycolipid acquisition by human glycolipid transfer protein dramatically alters intrinsic tryptophan fluorescence : Insights into glycolipid binding affinity. In: Journal of Biological Chemistry. 2009 ; Vol. 284, No. 20. pp. 13620-13628.
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T2 - Insights into glycolipid binding affinity

AU - Zhai, Xiuhong

AU - Malakhova, Margarita L.

AU - Pike, Helen M.

AU - Benson, Linda M.

AU - Bergen, Harold Robert (Bob) III

AU - Sugár, István P.

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AB - 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. ∼40% decrease in Trp intensity and ∼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 ∼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.

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