Glycolipid acquisition by human glycolipid transfer protein dramatically alters intrinsic tryptophan fluorescence: Insights into glycolipid binding affinity Journal Article
| Authors: | Zhai, X.; Malakhova, M. L.; Pike, H. M.; Benson, L. M.; Bergen, H. R. 3rd; Sugár, I. P.; Malinina, L.; Patel, D. J.; Brown, R. E. |
| Article Title: | Glycolipid acquisition by human glycolipid transfer protein dramatically alters intrinsic tryptophan fluorescence: Insights into glycolipid binding affinity |
| 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. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc. |
| Keywords: | carrier protein; unclassified drug; binding affinity; protein conformation; protein function; protein localization; proteins; metabolism; protein assembly; fluorescence; protein binding; tyrosine; wild type; physiology; chemistry; quantitative analysis; nucleotide sequence; carrier proteins; binding site; hydrogen bond; hydrogen bonding; dimerization; lipids; binding affinities; binding energy; sugars; conformational transition; amino acids; protein folding; x ray crystallography; concentration (parameters); high resolution; dissociation; acyl chain; phosphatidylcholine; ethanol; tryptophan; ceramide; amides; sugar (sucrose); lipid analysis; sugar; blue shift; dissociation constant; emission wavelength; ethanol solutions; glyco lipids; glycolipid transfer proteins; head groups; hydrogen bond networks; intermembrane transfer; intrinsic tryptophan fluorescences; local environments; membrane vesicles; soluble proteins; total emissions; tryptophan fluorescence; vesicle concentration; x-ray structure; hydrogen; hydrogen bonds; 2 oleoyl 1 palmitoylphosphatidylcholine; galactosylceramide; glucosylceramide; glycolipid; glycolipid transfer protein; lactosylceramide; lipid transfer protein; gltp protein, human; lipid composition; lipid transport; membrane vesicle; protein lipid interaction; glycolipids |
| Journal Title: | Journal of Biological Chemistry |
| Volume: | 284 |
| Issue: | 20 |
| ISSN: | 0021-9258 |
| Publisher: | American Society for Biochemistry and Molecular Biology |
| Date Published: | 2009-05-15 |
| Start Page: | 13620 |
| End Page: | 13628 |
| Language: | English |
| DOI: | 10.1074/jbc.M809089200 |
| PUBMED: | 19270338 |
| PROVIDER: | scopus |
| PMCID: | PMC2679463 |
| DOI/URL: | |
| Notes: | --- - "Cited By (since 1996): 4" - "Export Date: 30 November 2010" - "CODEN: JBCHA" - "Source: Scopus" |
