Human glycolipid transfer protein: probing conformation using fluorescence spectroscopy.
Glycolipid transfer protein (GLTP) is a soluble 24 kDa protein that selectively accelerates the intermembrane transfer of glycolipids in vitro. Little is known about the GLTP structure and dynamics. Here, we report the cloning of human GLTP and characterize the environment of the three tryptophans (Trps) of the protein using fluorescence spectroscopy. Excitation at 295 nm yielded an emission maximum (lambda(max)) near 347 nm, indicating a relatively polar average environment for emitting Trps. Quenching with acrylamide at physiological ionic strength or with potassium iodide resulted in linear Stern-Volmer plots, suggesting accessibility of emitting Trps to soluble quenchers. Insights into reversible conformational changes accompanying changes in GLTP activity were provided by addition and rapid dilution of urea while monitoring changes in Trp or 1-anilinonaphthalene-8-sulfonic acid fluorescence. Incubation of GLTP with glycolipid liposomes caused a blue shift in the Trp emission maximum but diminished the fluorescence intensity. The blue-shifted emission maximum, centered near 335 nm, persisted after separation of glycolipid liposomes from GLTP, consistent with formation of a GLTP-glycolipid complex at a glycolipid-liganding site containing Trp. The results provide the first insights into human GLTP structural dynamics by fluorescence spectroscopy, including global conformational changes that accompany GLTP folding into an active conformational state as well as more subtle conformational changes that play a role in GLTP-mediated transfer of glycolipids between membranes, and establish a foundation for future studies of membrane rafts using GLTP.
The Hormel Institute, University of Minnesota, 801 16th Avenue NE, Austin, Minnesota 55912-3698, USA.
Xin-Min
XM
Li
Margarita L
ML
Malakhova
Xin
X
Lin
Helen M
HM
Pike
Taeowan
T
Chung
Julian G
JG
Molotkovsky
Rhoderick E
RE
Brown
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics; D002417:Cattle; D003001:Cloning, Molecular; D006017:Glycolipids / Q000378:metabolism*; D006801:Humans; D008081:Liposomes; D051379:Mice; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods; D013552:Swine; D014364:Tryptophan / Q000737:chemistry; D014508:Urea / Q000737:chemistry
D002352:Carrier Proteins; C511922:GLTP protein, human; C511923:Gltp protein, mouse; D006017:Glycolipids; D008081:Liposomes; D011994:Recombinant Proteins; C030363:lipid transfer protein; D014364:Tryptophan; D014508:Urea