Human GLTP: Three distinct functions for the three tryptophans in a novel peripheral amphitropic fold.
Human glycolipid transfer protein (GLTP) serves as the GLTP-fold prototype, a novel, to our knowledge, peripheral amphitropic fold and structurally unique lipid binding motif that defines the GLTP superfamily. Despite conservation of all three intrinsic Trps in vertebrate GLTPs, the Trp functional role(s) remains unclear. Herein, the issue is addressed using circular dichroism and fluorescence spectroscopy along with an atypical Trp point mutation strategy. Far-ultraviolet and near-ultraviolet circular dichroism spectroscopic analyses showed that W96F-W142Y-GLTP and W96Y-GLTP retain their native conformation and stability, whereas W85Y-W96F-GLTP is slightly altered, in agreement with relative glycolipid transfer activities of >90%, ∼85%, and ∼45%, respectively. In silico three-dimensional modeling and acrylamide quenching of Trp fluorescence supported a nativelike folding conformation. With the Trp⁹⁶-less mutants, changes in emission intensity, wavelength maximum, lifetime, and time-resolved anisotropy decay induced by phosphoglyceride membranes lacking or containing glycolipid and by excitation at different wavelengths along the absorption-spectrum red edge indicated differing functions for W142 and W85. The data suggest that W142 acts as a shallow-penetration anchor during docking with membrane interfaces, whereas the buried W85 indole helps maintain proper folding and possibly regulates membrane-induced transitioning to a glycolipid-acquiring conformation. The findings illustrate remarkable versatility for Trp, providing three distinct intramolecular functions in the novel amphitropic GLTP fold.
The Hormel Institute, University of Minnesota, Austin, USA.
Ravi Kanth
RK
Kamlekar
Yongguang
Y
Gao
Roopa
R
Kenoth
Julian G
JG
Molotkovsky
Franklyn G
FG
Prendergast
Lucy
L
Malinina
Dinshaw J
DJ
Patel
William S
WS
Wessels
Sergei Y
SY
Venyaminov
Rhoderick E
RE
Brown
D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002942:Circular Dichroism; D005454:Fluorescence Polarization; D006017:Glycolipids / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D016296:Mutagenesis; D009154:Mutation; D017434:Protein Structure, Tertiary; D012995:Solubility; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014364:Tryptophan
D002352:Carrier Proteins; C511922:GLTP protein, human; D006017:Glycolipids; D014364:Tryptophan