Two-dimensional crystallization of histidine-tagged, HIV-1 reverse transcriptase promoted by a novel nickel-chelating lipid.
Recombinant proteins containing a short stretch of contiguous histidine residues (approximately 6) ("a His-tag") can be specifically bound to N-nitrilotriacetic-acid-chelated nickel ions, providing a convenient general method for their purification. A lipid derivatized with a nickel-chelating head group may provide a general approach to two-dimensional crystallization of the His-tagged proteins, using the lipid layer technique. We have designed a synthetic phospholipid that carries a chelated nickel ion (Ni-NTA-DOPE). His-tagged recombinant HIV-1 reverse transcriptase (HIV-RT) bound specifically to lipid layers containing Ni-NTA-DOPE and formed crystals within minutes from a dilute protein solution. Two-dimensional crystals preserved in negative stain diffracted strongly to approximately 21 A. The projection map computed from averaged Fourier transforms revealed a structure similar in size and shape to a selected projection view of the 3-D structure that was previously determined for HIV-RT by X-ray crystallography.
Kubalek
E W
EW
Howard Hughes Medical Institute, Stanford University, California.
Le Grice
S F
SF
Brown
P O
PO
D002614:Chelating Agents / Q000378:metabolism*; D003460:Crystallization; D018360:Crystallography, X-Ray; D054303:HIV Reverse Transcriptase; D006639:Histidine; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
C093198:1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine-N-(N-(5-amino-1-carboxypentyl(iminodiacetic acid)succinimide)); D002614:Chelating Agents; D010714:Phosphatidylethanolamines; D011993:Recombinant Fusion Proteins; D006639:Histidine; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D009571:Nitrilotriacetic Acid