Two-dimensional crystallization of histidine-tagged, HIV-1 reverse transcriptase promoted by a novel nickel-chelating lipid

J Struct Biol. 1994 Sep-Oct;113(2):117-23. doi: 10.1006/jsbi.1994.1039.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Chelating Agents / metabolism*
  • Crystallization
  • Crystallography, X-Ray
  • HIV Reverse Transcriptase
  • Histidine
  • Nitrilotriacetic Acid / analogs & derivatives*
  • Nitrilotriacetic Acid / metabolism
  • Phosphatidylethanolamines / metabolism*
  • RNA-Directed DNA Polymerase / chemistry*
  • RNA-Directed DNA Polymerase / isolation & purification
  • RNA-Directed DNA Polymerase / metabolism
  • Recombinant Fusion Proteins / chemistry*
  • Recombinant Fusion Proteins / isolation & purification
  • Recombinant Fusion Proteins / metabolism

Substances

  • 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine-N-(N-(5-amino-1-carboxypentyl(iminodiacetic acid)succinimide))
  • Chelating Agents
  • Phosphatidylethanolamines
  • Recombinant Fusion Proteins
  • Histidine
  • HIV Reverse Transcriptase
  • RNA-Directed DNA Polymerase
  • Nitrilotriacetic Acid