Paramagnetic relaxation enhancement of membrane proteins by incorporation of the metal-chelating unnatural amino acid 2-amino-3-(8-hydroxyquinolin-3-yl)propanoic acid (HQA)

J Biomol NMR. 2015 Apr;61(3-4):185-96. doi: 10.1007/s10858-014-9884-5. Epub 2014 Nov 28.

Abstract

The use of paramagnetic constraints in protein NMR is an active area of research because of the benefits of long-range distance measurements (>10 Å). One of the main issues in successful execution is the incorporation of a paramagnetic metal ion into diamagnetic proteins. The most common metal ion tags are relatively long aliphatic chains attached to the side chain of a selected cysteine residue with a chelating group at the end where it can undergo substantial internal motions, decreasing the accuracy of the method. An attractive alternative approach is to incorporate an unnatural amino acid that binds metal ions at a specific site on the protein using the methods of molecular biology. Here we describe the successful incorporation of the unnatural amino acid 2-amino-3-(8-hydroxyquinolin-3-yl)propanoic acid (HQA) into two different membrane proteins by heterologous expression in E. coli. Fluorescence and NMR experiments demonstrate complete replacement of the natural amino acid with HQA and stable metal chelation by the mutated proteins. Evidence of site-specific intra- and inter-molecular PREs by NMR in micelle solutions sets the stage for the use of HQA incorporation in solid-state NMR structure determinations of membrane proteins in phospholipid bilayers.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Alanine / analogs & derivatives*
  • Alanine / chemical synthesis
  • Alanine / chemistry
  • Amino Acid Sequence
  • Amino Acid Substitution
  • Amino Acids / chemical synthesis
  • Amino Acids / chemistry
  • Binding Sites / physiology
  • Electron Spin Resonance Spectroscopy / methods*
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Hydroxyquinolines / chemical synthesis
  • Hydroxyquinolines / chemistry*
  • Interleukin-8 / analysis*
  • Interleukin-8 / chemistry
  • Interleukin-8 / genetics
  • Membrane Proteins / analysis
  • Models, Molecular
  • Molecular Sequence Data
  • Nuclear Magnetic Resonance, Biomolecular / methods*
  • Propionates / chemical synthesis
  • Propionates / chemistry
  • Protein Binding / physiology
  • Protein Structure, Tertiary
  • Receptors, Interleukin-8A / analysis*
  • Receptors, Interleukin-8A / chemistry
  • Receptors, Interleukin-8A / genetics

Substances

  • 2-amino-3-(8-hydroxyquinolin-3-yl)propanoic acid
  • Amino Acids
  • Hydroxyquinolines
  • Interleukin-8
  • Membrane Proteins
  • Propionates
  • Receptors, Interleukin-8A
  • Alanine