A method for distance determination in proteins using a designed metal ion binding site and site-directed spin labeling: Evaluation with T4 lysozyme

John C Voss, Łukasz Salwiński, H. Ronald Kaback, Wayne L. Hubbell

Research output: Contribution to journalArticle

75 Scopus citations

Abstract

The use of molecular genetics to introduce both a metal ion binding site and a nitroxide spin label into the same protein opens the use of paramagnetic metalnitroxyl interactions to estimate intramolecular distances in a wide variety of proteins. In this report, a His-Xaa3-His metal ion binding motif was introduced at the N terminus of the long interdomain helix of T4 lysozyme (Lys-65 → His/Gln-69 → His) of three mutants, each containing a single nitroxide-labeled cysteine residue at position 71, 76, or 80. The results show that Cu(II)-induced relaxation effects on the nitroxide can be quantitatively analyzed in terms of interspin distance in the range of 10-25 Å using Redfield theory, as first suggested by Leigh [Leigh, J. S. (1970) J. Chem. Phys. 52, 2608-2612]. Of particular interest is the observation that distances can be determined both under rigid lattice conditions in frozen solution and in the presence of motion of the spins at room temperature under physiological conditions. The method should be particularly attractive for investigating structure in membrane proteins that are difficult to crystallize. In the accompanying paper, the technique is applied to a polytopic membrane protein, lactose permease.

Original languageEnglish (US)
Pages (from-to)12295-12299
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume92
Issue number26
DOIs
StatePublished - Dec 19 1995

Keywords

  • cysteine replacement
  • electron paramagnetic resonance
  • histidine residues
  • protein structure

ASJC Scopus subject areas

  • Genetics
  • General

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