Incorporation of spin-labelled amino acids into proteins

Abstract

The elucidation of structure and function of proteins and membrane proteins by EPR spectroscopy has become increasingly important in recent years as technological advances have been made in the design of spectrometers and in the chemistry of the nitroxide group. These new developments have increased the demand for tailor-made amino acids carrying a spin label on the one hand and for reliable methods for their incorporation into proteins on the other. Here we describe methods for site-specific spin labelling of proteins. It is shown that a combination of recombinant synthesis of proteins with chemically produced peptides (expressed protein ligation) allows the preparation of site-specifically spin-labelled proteins.

Accurate long‐range distance measurements in a doubly spin‐labeled protein by a four‐pulse, double electron–electron resonance method

“…The problem of absolute structure determination remains difficult as long as the label is attached by linkers of the given length, emphasizing the usefulness of conformationally rigid spin labels, such as used in synthetic peptides, [35] proteins, [36] and DNA or RNA systems. [34,37] The investigation of conformational changes or of interactions of protein subunits under different conditions suffers much less from the absolute distance uncertainty and has therefore been successfully addressed by the MTSL approach, as has been demonstrated in several studies.…”

Membrane Protein Structure and Dynamics Studied by Site-Directed Spin-Labeling ESR

“…In the case of pyrroline-based nitroxide amino acids, new strategies for their incorporation into peptides during SPPS have recently been successfully conducted. Additionally, coupling of a pyrroline nitroxide spin label to the -amino group of a lysine after complete assembly of the peptide has also been performed yielding a paramagnetic stable compound (Becker et al 2005).…”

“…The reaction can be performed even in the presence of other unprotected cysteines. Using this strategy, a spin-labeled RBD protein has been recently synthesized, showing a stable paramagnetic center detected by ESR (Becker et al 2005).…”

Site-Directed Spin Labeling and Electron Paramagnetic Resonance (EPR) Spectroscopy: A Versatile Tool to Study Protein-Protein Interactions

“…The reaction can be performed also in the presence of other unprotected cysteine residues because of a reversible reaction preceding an irreversible step. Using this methodology, a spin-labeled Ras binding domain has been synthesized, showing a stable paramagnetic center detected by EPR (Becker et al, 2005). …”