The use of EPR spectroscopy of site-selective nitroxide spin labeled proteins for measuring distances has recently been pioneered by Shin, Beth and Hubbell. We have investigated the advantages of using high frequency EPR to extract distances between biradical nitroxide spin labels. The 4K-li helical peptide used in our studies is an alanine based peptide where two cysteines have been introduced at different positions in the peptide sequence. The cysteine residues are then labeled with the methanethiosulfonate spin label (MTSSL). High frequency spectra have been obtained for a variety of doubly spin labeled peptides; AK6L is the singly labeled peptide, AK417 is labeled at positions (i,i+13), AK49 at positions (i,i+S), AK68 at positions (i,i+2), AK69 at positions (i,i+3), and AK48 at positions (i,i+4). There are 3.5 residues/turn of the helical peptide and one sees, therefore, the greatest effect on the nitroxide EPR spectrum for peptides AK48 (i,i+4) and AK69 (i,i+3), where the two spin labels are in close proximity (7-9 A separation). Analysis of the broadening and splitting of the high frequency EPR resonances enables the determination of the distance between the two nitroxide spin labels. The ?broadening/splitting of the EPR resonances is most pronounced on the '4N hyperfine resonances on the high field edge of the EPR powder pattern. The high frequency CW EPR spectra are sensitive to both the dipolar distance and the relative orientation of the electron g-tensors and the electron dipolar tensor.
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