Methionine-90-spin-labeled bovine alpha-lactalbumin: electron spin resonance and NMR distance measurements.
The unique methionine residue of bovine alpha-lactalbumin was modified by irreversible alkylation with the bromoacetamido nitroxide spin-label 4-(2-bromoacetamido)-2,2,6,6-tetramethylpiperidine-N-oxyl. The line shape of the electron spin resonance (ESR) spectrum was indicative of a fairly mobile spin-label and was sensitive to the calcium-induced conformational change. Paramagnetic broadening of the spin-label ESR lines by a Gd(III) ion substituted at the high-affinity calcium site of the protein yielded a distance between the spin-label and the metal-binding site of 8.0 +/- 1.0 A. The extent of the paramagnetic line broadening by the covalently attached nitroxide spin-label on the proton resonances of several amino acid residues of the protein at 500 MHz allowed estimation of intramolecular distances between the methionine-90 residue and several resolvable protons.
Musci
G
G
Department of Chemistry, Ohio State University, Columbus 43210.
Koga
K
K
Berliner
L J
LJ
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D003497:Cyclic N-Oxides; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D005682:Gadolinium / Q000494:pharmacology; D007768:Lactalbumin / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008715:Methionine; D011487:Protein Conformation; D013113:Spin Labels
D003497:Cyclic N-Oxides; D013113:Spin Labels; C028369:tempo 4-bromoacetamide; D007768:Lactalbumin; D008715:Methionine; D005682:Gadolinium; D002118:Calcium