Synthesis of peptide spin-labels that bind to neurophysin and their application to distance measurements within neurophysin complexes.
The synthesis of two spin-labels capable of binding to the hormone-binding site(s) of neurophysin is described. The two spin-labels are 4-(glycyl-L-phenylalanylamido)-2,2,6,6-tetramethylpiperidinyl-1-oxy and S-[[[3-(2,2,5,5-tetramethylpyrrolidine-1-oxy)amino]carbonyl]methyl]-L-cysteinyl -L-tyrosine amide; synthesis of the former is achieved by a novel route to circumvent problems associated with nitroxide instability under standard conditions of peptide deblocking. NMR studies of the effects of binding these spin-labels on relaxation rates of individual proton resonances of neurophysin were used to calculate correlation times and distances between the bound nitroxides and the observed protons. The results indicate that residue 3 of peptides bound to the strong site of neurophysin is greater than or equal to 14 A from Tyr-49 and argue against a distance of < 5 A between the ortho ring protons of Tyr-49 and those of residue 2 of peptides bound to the strong site. Alternatively, the data suggest that the previously observed nuclear Overhauser effect between these protons reflects spin diffusion at the strong site and a contribution of uncertain magnitude from a second but very weak binding site; this second site is close to Tyr-49 and is detected by the increased relaxation rate of Tyr-49 ring protons when 4-(glycyl-L-phenylalanylamido)-2,2,6,6-tetramethylpiperidinyl-1-oxy is displaced from the strong site by competing diamagnetic peptide. Additionally, the data indicate that residue 3 of bound peptides at the strong site is distant from His-80 but approximately 12 A from the amino terminus. The extended side chain of residue 1 of peptides at the strong site is calculated as less than or equal to 10 A from Tyr-49.
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003497:Cyclic N-Oxides / Q000138:chemical synthesis*; D009682:Magnetic Resonance Spectroscopy; D008433:Mathematics; D008722:Methods; D009481:Neurophysins; D011485:Protein Binding; D011487:Protein Conformation; D013113:Spin Labels
D003497:Cyclic N-Oxides; D009481:Neurophysins; D013113:Spin Labels; C028389:4-(glycyl-L-phenylalanylamido)-2,2,6,6-tetramethylpiperidinyl-1-oxy; C028390:S-(((3-(2,2,5,5-tetramethylpyrrolidine-1-oxy)amino)carbonyl)methyl)-L-cysteinyl-L-tyrosine amide