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The native state of apomyoglobin described by proton NMR spectroscopy: Interaction with the paramagnetic probe HyTEMPO and the fluorescent dye ANS
Author(s) -
Cocco Melanie J.,
Lecomte Juliette T.J.
Publication year - 1994
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560030211
Subject(s) - chemistry , side chain , crystallography , histidine , fluorescence , amide , stereochemistry , molecule , proton nmr , amino acid , polymer , physics , quantum mechanics , biochemistry , organic chemistry
Proton NMR experiments were carried out on apomyoglobin from sperm whale and horse skeletal muscle. Two small molecules, the paramagnetic relaxation agent 4‐hydroxy‐2,2,6,6‐tetramethylpiperidinyl‐l‐oxy (HyTEMPO) and the fluorescent dye 8‐anilino‐1‐naphthalenesulfonic acid (ANS), were used to alter and simplify the spectrum. Both were shown to bind in the heme pocket by docking onto the hydrophobic residues lining the distal side. Only 1 extensive region of the apoprotein structure, composed of hydrophobic residues, is not affected by HyTEMPO. It includes the 2 tryptophans (located in the A helix), other nonpolar residues of the A helix and side chains from the E, G, and GH helices. The spectral perturbations induced by ANS allowed assignment of the distal histidine (His‐64) in horse apomyoglobin. This residue was previously reported to titrate with a p K a below 5 and tentatively labeled as His‐82 on the basis of this value (Cocco MJ, Kao YH, Phillips AT, Lecomte JTJ, 1992, Biochemistry 31 :6481‐6491). The packing of the side chains and the low p K a of His‐64 reinforce the idea that the distal side of the binding site is folded in a manner closely related to that in the holoprotein. ANS was found to sharpen the protein signals and the improvement of the spectral resolution facilitated the assignment of backbone amide resonances. Secondary structure, as manifested in characteristic inter‐amide proton NOEs, was detected in the A, B, C, E, G, and H helices. The combined information on the hydrophobic cores and the secondary structure composes an improved representation of the native state of apomyoglobin.