Nuclear magnetic resonance characterization of aromatic residues of .alpha.-lactalbumins. Laser photochemically induced dynamic nuclear polarization nuclear magnetic resonance studies of surface exposure

Abstract

The alpha-lactalbumins are involved as modifier proteins in lactose biosynthesis. Lactalbumins from different mammalian species are cross-reactive with the galactosyltransferases from others because of their homologous primary and tertiary structure. We have studied the surface exposure of several alpha-lactalbumins (bovine, goat, human, guinea pig, and rabbit) by the laser photo-CIDNP technique, an NMR method which measures the access of a photoexcited flavin dye to surface exposed Tyr, Trp, and His residues. An exposed histidine-68 residue exists in the bovine and goat species but is missing in other species and is replaced with a His-10 in the guinea pig species. The only exposed tryptophan residue was Trp-104, which cross-relaxed to nearby Trp-60. Cross-polarization to the latter residue was proven by taking free induction decays after extremely short light pulses (0.1 s). Furthermore, this cross-polarized Trp-60 resonance was absent in the guinea pig species, which has a substitution at position 60. The chemical shifts of these Trp residues were extremely similar in all species but rabbit, which has substituted His for a Tyr at position 103, situated close to both Trp-104 and -60. Three of the four tyrosines in bovine, goat, human, and guinea pig alpha-lactalbumin were exposed (the latter species has an extra Tyr in its sequence which was apparently not accessible). The rabbit species contains only two Tyr residues, the one which was not accessible (Tyr-50) being common to all species.

Probing Protein Structure by Solvent Perturbation of NMR Spectra

“…Above 320K, new emissive peaks are progressively observed at 7.12 (320 K), 6.75 (327 K) and at 6.53 ppm (335 K). Surprisingly, the spectrum at 335 K is similar to that obtained previously by Kaptein and Berliner at the same pH but at room temperature (Berliner and Kaptein, 1981). A close investigation showed that the protein used in that study (no.…”

Expression, Folding, and Characterization of Small Proteins with Increasing Disulfide Complexity by a pT7-7-Derived Phagemid

“…Galactosyl transferase was isolated as previously described (12). Galactosyl transferase assays formatted for 96-well plates were performed as described by Fitzgerald et al (13).…”

CIDNP study of the aromatic side chain interactions in myotoxina

“…The basic process involves a radical pair formation step by reversible H-atom transfer (Muszkat and Weinstein, 1976) from a peptide side chain to a dye triplet. Protein CIDNP has been applied to peptides and proteins (see, e.g., Muszkat, 1977;Muszkat et al, , 1982Muszkat et al, , 1983Muszkat et al, , 1984aGitlin et al, 1989;Berliner and Kaptein, 1981;Berliner et al, 1990). It was applied some time ago in the study of the conformations of elapid snake cr-neurotoxins (Muszkat et al, 1984b;Muszkat, 1993) and of the membranotoxins (Muszkat et al, 1984b).…”

“…The prime experimental approach we use in these studies is protein CIDNP spectroscopy (photochemically induced dynamic nuclear polarization) (see, e.g., Muszkat, 1977;Muszkat et al, , 1982Muszkat et al, , 1983Muszkat et al, , 1984aBerliner and Kaptein, 1981;Berliner et al, 1990), which provides detailed structural and accessibility information, and ensures enhanced sensitivity specific for the CIDNP active side chains. Myotoxin a damages the sarcoplasmic reticulum (SR) membrane by associating with its two components, the Ca 2 § ATPase and the 53GP glycoprotein (Volpe et al, 1986;Utaisincharoen et al, 1991;Tu et al, 1992).…”