A study of beta-casein tertiary structure by intramolecular crosslinking and mass spectrometry.
The objective of this study was to obtain experimental evidence to extend the discussion on the 3-D structure of beta-casein (beta-CN). The approach involved the preparation of homobifunctional crosslinkers, bis(sulfosuccinimidyl) derivatives of dicarboxylic acids of several lengths, which specifically react with primary amines of lysinyl residues or the N-terminal in the protein. The intramolecular crosslinks formed were determined by enzymatic digestion and by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry combined with comparison against the theoretical digestion patterns. This procedure allowed the measurement of distances between the crosslinked residues. Ten different masses arising from 8 different specific intramolecular crosslinks were identified. Of these, 5 crosslinks were in good agreement with a published model (Kumosinski et al., 1993). Two other crosslinks each connected 2 residues that are much closer together, according to the model, than the maximum length of the crosslink. However, one of the crosslinks apparently connected 2 residues that are predicted by the model to be 16.7 A farther apart than the crosslink's stretched length. This disparity might be explained by structural flexibility. The structure expressed by the model is probably one of several energetically favorable conformations of the beta-CN molecule, whose structure is best described as rheomorphic rather than either a fixed structure or a random coil.
Department of Food Science, University of Guelph, ON, Canada N1G 2W1.
Y D
YD
Livney
A L
AL
Schwan
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Dalgleish
D000818:Animals; D002364:Caseins / Q000032:analysis* / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D013058:Mass Spectrometry / Q000662:veterinary; D008892:Milk / Q000737:chemistry*; D008970:Molecular Weight; D010316:Particle Size; D017434:Protein Structure, Tertiary; D021241:Spectrometry, Mass, Electrospray Ionization / Q000662:veterinary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000662:veterinary
D002364:Caseins