Detection and structural features of the betaB2-B3-crystallin heterodimer by radical probe mass spectrometry (RP-MS).
The predilection of the beta-crystallin B2 subunit to interact with the betaB3 subunit rather than self associate is evident by the detection of the betaB2-B3-crystallin heterodimer by native gel electrophoresis and electrospray ionisation time-of-flight (ESI-TOF) mass spectrometry under non denaturing conditions. The complex has been detected for the first time and its molecular mass is measured to be 47,450 +/- 1 Da. Radical probe mass spectrometry (RP-MS) was subsequently applied to investigate the nature of the heterodimer through the limited oxidation of the subunits in the complex. Two peptide segments of the betaB2 subunit and six of the betaB3 subunit were found to oxidise, with far greater oxidation observed within the betaB3 versus the betaB2 subunit. This, and the observation that the oxidation data of betaB2 subunit is inconsistent with the structure of the betaB2 monomer, demonstrates that the protection of betaB2 is conferred by its association with betaB3 subunit within the heterodimer where only the residues of, and towards, its N-terminal domain remain exposed to solvent. The results suggest that the betaB2 subunit adopts a more compacted form than in its monomeric form in order for much of its structure to be enveloped by the betaB3 subunit within the heterodimer.
Diemer
Hélène
H
Laboratoire de Spectrométrie de Masse BioOrganique, IPHC-DSA, Université Louis Pasteur, Centre National de la Recherche Scientifique UMR7178, Strasbourg, France.
Atmanene
Cédric
C
Sanglier
Sarah
S
Morrissey
Bethny
B
Van Dorsselaer
Alain
A
Downard
Kevin M
KM
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D055503:Protein Multimerization; D014357:Trypsin / Q000378:metabolism; D038206:beta-Crystallin B Chain / Q000737:chemistry* / Q000378:metabolism
D010446:Peptide Fragments; D038206:beta-Crystallin B Chain; D014357:Trypsin