The reaction of alpha-crystallin with the cross-linker 3,3'-dithiobis(sulfosuccinimidyl propionate) demonstrates close proximity of the C termini of alphaA and alphaB in the native assembly.
The chaperone-like activity of human lens alpha-crystallin in inhibiting the aggregation of denatured proteins suggests a role for alpha-crystallin in cataract prevention. Although a variety of techniques have generated structural information relevant to its chaperone-like activity, the size and heterogeneity of alpha-crystallin have prevented determination of its crystal structure. Even though synthetic cross-linkers have provided considerable information about protein structures, they have not previously been used to study the proximity and orientation of subunits within human alpha-crystallin. Cross-linkers provide structural insight into proteins by binding the side chains of amino acids within close proximity. To identify the cross-linked residues, the modified protein is digested and the resulting peptides are analyzed by mass spectrometry. Analysis of products from the reaction of alpha-crystallin with 3,3'dithiobis(sulfosuccinimidyl propionate), DTSSP, identified several modifications to both alphaA and alphaB. The most structurally informative of these modifications was a cross-link between lysine 166 of alphaA and lysine 175 of alphaB. This cross-link provides experimental evidence supporting theoretical structural models that place the C termini of alphaA and alphaB within close proximity in the native aggregate.
Swaim
Catherine L
CL
Department of Chemistry, University of Nebraska, Lincoln, NE 68588-0304. jsmith8@unl.edu
Smith
David L
DL
Smith
Jean B
JB
D000595:Amino Acid Sequence; D002648:Child; D003432:Cross-Linking Reagents / Q000737:chemistry*; D006801:Humans; D008239:Lysine / Q000737:chemistry; D025941:Protein Interaction Mapping; D013388:Succinimides / Q000737:chemistry*; D038202:alpha-Crystallin A Chain / Q000737:chemistry* / Q000378:metabolism; D038203:alpha-Crystallin B Chain / Q000737:chemistry* / Q000378:metabolism
D003432:Cross-Linking Reagents; D013388:Succinimides; D038202:alpha-Crystallin A Chain; D038203:alpha-Crystallin B Chain; C035759:3,3'-dithiobis(sulfosuccinimidyl propionate); D008239:Lysine