Probing α-crystallin structure using chemical cross-linkersand mass spectrometry

[摘要] Purpose: Alternatives to X-ray crystallographic techniques areneeded to probe the structure of the hetero-oligomeric lens proteinα-crystallin. We utilized mass spectrometry for 3 dimensionalanalysis (MS3D) to study the quaternary structural characteristics ofthis important lens protein and molecular chaperone.Methods: We have employed two types of chemical cross-linkers toprobe key protein-protein and protein-solvent interactions ofα-crystallin using MS3D. Native α-crystallin was exposed to3,3'-dithiobis[sulfosuccinimidyl propionate] (DTSSP) and the commonfixative, formaldehyde. The reaction products were denatured andenriched in cross-linked and modified species using size exclusionchromatography. Tryptic digests of these fractions were purified usingreverse phase HPLC and analyzed by both electrospray and matrix assistedlaser desorption mass spectrometry. Comprehensive spectra for eachC18 fraction were screened for ions with mass unique to eachchemical treatment and candidate sequences matching the experimentaldata were assigned using MS3D "Links" and "ASAP" software. Selected ionswere sequenced by collision induced dissociation.Results: Peptides including residues 164-175 of αB-crystallinand residues 1-99 of αA-crystallin were modified by formaldehydeand partially hydrolyzed DTSSP. Peptides containing modified lysines 11,78, and 99 of αA-crystallin were sequenced and the modified aminoacids identified. In addition, ions corresponding to intramolecularand/or intermolecular cross-links were assigned a sequence based on twocriteria. First, the mass values observed were unique to a singlecross-linking experiment and were not present in a control where nocross-linker was utilized. Second, two unique ions detected fromdifferent cross-linking experiments were correlated in that thestructures assigned to the masses were equivalent apart from thestructure of the cross-linker. One such correlation was found involvinglysine121, within the "highly conserved α-crystallin domain"of αB-crystallin, cross-linked to either lysine11 orlysine99 of αA-crystallin. Another two independentcorrelations involving lysine72 of αB-crystallin were foundthat indicate cross-linking of two subunits of αB-crystallinthrough this same residue.Conclusions: Sequences of peptides modified by partially hydrolyzedDTSSP and formaldehyde provide experimental evidence for models ofα-crystallin quaternary structure that suggest a similar tertiaryfold for both αA-crystallin and αB-crystallin. Analogous tomultiple phosphorylations along the N-terminus of αB-crystallin,our data indicate that the same region of αA-crystallin, up to andincluding lysine99 is also relatively accessible to modificationdespite its hydrophobicity. Mass correlation between experiments usingdifferent reagents suggests that cross-linking occurred betweenN-termini of adjacent subunits of αB-crystallin in the nativecomplex in support of the amphiphilic, toroidal, or "open micelle"models. In addition, multiple cross-links involving lysine121 ofthe so called "dimer interface" region within the "highly conservedα-crystallin domain" indicate that this region is a site ofinter-subunit contacts in the native context.

[学科分类] 生物化学/生物物理