Probing alpha-crystallin structure using chemical cross-linkers and mass spectrometry.
OBJECTIVE
Alternatives to X-ray crystallographic techniques are needed to probe the structure of the hetero-oligomeric lens protein alpha-crystallin. We utilized mass spectrometry for 3 dimensional analysis (MS3D) to study the quaternary structural characteristics of this important lens protein and molecular chaperone.


METHODS
We have employed two types of chemical cross-linkers to probe key protein-protein and protein-solvent interactions of alpha-crystallin using MS3D. Native alpha-crystallin was exposed to 3,3'-dithiobis[sulfosuccinimidyl propionate] (DTSSP) and the common fixative, formaldehyde. The reaction products were denatured and enriched in cross-linked and modified species using size exclusion chromatography. Tryptic digests of these fractions were purified using reverse phase HPLC and analyzed by both electrospray and matrix assisted laser desorption mass spectrometry. Comprehensive spectra for each C18 fraction were screened for ions with mass unique to each chemical treatment and candidate sequences matching the experimental data were assigned using MS3D "Links" and "ASAP" software. Selected ions were sequenced by collision induced dissociation.


RESULTS
Peptides including residues 164-175 of alphaB-crystallin and residues 1-99 of alphaA-crystallin were modified by formaldehyde and partially hydrolyzed DTSSP. Peptides containing modified lysines 11, 78, and 99 of alphaA-crystallin were sequenced and the modified amino acids identified. In addition, ions corresponding to intramolecular and/or intermolecular cross-links were assigned a sequence based on two criteria. First, the mass values observed were unique to a single cross-linking experiment and were not present in a control where no cross-linker was utilized. Second, two unique ions detected from different cross-linking experiments were correlated in that the structures assigned to the masses were equivalent apart from the structure of the cross-linker. One such correlation was found involving lysine121, within the "highly conserved alpha-crystallin domain" of alphaB-crystallin, cross-linked to either lysine11 or lysine99 of alphaA-crystallin. Another two independent correlations involving lysine72 of alphaB-crystallin were found that indicate cross-linking of two subunits of alphaB-crystallin through this same residue.


CONCLUSIONS
Sequences of peptides modified by partially hydrolyzed DTSSP and formaldehyde provide experimental evidence for models of alpha-crystallin quaternary structure that suggest a similar tertiary fold for both alphaA-crystallin and alphaB-crystallin. Analogous to multiple phosphorylations along the N-terminus of alphaB-crystallin, our data indicate that the same region of alphaA-crystallin, up to and including lysine99 is also relatively accessible to modification despite its hydrophobicity. Mass correlation between experiments using different reagents suggests that cross-linking occurred between N-termini of adjacent subunits of alphaB-crystallin in the native complex in support of the amphiphilic, toroidal, or "open micelle" models. In addition, multiple cross-links involving lysine121 of the so called "dimer interface" region within the "highly conserved alpha-crystallin domain" indicate that this region is a site of inter-subunit contacts in the native context.
Peterson
James J
JJ
Kansas State University, Department of Biology, Manhattan 66506, USA.
Young
Malin M
MM
Takemoto
Larry J
LJ
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D005557:Formaldehyde / Q000494:pharmacology; D007908:Lens, Crystalline / Q000737:chemistry / Q000196:embryology; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010446:Peptide Fragments / Q000737:chemistry; D020836:Protein Structure, Quaternary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013388:Succinimides / Q000494:pharmacology; D038201:alpha-Crystallins / Q000737:chemistry* / Q000187:drug effects / Q000302:isolation & purification
D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D013388:Succinimides; D038201:alpha-Crystallins; D005557:Formaldehyde; C035759:3,3'-dithiobis(sulfosuccinimidyl propionate)