Mechanism of the Very Efficient Quenching of Tryptophan Fluorescence in Human γD- and γS-Crystallins: The γ-Crystallin Fold May Have Evolved To Protect Tryptophan Residues from Ultraviolet Photodamage†

Proteins exposed to UV radiation are subject to irreversible photodamage through covalent modification of tryptophans (Trps) and other UV-absorbing amino acids. Crystallins, the major protein components of the vertebrate eye lens that maintain lens transparency, are exposed to ambient UV radiation throughout life. The duplicated β-sheet Greek key domains of β- and γ-crystallins in humans and all other vertebrates each have two conserved buried Trps. Experiments and computation showed that the fluorescence of these Trps in human γD-crystallin is very efficiently quenched in the native state by electrostatically enabled electron transfer to a backbone amide [Chen et al. (2006) Biochemistry 45, 11552−11563]. This dispersal of the excited state energy would be expected to minimize protein damage from covalent scission of the excited Trp ring. We report here both experiments and computation showing that the same fast electron transfer mechanism is operating in a different crystallin, human γS-crystallin. Examination of solved structures of other crystallins reveals that the Trp conformation, as well as favorably oriented bound waters, and the proximity of the backbone carbonyl oxygen of the n − 3 residues before the quenched Trps (residue n), are conserved in most crystallins. These results indicate that fast charge transfer quenching is an evolved property of this protein fold, probably protecting it from UV-induced photodamage. This UV resistance may have contributed to the selection of the Greek key fold as the major lens protein in all vertebrates.National Eye Institute (Grant EY 015834

Development of a DNA microarray for detection of expressed equine classical MHC class I sequences in a defined population

Development of an accurate and efficient molecular-based equine MHC class I typing method would facilitate the study of T lymphocyte immune responses in horses. Here, a DNA microarray was designed to detect expressed classical MHC class I genes comprising serologically defined equine leukocyte antigen (ELA)-A haplotypes represented in a closed Arabian horse breeding herd. Initially, cloning and sequencing of RT-PCR products were used to identify sequences associated with the ELA-A1, A4, and W11 haplotypes, and one undefined haplotype, in six horses. Subsequently, sequence-specific, conserved (positive control), and random nucleotide (negative control) 23- to 27-mer oligonucleotide microarray probes were designed and spotted onto an epoxy-coated masked slide using a robotic arrayer. Bulk RT-PCR products from each horse were biotinylated by nick translation, hybridized to the array, and detected using tyramide signal amplification. The microarray consistently detected eight of nine classical MHC class I transcripts and allowed ELA haplotypic associations to be made. Cloning and sequencing of RT-PCR products were then performed in a group of ELA disparate horses and ponies, in which six novel sequences were identified. This group was used to determine the specificity of the array. Overall, the microarray was more efficient than cloning and sequencing for detecting expressed classical MHC class I sequences in this defined population of horses, and was significantly more specific than serology. These results confirmed the utility of a microarray-based method for high-resolution MHC class I typing in the horse. With additional probes the array could be useful in a broader population