The ability to image biochemical and phenotypical changes at the molecular level is imperative for the investigation and understanding of the molecular mechanisms that govern health and disease. Since 1962, the green fluorescent protein (GFP) has been the tool of choice for imaging tens of thousands of otherwise invisible proteins. Despite its role as one of the most important tools in contemporary bioscience, the sheer size of GFP (238 amino acids) can limit its applications. The labeling of shorter genetically expressed peptide tags (∼10 amino acids) with fluorogenic small molecules has emerged as a promising alternative.
This trend has prompted our group to screen millions of peptides from combinatorial one-bead one-compound (OBOC) libraries. In our case, these libraries contain 130 mM TentaGel beads each bearing ∼300 pmol of an unknown peptide. These libraries can be reacted with a fluorogenic small molecule and any peptide reacting with the molecule will cause the bead to fluoresce (termed a “hit”). Isolation of the bead hits allows the peptide to be sequenced, and further evaluated for use in protein visualization.
Our advances in synthesis and sequencing methodology has expanded the power of OBOC libraries to include short cysteine-rich peptides for applications such as biarsenical binding tags, metal-chelating species, and cysteine-rich pharmaceuticals. Our screens of cysteine-rich peptides have led to the discovery of several new reactive biarsenical motifs. Certainly these motifs serve to identify endogenous proteins targets. And, with optimization, could serve as better tags for fluorescent labeling in living cells.
We have also identified the first peptide tags that react selectively with 1,4-dicarbonyls to give fluorescent isoindoles. Currently, the biarsenical-tetracysteine system is state of the art for labeling living cells, but hinges non-covalent bonding to cytotoxic arsenic. These peptide tags that react selectively and covalently with 3-benzoyl-2-quinolinecarboxaldehyde (BQCA) represent progress towards the betterment of protein labeling, and ultimately, an understanding of the macromolecules that define us.
|Advisor:||Vranken, David L. Van|
|Commitee:||Prescher, Jennifer A., Rychnovsky, Scott D.|
|School:||University of California, Irvine|
|Department:||Chemistry - Ph.D.|
|School Location:||United States -- California|
|Source:||DAI-B 72/03, Dissertation Abstracts International|
|Subjects:||Cellular biology, Biochemistry|
|Keywords:||Biarsenical binding tags, Combinatorial libraries, Cysteine-rich pharmaceuticals, Fusion, Mcgee, Metal-chelating species, Peptides|
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