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1.
Proc Natl Acad Sci U S A ; 103(7): 2052-6, 2006 Feb 14.
Article in English | MEDLINE | ID: mdl-16461889

ABSTRACT

Fluorescent proteins have proven to be excellent reporters and biochemical sensors with a wide range of applications. In a split form, they are not fluorescent, but their fluorescence can be restored by supplementary protein-protein or protein-nucleic acid interactions that reassemble the split polypeptides. However, in prior studies, it took hours to restore the fluorescence of a split fluorescent protein because the formation of the protein chromophore slowly occurred de novo concurrently with reassembly. Here we provide evidence that a fluorogenic chromophore can self-catalytically form within an isolated N-terminal fragment of the enhanced green fluorescent protein (EGFP). We show that restoration of the split protein fluorescence can be driven by nucleic acid complementary interactions. In our assay, fluorescence development is fast (within a few minutes) when complementary oligonucleotide-linked fragments of the split EGFP are combined. The ability of our EGFP system to respond quickly to DNA hybridization should be useful for detecting the kinetics of many other types of pairwise interactions both in vitro and in living cells.


Subject(s)
DNA/chemistry , Green Fluorescent Proteins/chemistry , Green Fluorescent Proteins/genetics , Spectrometry, Fluorescence/methods , Fluorescence , Nucleic Acid Hybridization , Oligonucleotides/chemistry , Protein Folding , Sequence Deletion
2.
Structure ; 10(4): 557-67, 2002 Apr.
Article in English | MEDLINE | ID: mdl-11937060

ABSTRACT

S100A6 is a member of the S100 family of Ca(2+) binding proteins, which have come to play an important role in the diagnosis of cancer due to their overexpression in various tumor cells. We have determined the crystal structures of human S100A6 in the Ca(2+)-free and Ca(2+)-bound states to resolutions of 1.15 A and 1.44 A, respectively. Ca(2+) binding is responsible for a dramatic change in the global shape and charge distribution of the S100A6 dimer, leading to the exposure of two symmetrically positioned target binding sites. The results are consistent with S100A6, and most likely other S100 proteins, functioning as Ca(2+) sensors in a way analogous to the prototypical sensors calmodulin and troponin C. The structures have important implications for our understanding of target binding and cooperativity of Ca(2+) binding in the S100 family.


Subject(s)
Calcium/metabolism , Cell Cycle Proteins , Protein Structure, Tertiary , S100 Proteins/chemistry , S100 Proteins/metabolism , Amino Acid Sequence , Crystallography, X-Ray , Dimerization , Humans , Models, Molecular , Molecular Sequence Data , Protein Binding , S100 Calcium Binding Protein A6 , S100 Proteins/genetics
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