Substitution of glutamic acids for the conserved lysines in the alpha domain affects metal binding in both the alpha and beta domains of mammalian metallothionein.

Lysine residues are highly conserved in mammalian metallothioneins (MTs). Recombinant mutant Chinese hamster MT2 in which all of the lysines (K) in the alpha-domain were substituted by glutamic acids (E) was assayed with, expressed in and purified from a cadmium sensitive strain of yeast Saccharomyces cerevisiae. Circular dichroism analyses of the mutated protein, mutein K43,51,56E, revealed that the overall structure remained unchanged. However, a 1-D 113Cd NMR study detected significant differences in the chemical shifts of the corresponding resonances between wild type protein and the recombinant mutein. Reduction of integrated intensity in the NMR spectra was also observed for resonances from the four-metal cluster (I and V-VII) in the alpha-domain of the mutein. At various temperatures, facile intermolecular exchange of metals in the beta-domain of the mutein was also observed, which was unexpected and was different from wild type. Our results thus demonstrate that replacing all three lysines by glutamic acids in the alpha-domain changed metal-thiolate interactions in both domains of the recombinant mutein. This may explain the reduced ability of the mutein to convey cadmium resistance. We propose that while the lysine residues in the alpha-domain of wild type MT are not critical for maintaining protein structure, they play a role in regulating the microenvironment and stability of both metal-binding clusters, a feature critical to metal detoxification.

Replacement of all alpha-domain lysines with glutamates reduces metallothionein detoxification function.

Mammalian metallothioneins (MTs) possess eight highly conserved lysine residues, including three in each of two metal binding domains. We used site-directed mutagenesis to replace these intradomain lysines in Chinese hamster ovary MT2 with glutamic acid and/or glutamine. These mutant MTs were expressed in a metal sensitive yeast host. One mutant which had all three lysines in the alpha-domain replaced by glutamates (K43,51,56E) exhibited a reduced ability, relative to native MT, to protect yeast transformants against otherwise toxic levels of cadmium. This triply substituted mutant also exhibited anomalous migration on a non-denaturing gel relative to wild type MT and other MT lysine mutants, suggesting that the intradomain lysines are important in maintaining the conformational integrity of MT.

Complete cDNA sequence of a human dioxin-inducible mRNA identifies a new gene subfamily of cytochrome P450 that maps to chromosome 2.

Previously, levels of a novel human mRNA, detected by a recombinant cDNA designated clone 1, were shown to be increased 50-fold in response to treatment of a keratinocyte cell line with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), in part as a function of increased rates of gene transcription (Sutter, T.R., Guzman, K., Dold, K.M., and Greenlee, W.F. (1991) Science 254, 415-418). Here we report the complete corresponding 5.1-kilobase cDNA sequence. A single open reading frame that predicts a protein of 543 amino acid residues was determined by computer-assisted analysis of the cDNA sequence. This predicted protein identifies a new gene subfamily of cytochrome P450, cytochrome P4501B1 (CYP1B1), that maps to human chromosome 2. Southern blot analysis of genomic DNA indicates that the human CYP1B subfamily is likely to contain only this single gene. Northern blot analysis of RNA isolated from primary cultures of normal human epidermal keratinocytes showed approximately 100-fold increased levels of the CYP1B1 mRNA after treatment with 10 nM TCDD for 24 h. Low levels of constitutive CYP1B1 mRNA were detected in 15 different human tissue samples. These results indicate that CYP1B1 is expressed in many normal human tissues and advance our understanding of the complexity of a gene family of cytochromes P450 whose expression is altered by TCDD.

Metallothionein detoxification function is impaired by replacement of both conserved lysines with glutamines in the hinge between the two domains.

Mammalian metallothioneins (MTs) possess eight highly conserved lysine residues, two of which constitute the hinge between two metal binding domains. By site-directed mutagenesis and recombinant DNA techniques, we replaced the interdomain lysines in Chinese hamster ovary MT2 with all possible combinations of glutamic acid and/or glutamine. The resultant MTs were expressed and assayed for detoxification function in a transformed yeast system. Results showed that these mutant MTs, like the native protein, bound seven atoms of divalent metal per molecule and conferred cadmium resistance to a metal-sensitive yeast host. Replacement of one or both of the lysines in the interdomain region was inconsequential to the structure and function of MT, unless both substituted residues were uncharged. When both lysines were replaced by glutamine (K30,31Q), a reduction in the ability of MT to protect yeast transformants against otherwise toxic levels of cadmium was observed. This diminished metal detoxification capacity was due to a decrease in the steady-state level of MT. These results suggest that at least one charged amino acid must be present in the hinge for the proper expression of MT.

Chemical structure of the hexapeptide chromophore of the Aequorea green-fluorescent protein.

The green-fluorescent proteins (GFP) are a unique class of proteins involved in bioluminescence of many cnidaria. The GFPs serve as energy-transfer acceptors, receiving energy from either a luciferase-oxyluciferin complex or a Ca(2+)-activated photoprotein, depending on the organism. Upon mechanical stimulation of the organism, GFP emits green light spectrally identical to its fluorescence emission. These highly fluorescent proteins are unique due to the nature of the covalently attached chromophore, which is composed of modified amino acid residues within the polypeptide. This report describes the characterization of the Aequorea victoria GFP chromophore which is released as a hexapeptide upon digestion of the protein with papain. The chromophore is formed upon cyclization of the residues Ser-dehydroTyr-Gly within the polypeptide. The chromophore structure proposed here differs from that described by Shimomura [(1979) FEBS Lett. 104, 220] in a number of ways.