Functional expression in Escherichia coli of the mitotic regulator proteins p24ran and p45rcc1 and fluorescence measurements of their interaction.
The gene products for the mitotic regulator genes RCC1 and Ran, p45rcc1 and p24ran, were expressed in Escherichia coli, purified in large amounts, and characterized for their biochemical properties. p24ran binds guanine nucleotide as a 1:1 complex, which is only slowly released from the protein. p45rcc1 catalyzes the exchange of nucleotide bound to the guanine nucleotide binding protein p24ran in the same way as the protein purified from HeLa cells. Likewise, the nucleotide dissociation from HeLa cell-derived p24ran protein is equally efficient with recombinant and nonrecombinant proteins. The recombinant proteins form a strong complex which contains no bound nucleotide. The kinetics of nucleotide exchange on p24ran in the presence or absence of p45rcc1 can be conveniently monitored either by the direct tryptophan fluorescence of p24ran or by fluorescence energy transfer measurements involving fluorescent nucleotides.
Klebe
C
C
Abteilung Biophysik, Max-Planck-Institut für medizinische Forschung, Heidelberg, FRG.
Nishimoto
T
T
Wittinghofer
F
F
D001483:Base Sequence; D018797:Cell Cycle Proteins; D003001:Cloning, Molecular; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D015870:Gene Expression; D005809:Genes, Regulator; D020662:Guanine Nucleotide Exchange Factors; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008938:Mitosis; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D013050:Spectrometry, Fluorescence; D020931:ran GTP-Binding Protein
D018797:Cell Cycle Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D020662:Guanine Nucleotide Exchange Factors; D009687:Nuclear Proteins; C062521:RCC1 protein, human; D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D020931:ran GTP-Binding Protein