Molecular and structural characterization of the heat-resistant thyroxine-binding globulin-Chicago.
Thyroxine-binding globulin (TBG) is the main transport protein for thyroxine (T4) in blood. It shares considerable sequence homology with alpha 1-antitrypsin (AT) and other members of the serine proteinase inhibitor (serpin) superfamily of proteins. The crystallographic structure of AT has been determined and was found to represent the archetype of the serpins. This model has been used for structure-function correlations of TBG. Sequence analysis of the heat-resistant variant TBG-Chicago (TBG-CH) revealed a substitution of the normal tyrosine 309 with phenylalanine. For further analysis, vectors containing the coding regions of normal TBG (TBG-N) and TBG-CH were constructed, transcribed in vitro, and expressed in Xenopus oocytes. Both TBGs were secreted into the culture medium and could not be distinguished by gel electrophoresis. Scatchard analysis of T4 binding to TBG-N and -CH revealed no significant differences in binding affinity. The rate of heat denaturation of TBGs was determined by measurement of residual T4 binding capacity after incubation at 60 degrees C for various periods of time. The half-life values of denaturation of TBG-N and -CH were 7 and 132 min, respectively. The tyrosine 309 to phenylalanine substitution of TBG-CH involves a highly conserved phenylalanine residue of the serpins. The respective phenylalanine 312 of AT ties the alpha-helix hI1 to the molecule, thus stabilizing the tertiary structure. A substitution with tyrosine would disrupt this interaction. Accordingly, stabilization of the TBG molecule by replacement of tyrosine with phenylalanine in position 309 causes the increased heat stability of TBG-CH.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D005260:Female; D014644:Genetic Variation; D015698:Genomic Library; D006801:Humans; D007962:Leukocytes / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D009865:Oocytes / Q000502:physiology; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D012156:Reticulocytes / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013974:Thyroxine / Q000378:metabolism; D013975:Thyroxine-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D014158:Transcription, Genetic; D014982:Xenopus laevis; D000515:alpha 1-Antitrypsin / Q000737:chemistry
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D013975:Thyroxine-Binding Proteins; D000515:alpha 1-Antitrypsin; C050338:thyroxine-binding globulin Chicago; D013974:Thyroxine