MOLECULAR AND STRUCTURAL CHARACTERIZATION OF THE HEAT-RESISTANT THYROXINE-BINDING GLOBULIN CHICAGO

Thyroxine-binding globulin (TBG) is the main transport protein for thy roxine (T-4) in blood, It shares considerable sequence homology with a lpha(1)-antitrypsin (AT) and other members of the serine proteinase in hibitor (serpin) superfamily of proteins. The crystallographic structu re of AT has been determined and was found to represent the archetype of the serpins, This model has been used for structure-function correl ations of TBG. Sequence analysis of the heat-resistant variant TBG-Chi cago (TBG-CH) revealed a substitution of the normal tyrosine 309 with phenylalanine. For further analysis, vectors containing the coding reg ions 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 electrophore sis. Scatchard analysis of T-4 binding to TBG-N and -CH revealed no si gnificant differences in binding affinity. The rate of heat denaturati on of TBGs was determined by measurement of residual T-4 binding capac ity 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 mo lecule, thus stabilizing the tertiary structure. A substitution with t yrosine would disrupt this interaction, Accordingly, stabilization of the TBG molecule by replacement of tyrosine with phenylalanine in posi tion 309 causes the increased heat stability of TBG-CH.