THE EXPRESSION, CHARACTERIZATION, AND CRYSTALLIZATION OF WILD-TYPE AND SELENOMETHIONYL HUMAN CHORIONIC-GONADOTROPIN

Although the glycoprotein hormone hCG was crystallized over 4 yr ago, it is only now that three-dimensional structural information is availa ble. This manuscript reports the method for successful production of m odified expressed hormone, the characteristics of the crystallized pro tein, and unexpected observations during the crystallization process. Two different routes of solution to the structure of hCG were followed . The first was based on the traditional method of heavy atom isomorph ous replacement, and the second was the more novel method of expressin g the protein with selenomethionine substituting for methionine and ap plying multiwavelength anomalous diffraction analysis. Selenomethionyl hCG was employed to successfully grow the crystals used for the solut ion of the structure of hCG after partial deglycosylation by hydrogen fluoride (HF) treatment. The selenomethionyl hCG proved to be more hyd rophobic than the expressed form of native hCG. Furthermore, expressed forms of hCG that were deglycosylated by HF proved to be more intact and less susceptible to peptide bond cleavages during the crystallizat ion process than the urinary form of HF-treated hCG studied previously . It was found that addition of reducing agent during the crystallizat ion period was necessary for the growth of crystals of HF-treated sele nomethionyl hCG suitable for diffraction studies. Growth of crystals o f HF-treated expressed hCG were accelerated by the addition of dithiot hreitol, but would successfully grow without reductant. HPLC analysis of the HF-treated hormones before and during the crystallization proce ss was used to identify alterations in the molecules, including oxidat ion and aggregation, both of which may affect the growth of crystals.