X-RAY ANALYSIS OF HEAT-RESISTANT MUTANTS OF HU PROTEIN Takahiro Tominaga*, Shunsuke Kawamura, Makoto Kimura, Atsushi Nakagawa*, Isao Tanaka*, *Division of Biological Science, Graduate School of Science, Hokkaido University, Sapporo, 060, Japan, Laboratory of Biochemistry, Faculty of Agriculture, Kyushu University, Fukuoka, 812, Japan

HU protein is ubiquitous in eubacterial kingdom. This small basic protein of molecular weight 9,500 is also known as DNA bending protein. Its amino acid sequence is highly conserved, e.g., B.stearothermophilus and B.subtilis have only 12 different residues. However the HU proteins of the two species have quite different thermal denaturation temperature (B.st 65deg.C, B.su 48deg.C ). To elucidate the relationship between structure and thermostability of B.st and B.su HUs, we constructed T13A, T33L, E34D, and K38N mutants where the amino acids in BstHU were changed to the corresponding ones in BsuHU. Mutant proteins were expressed, and purified. Tm values of each mutant were determined by circular dichroism (CD) spectra. The change of Tm value ([[Delta]]Tm) from native BstHU was +3.1deg.C(T13A), +1.7deg.C(T33L), -2.0deg.C(E34D), and -4.0deg.C(K38N) respectively. Each mutant was crystallized for X-ray structure analysis. The crystallization conditions of each mutant were 40% MPD, 80mM phosphate(pH 8.0), and protein concentration was 25mg/ml. E34D mutant was crystallized as above condition plus 6% dioxane. X-ray experiments were done for T13A mutant, and this crystal diffracts at least 2.5Å. Cell parameters are a=65.5 b=37.3 c=65.5 and ß=114.5 (that is isomorphous to native B.st HU crystal), and overall merging-R was 3.8%. Structure determination was done by a molecular replacement. For model structure, we used the coordinates of native B.st HU. Refinement of this mutant structure was done with X-plor and current R-factor is 20%. We discuss the difference of thermal stability between native and mutant based on the structure.