Reece L J, Nichols R, Ogden R C, Howell E E
Department of Biochemistry, University of Tennessee, Knoxville 37996-0840.
Biochemistry. 1991 Nov 12;30(45):10895-904. doi: 10.1021/bi00109a013.
R67 dihydrofolate reductase (DHFR) is a novel protein that provides clinical resistance to the antibacterial drug trimethoprim. The crystal structure of a dimeric form of R67 DHFR indicates the first 16 amino acids are disordered [Matthews et al. (1986) Biochemistry 25, 4194-4204]. To investigate whether these amino acids are necessary for protein function, the first 16 N-terminal residues have been cleaved off by chymotrypsin. The truncated protein is fully active with kcat = 1.3 s-1, Km(NADPH) = 3.0 microM, and Km(dihydrofolate) = 5.8 microM. This result suggests the functional core of the protein resides in the beta-barrel structure defined by residues 27-78. To study this protein further, synthetic genes coding for full-length and truncated R67 DHFRs were constructed. Surprisingly, the gene coding for truncated R67 DHFR does not produce protein in vivo or confer trimethoprim resistance upon Escherichia coli. Therefore, the relative stabilities of native and truncated R67 DHFR were investigated by equilibrium unfolding studies. Unfolding of dimeric native R67 DHFR is protein concentration dependent and can be described by a two-state model involving native dimer and unfolded monomer. Using absorbance, fluorescence, and circular dichroism techniques, an average delta GH2O of 13.9 kcal mol-1 is found for native R67 DHFR. In contrast, an average delta GH2O of 11.3 kcal mol-1 is observed for truncated R67 DHFR. These results indicate native R67 DHFR is 2.6 kcal mol-1 more stable than truncated protein. This stability difference may be part of the reason why protein from the truncated gene is not found in vivo in E. coli.
R67二氢叶酸还原酶（DHFR）是一种新型蛋白质，它赋予了对抗菌药物甲氧苄啶的临床抗性。R67 DHFR二聚体形式的晶体结构表明，前16个氨基酸是无序的[马修斯等人（1986年），《生物化学》25卷，4194 - 4204页]。为了研究这些氨基酸对于蛋白质功能是否必要，用胰凝乳蛋白酶切除了前16个N端残基。截短的蛋白质具有完全活性，催化常数（kcat） = 1.3 s-1，NADPH的米氏常数（Km） = 3.0微摩尔，二氢叶酸的米氏常数（Km） = 5.8微摩尔。这一结果表明，该蛋白质的功能核心位于由27 - 78位残基定义的β桶结构中。为了进一步研究这种蛋白质，构建了编码全长和截短的R67 DHFR的合成基因。令人惊讶的是，编码截短的R67 DHFR的基因在体内不产生蛋白质，也不能赋予大肠杆菌对甲氧苄啶的抗性。因此，通过平衡去折叠研究考察了天然和截短的R67 DHFR的相对稳定性。二聚体天然R67 DHFR的去折叠依赖于蛋白质浓度，并且可以用涉及天然二聚体和去折叠单体的两态模型来描述。使用吸光度、荧光和圆二色性技术，发现天然R67 DHFR的平均水相吉布斯自由能变化（ΔGH2O）为13.9千卡/摩尔。相比之下，截短的R67 DHFR的平均水相吉布斯自由能变化（ΔGH2O）为11.3千卡/摩尔。这些结果表明，天然R67 DHFR比截短的蛋白质稳定2.6千卡/摩尔。这种稳定性差异可能是截短基因的蛋白质在大肠杆菌体内未被发现的部分原因。
