Williams Neal K, Liepinsh Edvards, Watt Stephen J, Prosselkov Pavel, Matthews Jacqueline M, Attard Phil, Beck Jennifer L, Dixon Nicholas E, Otting Gottfried
Research School of Chemistry, Australian National University, Canberra, ACT 0200, Australia.
J Mol Biol. 2005 Mar 4;346(4):1095-108. doi: 10.1016/j.jmb.2004.12.037. Epub 2005 Jan 18.
A mutant version of the N-terminal domain of Escherichia coli DnaB helicase was used as a model system to assess the stabilization against unfolding gained by covalent cyclization. Cyclization was achieved in vivo by formation of an amide bond between the N and C termini with the help of a split mini-intein. Linear and circular proteins were constructed to be identical in amino acid sequence. Mutagenesis of Phe102 to Glu rendered the protein monomeric even at high concentration. A difference in free energy of unfolding, DeltaDeltaG, between circular and linear protein of 2.3(+/-0.5) kcal mol(-1) was measured at 10 degrees C by circular dichroism. A theoretical estimate of the difference in conformational entropy of linear and circular random chains in a three-dimensional cubic lattice model predicted DeltaDeltaG=2.3 kcal mol(-1), suggesting that stabilization by protein cyclization is driven by the reduced conformational entropy of the unfolded state. Amide-proton exchange rates measured by NMR spectroscopy and mass spectrometry showed a uniform, approximately tenfold decrease of the exchange rates of the most slowly exchanging amide protons, demonstrating that cyclization globally decreases the unfolding rate of the protein. The amide proton exchange was found to follow EX1 kinetics at near-neutral pH, in agreement with an unusually slow refolding rate of less than 4 min(-1) measured by stopped-flow circular dichroism. The linear and circular proteins differed more in their unfolding than in their folding rates. Global unfolding of the N-terminal domain of E.coli DnaB is thus promoted strongly by spatial separation of the N and C termini, whereas their proximity is much less important for folding.
大肠杆菌DnaB解旋酶N端结构域的突变体被用作模型系统，以评估通过共价环化获得的抗解折叠稳定性。借助分裂型小内含肽，通过在N端和C端之间形成酰胺键，在体内实现环化。构建的线性和环状蛋白质在氨基酸序列上相同。将Phe102突变为Glu，即使在高浓度下该蛋白质也呈单体状态。在10℃下，通过圆二色性测量环状和线性蛋白质之间的解折叠自由能差ΔΔG为2.3（±0.5）kcal/mol。在三维立方晶格模型中对线性和环状无规链构象熵差的理论估计预测ΔΔG = 2.3 kcal/mol，这表明蛋白质环化导致的稳定性是由未折叠状态下构象熵的降低驱动的。通过核磁共振光谱和质谱测量的酰胺质子交换率表明，最慢交换的酰胺质子的交换率均匀地降低了约10倍，这表明环化总体上降低了蛋白质的解折叠速率。发现在接近中性pH条件下，酰胺质子交换遵循EX1动力学，这与通过停流圆二色性测量的异常缓慢的重折叠速率（小于4 min⁻¹）一致。线性和环状蛋白质在解折叠方面的差异比在折叠速率方面的差异更大。因此，大肠杆菌DnaB N端结构域的全局解折叠受到N端和C端空间分离的强烈促进，而它们的接近程度对折叠的重要性要小得多。
