The Scripps Research Institute, La Jolla, CA 92037, USA.
FEBS Lett. 2011 Nov 16;585(22):3528-32. doi: 10.1016/j.febslet.2011.10.014. Epub 2011 Oct 20.
Dihydrofolate reductase (DHFR) is a well-studied drug target and a paradigm for understanding enzyme catalysis. Preparation of pure DHFR samples, in defined ligand-bound states, is a prerequisite for in vitro studies and drug discovery efforts. We use NMR spectroscopy to monitor ligand content of human and Escherichia coli DHFR (ecDHFR), which bind different co-purifying ligands during expression in bacteria. An alternate purification strategy yields highly pure DHFR complexes, containing only the desired ligands, in the quantities required for structural studies. Interestingly, ecDHFR is bound to endogenous THF while human DHFR is bound to NADP. Consistent with these findings, a designed "humanized" mutant of ecDHFR switches binding specificity in the cell.
二氢叶酸还原酶（DHFR）是一个研究得很好的药物靶点，也是理解酶催化的范例。制备纯 DHFR 样品，并使其处于特定配体结合状态，是体外研究和药物发现工作的前提。我们使用 NMR 光谱法监测人源和大肠杆菌源 DHFR（ecDHFR）的配体含量，在细菌中表达时，它们会结合不同的共纯化配体。另一种纯化策略可产生高纯度的 DHFR 复合物，仅含有所需的配体，且数量足以满足结构研究的需要。有趣的是，ecDHFR 与内源性 THF 结合，而人源 DHFR 与 NADP 结合。与这些发现一致的是，ecDHFR 的一种设计“人源化”突变体在细胞中改变了结合特异性。
