Marvin J S, Hellinga H W
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
Nat Struct Biol. 2001 Sep;8(9):795-8. doi: 10.1038/nsb0901-795.
Traditional approaches for increasing the affinity of a protein for its ligand focus on constructing improved surface complementarity in the complex by altering the protein binding site to better fit the ligand. Here we present a novel strategy that leaves the binding site intact, while residues that allosterically affect binding are mutated. This method takes advantage of conformationally distinct states, each with different ligand-binding affinities, and manipulates the equilibria between these conformations. We demonstrate this approach in the Escherichia coli maltose binding protein by introducing mutations, located at some distance from the ligand binding pocket, that sterically affect the equilibrium between an open, apo-state and a closed, ligand-bound state. A family of 20 variants was generated with affinities ranging from an approximately 100-fold improvement (7.4 nM) to an approximately two-fold weakening (1.8 mM) relative to the wild type protein (800 nM).
传统提高蛋白质对其配体亲和力的方法侧重于通过改变蛋白质结合位点以更好地契合配体，从而在复合物中构建更好的表面互补性。在此，我们提出一种新颖的策略，即保持结合位点不变，同时对变构影响结合的残基进行突变。该方法利用构象不同的状态，每种状态具有不同的配体结合亲和力，并操纵这些构象之间的平衡。我们通过引入位于距配体结合口袋一定距离处的突变，在大肠杆菌麦芽糖结合蛋白中证明了这种方法，这些突变在空间上影响开放的无配体状态和封闭的配体结合状态之间的平衡。相对于野生型蛋白（800 nM），产生了一个包含20个变体的家族，其亲和力范围从提高约100倍（7.4 nM）到减弱约两倍（1.8 mM）。
