Albeck S, Schreiber G
Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel.
Biochemistry. 1999 Jan 5;38(1):11-21. doi: 10.1021/bi981772z.
BLIP is a secreted protein from Streptomyces clavuligerus that inhibits a wide range of beta-lactamases. Here we investigate the tight interaction of BLIP, expressed heterologousely in E. coli, with TEM-1. Kinetic and thermodynamic constants were determined using methods with the proteins either in a homogeneous or in a heterogeneous phase. While values of Delta DeltaG(mut-wt) are similar whether measured by fluorescence quench, enzyme inhibition, or surface plasmon resonance, absolute values of DeltaG and kinetic constants vary. Association and dissociation rate constants of 10(5) M-1 s-1 and 10(-)4 s-1, respectively, and a nanomolar affinity were determined for the wild-type proteins. The highest affinity is measured at pH 7.5, with a decreasing association rate constant at higher pH values, and an increasing dissociation rate constant at lower pH values. The marginal effect of salt on the kinetics of binding, as well as the calculated surface potentials, suggests a limited role for electrostatic forces in guiding this reaction. Still, mutations of interfacial residues affect the rate of association significantly, so that an increase in the net negative charge on either protein reduces the association rate constant. We show that simple electrostatic rules can explain this behavior. BLIP inhibits the catalytic activity of TEM-1 by binding its active site. Yet, mutations of active site residues on TEM-1 only have a moderate though cooperative effect on the binding energy. This can be explained in light of the peripheral location of the active site in the interface between the two proteins.
BLIP是来自棒状链霉菌的一种分泌蛋白，可抑制多种β-内酰胺酶。在此，我们研究了在大肠杆菌中异源表达的BLIP与TEM-1之间的紧密相互作用。使用蛋白质处于均相或异相的方法测定了动力学和热力学常数。虽然通过荧光猝灭、酶抑制或表面等离子体共振测量的ΔΔG（突变体-野生型）值相似，但ΔG的绝对值和动力学常数有所不同。野生型蛋白质的缔合和解离速率常数分别为10⁵ M⁻¹ s⁻¹和10⁻⁴ s⁻¹，亲和力为纳摩尔级。在pH 7.5时测得的亲和力最高，在较高pH值时缔合速率常数降低，在较低pH值时解离速率常数增加。盐对结合动力学的边际效应以及计算出的表面电位表明静电力在引导该反应中作用有限。然而，界面残基的突变会显著影响缔合速率，因此任何一种蛋白质上净负电荷的增加都会降低缔合速率常数。我们表明简单的静电规则可以解释这种行为。BLIP通过结合TEM-1的活性位点来抑制其催化活性。然而，TEM-1上活性位点残基的突变对结合能仅具有适度但协同的影响。鉴于活性位点在两种蛋白质之间界面的外围位置，这一点可以得到解释。
