Goobes Rivka, Goobes Gil, Shaw Wendy J, Drobny Gary P, Campbell Charles T, Stayton Patrick S
Department of Bioengineering, University of Washington, Seattle, Washington 98195, USA.
Biochemistry. 2007 Apr 24;46(16):4725-33. doi: 10.1021/bi602345a. Epub 2007 Mar 29.
Salivary statherin is a highly acidic, 43 amino acid residue protein that functions as an inhibitor of primary and secondary crystallization of the biomineral hydroxyapatite. The acidic domain at the N-terminus was previously shown to be important in the binding of statherin to hydroxyapatite surfaces. This acidic segment is followed by a basic segment whose role is unclear. In this study, the role of the basic amino acids in the hydroxyapatite adsorption thermodynamics has been determined using isothermal titration calorimetry and equilibrium adsorption isotherm analysis. Single point mutations of the basic side chains to alanine lowered the binding affinity to the surface but did not perturb the maximal surface coverage and the adsorption enthalpy. The structural and dynamic properties of the single point mutants as characterized by solid-state NMR techniques were not altered either. Simultaneous replacement of all four basic amino acids with alanine lowered the adsorption equilibrium constant by 5-fold and the maximal surface coverage by nearly 2-fold. The initial exothermic phase of adsorption exhibited by native statherin is preserved in this mutant, along with the alpha-helical structure and the dynamic properties of the N-terminal domain. These results help to refine the two binding site model of statherin adsorption proposed earlier in our study of wild-type statherin (Goobes, R., Goobes, G., Campbell, C.T., and Stayton, P.S. (2006) Biochemistry 45, 5576-5586). The basic charges function to reduce protein-protein charge repulsion on the HAP surface, and in their absence, there is a considerable decrease in statherin packing density on the surface at binding saturation.
唾液磷蛋白是一种高度酸性的、由43个氨基酸残基组成的蛋白质，其功能是作为生物矿物质羟基磷灰石初级和次级结晶的抑制剂。先前已表明，N端的酸性结构域在磷蛋白与羟基磷灰石表面的结合中起重要作用。该酸性片段之后是一个作用尚不清楚的碱性片段。在本研究中，使用等温滴定量热法和平衡吸附等温线分析确定了碱性氨基酸在羟基磷灰石吸附热力学中的作用。碱性侧链单点突变为丙氨酸降低了对表面的结合亲和力，但未干扰最大表面覆盖率和吸附焓。通过固态核磁共振技术表征的单点突变体的结构和动力学性质也未改变。将所有四个碱性氨基酸同时替换为丙氨酸使吸附平衡常数降低了5倍，最大表面覆盖率降低了近2倍。天然磷蛋白表现出的初始放热吸附阶段在该突变体中得以保留，同时保留了N端结构域的α螺旋结构和动力学性质。这些结果有助于完善我们在野生型磷蛋白研究中（Goobes, R., Goobes, G., Campbell, C.T., and Stayton, P.S. (2006) Biochemistry 又译《生物化学》45, 5576 - 5586）早期提出的磷蛋白吸附的双结合位点模型。碱性电荷的作用是减少HAP表面上蛋白质 - 蛋白质之间的电荷排斥力，在没有碱性电荷的情况下，结合饱和时表面上磷蛋白的堆积密度会显著降低。
