Katz B A, Cass R T
Arris Pharmaceutical Corporation, South San Francisco, California 94080, USA.
J Biol Chem. 1997 May 16;272(20):13220-8. doi: 10.1074/jbc.272.20.13220.
The pH dependences of the affinities for streptavidin of linear and cyclic peptide ligands containing the HPQ sequence discovered by phage display were determined by plasmon resonance measurements. At pH values ranging from 3.0 to 9.0, the Kd values for Ac-AEFSHPQNTIEGRK-NH2, cyclo-Ac-AE[CHPQGPPC]IEGRK-NH2, and cyclo-Ac-AE[CHPQFC]IEGRK-NH2, were determined by competition, and those for cyclo-[5-S-valeramide-HPQGPPC]K-NH2 were determined directly by equilibrium affinity measurements. The Kd values of the ligands increase by an average factor of 3.0 +/- 0.8 per decrease in pH unit between pH approximately 4.5 and pH approximately 6.3. Below pH approximately 4.5 there is a smaller increase in Kd values, and above pH approximately 6.3 the Kd values become relatively pH-independent. We determined the crystal structures of complexes of streptavidin with cyclo-[5-S-valeramide-HPQGPPC]K-NH2 at pH 1.5, 2.5, 3.0, and 3.5, with cyclo-Ac-[CHPQFC]-NH2 at pH 2.0, 3.0, 3.6, 4.2, 4.8, and 11.8, with cyclo-Ac-[CHPQGPPC]-NH2 at pH 2.5, 2.9, and 3.7, and with FSHPQNT at pH 4.0 and compared the structures with one another and with those previously determined at other pH values. At pH values from 3.0 to 11.8, the electron density for the peptide His side chain is strong, flat, and well defined. A hydrogen bond between the Ndelta1 atom of the His and the peptide Gln amide group indicates the His of the bound peptide in the crystals is uncharged at pH >/= 3.0. By determining selected structures in two different space groups, I222 with two crystallographically inequivalent ligand sites and I4122 with one site, we show that below pH approximately 3.0, the pKa of the bound peptide His in the crystals is influenced by crystal packing interactions. The presence of the Ndelta1His-NGln hydrogen bond along with pH dependences of the peptide affinities suggest that deprotonation of the peptide His is required for high affinity binding of HPQ-containing peptides to streptavidin both in the crystals and in solution.
通过等离子体共振测量，确定了噬菌体展示所发现的含HPQ序列的线性和环状肽配体与链霉亲和素亲和力的pH依赖性。在pH值范围为3.0至9.0时，通过竞争法测定了Ac-AEFSHPQNTIEGRK-NH2、环Ac-AE[CHPQGPPC]IEGRK-NH2和环Ac-AE[CHPQFC]IEGRK-NH2的解离常数（Kd）值，通过平衡亲和力测量直接测定了环[5-S-戊酰胺-HPQGPPC]K-NH2的Kd值。在pH约4.5至pH约6.3之间，pH每降低一个单位，配体的Kd值平均增加3.0±0.8倍。在pH约4.5以下，Kd值增加较小，在pH约6.3以上，Kd值相对与pH无关。我们测定了链霉亲和素与环[5-S-戊酰胺-HPQGPPC]K-NH2在pH 1.5、2.5、3.0和3.5时、与环Ac-[CHPQFC]-NH2在pH 2.0、3.0、3.6、4.2、4.8和11.8时、与环Ac-[CHPQGPPC]-NH2在pH 2.5、2.9和3.7时以及与FSHPQNT在pH 4.0时的复合物晶体结构，并将这些结构相互比较以及与之前在其他pH值下测定的结构进行比较。在pH值从3.0至11.8时，肽His侧链的电子密度强、平坦且明确。His的Nδ1原子与肽Gln酰胺基团之间的氢键表明，在pH≥3.0时，晶体中结合肽的His不带电荷。通过在两个不同空间群中测定选定结构，即具有两个晶体学不等价配体位点的I222和具有一个位点的I4122，我们表明，在pH约3.0以下，晶体中结合肽His的pKa受晶体堆积相互作用影响。Nδ1His-NGln氢键的存在以及肽亲和力的pH依赖性表明，肽His去质子化是含HPQ肽在晶体和溶液中与链霉亲和素高亲和力结合所必需的。
