Andersson Linda K, Caspersson Maud, Baltzer Lars
Department of Chemistry, Göteborg University, Sweden.
Chemistry. 2002 Aug 16;8(16):3687-97. doi: 10.1002/1521-3765(20020816)8:16<3687::AID-CHEM3687>3.0.CO;2-8.
Five 42-residue polypeptides have been designed to fold into hairpin helix-loop-helix motifs that dimerize to form four-helix bundles, and to serve as protein scaffolds for the elucidation at the molecular level of the principles that control and fine-tune lysine and ornithine reactivities in a protein context. Site-selective control of Lys and Orn reactivity provides a mechanism for addressing directly individual residues and is a prerequisite for the site-selective functionalization of folded proteins. Several lysine and one ornithine residues were introduced on the surface and in the hydrophobic core of the folded motif. The reactivity of each residue was determined by measuring the degree of acylation of the trypsin cleaved fragments by HPLC and mass spectrometry. The most reactive residues were Orn34 and Lys19, both of which were located in d positions in the heptad repeat, and therefore in hydrophobic environments. Upon reaction of the helix-loop-helix dimer KA-I with one equivalent of mono-p-nitrophenyl fumarate, Orn34 was acylated approximately three times more efficiently than Lys19, whereas Lys10 (b position), Lys15 (g position), and Lys33 (c position) remained unmodified. In the sequence KA-I-A(15) Lys15 was replaced by an alanine residue and the selectivity of Orn34 over Lys19 increased to approximately a factor of six, probably because Lys15 had the capacity to reduce the pK(a) value of Lys19 and 85 % of site-selectively monoacylated product was obtained. The pH dependence of the acylation reaction was determined and showed that the pK(a) of the reactive residues were 9.3, more than a pK(a) unit below the magnitude of the corresponding residue in a solvent exposed position. Introducing Lys and Orn residues into a or d positions of the heptad repeat therefore serves as a mechanism of depressing their pK(a) to increase their reactivity site selectively. Extensive NMR and CD spectroscopic analyses showed that the sequences fold according to prediction.
已设计出五种由42个残基组成的多肽，使其折叠成发夹状螺旋-环-螺旋基序，这些基序二聚化形成四螺旋束，并作为蛋白质支架，用于在分子水平阐明在蛋白质环境中控制和微调赖氨酸和鸟氨酸反应性的原理。赖氨酸和鸟氨酸反应性的位点选择性控制提供了一种直接针对单个残基的机制，并且是折叠蛋白位点选择性功能化的先决条件。在折叠基序的表面和疏水核心引入了几个赖氨酸和一个鸟氨酸残基。通过高效液相色谱（HPLC）和质谱法测量胰蛋白酶切割片段的酰化程度，确定每个残基的反应性。反应性最高的残基是鸟氨酸34（Orn34）和赖氨酸19（Lys19），它们都位于七肽重复序列的d位，因此处于疏水环境中。当螺旋-环-螺旋二聚体KA-I与一当量的单对硝基苯基富马酸酯反应时，鸟氨酸34的酰化效率比赖氨酸19高约三倍，而赖氨酸10（b位）、赖氨酸15（g位）和赖氨酸33（c位）未被修饰。在序列KA-I-A(15)中，赖氨酸15被丙氨酸残基取代，鸟氨酸34对赖氨酸19的选择性增加到约六倍，这可能是因为赖氨酸15能够降低赖氨酸19的pK(a)值，并且获得了85%的位点选择性单酰化产物。测定了酰化反应的pH依赖性，结果表明反应性残基的pK(a)为9.3，比溶剂暴露位置相应残基的pK(a)值低一个多单位。因此，将赖氨酸和鸟氨酸残基引入七肽重复序列的a或d位可作为一种机制，降低其pK(a)值以选择性地增加其反应性。广泛的核磁共振（NMR）和圆二色（CD）光谱分析表明，这些序列按预测折叠。
