Characterization of the "helix clamp" motif of HIV-1 reverse transcriptase using MALDI-TOF MS and surface plasmon resonance.
Characterization of the "helix clamp" motif of HIV-1 reverse transcriptase using MALDI-TOF MS and surface plasmon resonance.
复制标题
使用 MALDI-TOF MS 和表面等离子共振表征 HIV-1 逆转录酶的“螺旋夹”基序。
DOI：
10.1021/ac991429f
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发表时间：
2000
影响因子：
7.4
通讯作者：
Woods,AS
中科院分区：
文献类型：
--
作者：
Lin,S;Long,S;Ramirez,SM;Cotter,RJ;Woods,AS
人类免疫缺陷病毒 1 型逆转录酶 (HIV-1 RT) 晶体结构中的螺旋-转角-螺旋基序被认为是几种核苷酸聚合酶之间的保守核酸结合域 (Hermann, T.; Meier, T.; Götte, M.; Heumann, H.Nucleic Acids Res.1994,22, 4625−4633)。该结构域的序列与 HIV-1 RT 的 259KLVGKL(X)16KLLR284 同源，其充当抓住模板-引物 (T-P) 复合物的“螺旋夹”。我们使用 MALDI-TOF MS 和表面等离子共振 (BIAcore) 表征了螺旋夹基序。我们的研究表明，“螺旋夹”具有核酸结合功能，但可能不是序列特异性的。该证据表明螺旋夹和寡核苷酸主链之间的离子相互作用不仅仅负责结合。蛋白质的二级和三级结构也可能在核酸结合中发挥重要作用。单链寡核苷酸与螺旋夹结合的缔合常数 ka 和解离常数 kd 分别确定为 7.03 × 103M-1s-1 和 1.22 × 103s-1。
A helix-turn-helix motif in the crystal structure of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) was proposed to be a conserved nucleic acid binding domain among several nucleotide polymerizing enzymes (Hermann, T.; Meier, T.; Götte, M.; Heumann, H.Nucleic Acids Res.1994,22, 4625−4633). The sequence of this domain is homologous to259KLVGKL(X)16KLLR284of HIV-1 RT, which acts as a “helix clamp” grasping the template−primer (T−P) complex. We characterized the helix clamp motif using MALDI-TOF MS and surface plasmon resonance (BIAcore). Our studies showed that the “helix clamp” has a nucleic acid binding function that may not be sequence specific. This evidence suggests that ionic interactions between the helix clamp and oligonucleotide backbone are not solely responsible for binding. Secondary and tertiary structures of the protein may also play a significant role in nucleic acid binding. The association and dissociation constants,kaandkd, for the binding of single-stranded oligonucleotide to the helix clamp were determined to be 7.03 × 103M-1s-1and 1.22 × 103s-1, respectively.