STRUCTURE-FUNCTION STUDIES OF A SWITCHABLE ION CHANNEL

可切换离子通道的结构-功能研究

• 批准号: 3279616
• 负责人: FRANCOISE LEVINTHAL
• 金额: $ 19.74万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-12-01 至 1992-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3279616
• 关键词: binding proteins; colicines; computer simulation; cysteine; electron spin resonance spectroscopy; electrophysiology; fluorescence spectrometry; genetic manipulation; laboratory rabbit; membrane activity; membrane channels; membrane model; membrane permeability; membrane potentials; membrane structure; membrane transport proteins; methionine; phospholipids; point mutation; protein engineering; protein sequence; protein structure function; site directed mutagenesis

The ion channel region of the colicin El protein will be studied as a model for voltage switchable ion channels found in all organisms. Site-directed mutagenesis with chemically synthesized oligonucleotides have been used to introduce methionine codons in the gene in order to provide new cyanogen bromide cleavage sites and produce short C-terminal fragments which can be tested for their channel forming activity. At present, a C-terminal fragment of only 88 amino acids has been found to be active in producing channels in a phospholipid membrane while one with only 75 amino acids was not active. Additional methionine codons will be introduced to establish the critical minimum length of the active peptide. We also have determined stochiometrically that only on peptide molecule is enough to make the channel and that in the experimental conditions used, this molecule is a monomer. The one cysteine residue in the wild type molecule has been changed to glycine and nine new cysteine codons introduced, one at a time. Many more of these amino acid changes will be made in the part involved in the channel formation and the proteins produced will be studied by attaching specific chemical groups to the SH groups located throughout the peptide chains. The effects of these sulfhydryl reagents will be examined by either electron- spin resonance spectroscopy if the attached molecule's have an electron spin or they will be studied by determining the alterations in th conductance of single channels when the attached molecules are big since the channel will then be blocked if a large group is pointing inward. Using the information obtained from these modified proteins, one can determine which residues in the peptide point into the lumen and which point out into the lipid. In this way, one can determine which parts of th protein are in the form of a beta-sheet where the residues would point alternatively in or out with a repeat period of 2 and which are in the form of an alpha-helix in which case the repeat period would be 3.6 residues. These experimental measurements will be used to help in deducing the structure of the channel. New channels could be produced by altering the amino acids facing the lumen. In all cases, extensive computations will be done in order to make theoretical estimates of the channel conductance and their changes and these will be compared with the observed values.

将蛋白质蛋白的离子通道区域研究 在所有中发现的电压可切换离子通道的模型 有机体。 定点诱变，化学 合成的寡核苷酸已用于引入 基因中的蛋氨酸密码子为了提供新的氰基 溴化物裂解位点并产生短的C末端片段 可以测试其通道形成活动。 在 目前，仅88个氨基酸的C末端片段已经 发现在磷脂中产生通道有效 膜虽然只有75个氨基酸，但没有活性。 将引入其他蛋氨酸密码子来建立 活动肽的临界最小长度。 我们也有 固定测定只有在肽分子上是 足以制定通道，并且在实验中 使用的条件，该分子是单体。 野生型分子中的一个半胱氨酸残基已经 更改为甘氨酸和九个新的半胱氨酸密码子，一个 一次。 将进行更多这些氨基酸的变化 在通道形成和蛋白质中涉及的部分 通过将特定的化学基团附加到 SH组遍布整个肽链。 效果 在这些电子 旋转共振光谱如果附着的分子具有 电子旋转，否则将通过确定 当单个通道电导率的改变时 附着的分子很大，因为该通道将被阻止 如果一群人向内指向。 使用信息 从这些修饰的蛋白质中获得，可以确定哪个 肽点中的残留物进入管腔，并指出 进入脂质。 这样，可以确定TH的哪个部分 蛋白质是β片的形式，残留物将在其中 替代重复时期为2，然后将其指向2 以α-螺旋的形式，在这种情况下重复周期 将是3.6个残留物。 这些实验测量将用于帮助推导 通道的结构。 新渠道可以由 改变面向管腔的氨基酸。 在所有情况下，广泛 将进行计算以进行理论估计 渠道电导及其变化，这些将是 与观察到的值相比。