e********r
发帖数: 147 | 1 最近我们实验室发现一个小分子（核苷类物质）能够特异性地结合一个蛋白（受体），
结果非常有意思。目前正在准备精细分析具体的结合位点。如果通过分段表达的方式，
把结合位点定在某一个蛋白区域以后，后面肿么做或采取什么方法才能定到具体的氨基
酸上呢？（当然丙氨酸扫描就算了吧，好几百个氨基酸呢......） |
v**********m
发帖数: 5516 | 2 化学修饰。
【在 e********r 的大作中提到】
: 最近我们实验室发现一个小分子（核苷类物质）能够特异性地结合一个蛋白（受体），
: 结果非常有意思。目前正在准备精细分析具体的结合位点。如果通过分段表达的方式，
: 把结合位点定在某一个蛋白区域以后，后面肿么做或采取什么方法才能定到具体的氨基
: 酸上呢？（当然丙氨酸扫描就算了吧，好几百个氨基酸呢......）
|
b******y
发帖数: 627 | 3 Is the receptor structure or its homolog's available? If so, do conservation
mapping to its surface. I would predict the binding pocket is 1) conserved;
2) positively charged especially Arginine; 3) some aromatics for pi-pi
interaction with bases of nucleic acid. This is the cheapest and first step
for whatever following up experiments.
If no structure available whatsoever, do a structure with/without ligand.
For biochemical experiments, UV-crosslinking and then MS-ID isn't a bad
option.
【在 e********r 的大作中提到】
: 最近我们实验室发现一个小分子（核苷类物质）能够特异性地结合一个蛋白（受体），
: 结果非常有意思。目前正在准备精细分析具体的结合位点。如果通过分段表达的方式，
: 把结合位点定在某一个蛋白区域以后，后面肿么做或采取什么方法才能定到具体的氨基
: 酸上呢？（当然丙氨酸扫描就算了吧，好几百个氨基酸呢......）
|
h****f
发帖数: 2 | 4 蛋白（受体）能表达纯化吗 核磁滴定可以看到具体哪些残基参与结合 或者干脆得到复
合物晶体结构 但这些前提都是蛋白可以表达纯化。 |
e********r
发帖数: 147 | 5 谢谢！信息量很大，看来还不是件太容易的事。类似蛋白的结构是有的，我们照您说的
预测一下看看有无可能。另，正在搞清what is uv-crosslinking with MS-ID
conservation
conserved;
step
【在 b******y 的大作中提到】
: Is the receptor structure or its homolog's available? If so, do conservation
: mapping to its surface. I would predict the binding pocket is 1) conserved;
: 2) positively charged especially Arginine; 3) some aromatics for pi-pi
: interaction with bases of nucleic acid. This is the cheapest and first step
: for whatever following up experiments.
: If no structure available whatsoever, do a structure with/without ligand.
: For biochemical experiments, UV-crosslinking and then MS-ID isn't a bad
: option.
|
e********r
发帖数: 147 | 6 胞内区是可以被纯化出来的。正在查阅啥叫核磁滴定......，谢谢
【在 h****f 的大作中提到】
: 蛋白（受体）能表达纯化吗 核磁滴定可以看到具体哪些残基参与结合 或者干脆得到复
: 合物晶体结构 但这些前提都是蛋白可以表达纯化。
|
b******y
发帖数: 627 | 7 A protein of a few hundred amino acid residuals is sort of on the upper side
of NMR studies. Only hard core NMR labs should do it.
Just give you a bit info. You need to label the protein with N15 and C13 in
D2O given its size. Assign all the amide peaks on a 2-D HSQC or HMQC (again
hundreds of them, of course). Then take a series of 2-D HSQC or HMQC with
increasing concentrations of the ligand. Presumably the amide close to the
ligand will change and those far away remain unaltered. As such, you will
know where the binding pocket is.
Unless NMR is handy for you, I don't recommend this to you. |
b******y
发帖数: 627 | 8 About UV crosslinking, many nucleic acid can be radicalized upon UV
treatment. Such radicals is highly reactive to active residuals in the
vicinity. Afterwards, digestion for MS. Abnormal peptides upon crosslinking
in comparison with those without crosslinking can tell you which peptide(s)
are close to the ligand.
【在 e********r 的大作中提到】
: 谢谢！信息量很大，看来还不是件太容易的事。类似蛋白的结构是有的，我们照您说的
: 预测一下看看有无可能。另，正在搞清what is uv-crosslinking with MS-ID
:
: conservation
: conserved;
: step
|
K******S
发帖数: 10109 | 9 如果附近有做mass spec的，这是经典的H/D exchange实验
：最近我们实验室发现一个小分子（核苷类物质）能够特异性地结合一个蛋白（受体）
，结果非常有意思。目前正在准备精细分析具体的结合位点。如果通过分段表达的方式
，把结合位点定在某一个蛋白区域以后，后面肿么做或采取什么方法才能定到具体的氨
基酸上呢？（当然丙氨酸扫描就算了吧，好几百个氨基酸呢......） |
b******y
发帖数: 627 | 10 H/D exchange is a good one. |
f*****e
发帖数: 19 | 11 楼主那几百个氨基酸会死人的...而且assign每个峰时间也很长啊...
话说可以简单看看有没有可能结合一些特异的氨基酸,比如trp, gly神马的,在N15-HSQC
的谱上很明显,可以缩小范围,呵呵
有米的话还可以标记特殊的氨基酸,分别表达蛋白,然后做HSQC,挨个试...
side
in
again
【在 b******y 的大作中提到】
: A protein of a few hundred amino acid residuals is sort of on the upper side
: of NMR studies. Only hard core NMR labs should do it.
: Just give you a bit info. You need to label the protein with N15 and C13 in
: D2O given its size. Assign all the amide peaks on a 2-D HSQC or HMQC (again
: hundreds of them, of course). Then take a series of 2-D HSQC or HMQC with
: increasing concentrations of the ligand. Presumably the amide close to the
: ligand will change and those far away remain unaltered. As such, you will
: know where the binding pocket is.
: Unless NMR is handy for you, I don't recommend this to you.
|
