q******g 发帖数: 3858 | 1 发现一个新的化合物,可以特异性地抑制某个转运蛋白的活性。我们是用oocytes来表
达该蛋白,然后做的转运试验。我们投了nature chemical biology,但编辑说我们缺少
证据来证明蛋白和化合物直接结合。不知道什么样的实验才能说明他们直接结合?
另外,还有什么杂志可以投呢?
谢谢大家了。 |
g*********5 发帖数: 2533 | 2 octet.
transfect the plasmids encode your protein with tag, use pall biology tips,
and loading protein on tips, and put the chemical in 96 wells, octec could
detect the direct interaction.
【在 q******g 的大作中提到】![](/moin_static193/solenoid/img/up.png) : 发现一个新的化合物,可以特异性地抑制某个转运蛋白的活性。我们是用oocytes来表 : 达该蛋白,然后做的转运试验。我们投了nature chemical biology,但编辑说我们缺少 : 证据来证明蛋白和化合物直接结合。不知道什么样的实验才能说明他们直接结合? : 另外,还有什么杂志可以投呢? : 谢谢大家了。
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s*******7 发帖数: 399 | 3 楼上提供的生物化学方法固然是妙,但阿戈感觉编辑的意思想要适合生物物理角度的方
法证明。
nchb对这一类文章,有时候不但要求化合物是直接阻断,还要求提供详细的阻断机制,
甚至要求这套机制又是如何决定或操纵(而不是间接影响)目标蛋白的功能,这样在他
们看来才算是一个完整的story。
俺觉得吧,根据楼主表达的内容,编辑是要作者说明化合物对转运蛋白的抑制效果是直
接性的阻断作用(block)而不是间接性的调控(modification)。
有的化合物,即便你结合上去蛋白了,但对功能依然是间接调控。
但如果能证明化合物在蛋白的结合点(binding site)正好位于转运蛋白的功能部分(
转运途径)之中,那这个将是最具说服性的数据(=直接block)。对此,如果没有现成
的目标蛋白或者同源蛋白结构的话,那下面就是体力活了:大规模突变做Double-
Mutant Cycle Analysis。 |
m******5 发帖数: 1383 | 4 楼主是专门做转运的?
可以考虑做 Invitro-nuclear import assay
就是用纯化的转运体系在digitonin permeablized 的Hela 细胞核上做。
因为整个体系都是体外重建的,理论上不会出现非直接影响的情况
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3261132/
不过好奇地问一下,这个化合物阻断核转运你们是propose和哪个转运蛋白结合?
importin beta? alpha? or exportin?
如果真的只特异某个subtype的importin的活性的话还是很有意思的,不过鉴于楼主能
在oocyte里做出来,可能性不大。 |
q******g 发帖数: 3858 | 5 不好意思,没有说清楚中文名称,其实是amino acid transporter. 目前还没有晶体结
构。
我们已经知道一些位点突变可以影响活性,准备用突变体+化合物做些转运实验。但是
不知道是否足够。 |
q******g 发帖数: 3858 | |
L*********y 发帖数: 322 | 7
You might want to do more function-structural analysis of this transporter
to back up your data.
For example, within this transporter, can you identify the key amino acid
residues or protein domains that affect the compound binding? Second,
can you synthesize a fluorophore or radiation conjugated version of this
new compound you discovered? If yes, you should be able to test its
interaction with your transporter by IP.
Third, Can you do some in vivo experiments to support you in vitro findings?
Say, knocking down the amino acid transporter in vivo will attenuate the
inhibitory effects of the compound.
【在 q******g 的大作中提到】![](/moin_static193/solenoid/img/up.png) : 发现一个新的化合物,可以特异性地抑制某个转运蛋白的活性。我们是用oocytes来表 : 达该蛋白,然后做的转运试验。我们投了nature chemical biology,但编辑说我们缺少 : 证据来证明蛋白和化合物直接结合。不知道什么样的实验才能说明他们直接结合? : 另外,还有什么杂志可以投呢? : 谢谢大家了。
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q******g 发帖数: 3858 | 8 For example, within this transporter, can you identify the key amino acid
residues or protein domains that affect the compound binding?
We will examine the amino acid uptake in oocytes expressing mutant with
compound.
Second, can you synthesize a fluorophore or radiation conjugated version of
this new compound you discovered? If yes, you should be able to test its
interaction with your transporter by IP.
This is a revesible inhibitor for transporter. I am afraid that compound
will not bind to the transporter in lysis buffer.
Third, Can you do some in vivo experiments to support you in vitro findings?
Say, knocking down the amino acid transporter in vivo will attenuate the
inhibitory effects of the compound.
we have the knockdown cell lines. I will try it. but it still cannot show
the direct interaction. just for backup I guess.
thanks Loveanthony.
findings?
【在 L*********y 的大作中提到】![](/moin_static193/solenoid/img/up.png) : : You might want to do more function-structural analysis of this transporter : to back up your data. : For example, within this transporter, can you identify the key amino acid : residues or protein domains that affect the compound binding? Second, : can you synthesize a fluorophore or radiation conjugated version of this : new compound you discovered? If yes, you should be able to test its : interaction with your transporter by IP. : Third, Can you do some in vivo experiments to support you in vitro findings? : Say, knocking down the amino acid transporter in vivo will attenuate the
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L*********y 发帖数: 322 | 9
of
What is the Kd of this compound? Does the inhibition depend on the presence
of the cargo carried by the transporter? You don't have to do this
experiments in lysis buffer. The transporter protein, ideally with a tag can
be prepared from oocyte, then you throw the labelled compound in the oocyte
system. After pulling-down the transporter, the binding can be analyzed
through assaying the amount of compound incorporated into this transporter.
findings?
I agree it is not a direct biochemical experiment to test the DIRECT binding
. However, the editors apparently ask you to show more convincing data to
support you original findings. The in vivo experiment, I think, is key to
showing the specificity and physiological relevance of the compound-
transporter interaction.
【在 q******g 的大作中提到】![](/moin_static193/solenoid/img/up.png) : For example, within this transporter, can you identify the key amino acid : residues or protein domains that affect the compound binding? : We will examine the amino acid uptake in oocytes expressing mutant with : compound. : Second, can you synthesize a fluorophore or radiation conjugated version of : this new compound you discovered? If yes, you should be able to test its : interaction with your transporter by IP. : This is a revesible inhibitor for transporter. I am afraid that compound : will not bind to the transporter in lysis buffer. : Third, Can you do some in vivo experiments to support you in vitro findings?
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w**********c 发帖数: 6 | 10 蛋白如果能在E.coli里表达,直接拿到蛋白,测小分子和蛋白结合的方法太多了,ITC
, SPR,Thermal shift,FA。NCB的editor是想看这些结果,最直接的是co-
crystallization实在不行NMR |
s*******7 发帖数: 399 | 11 楼上,我估计楼主实验室的条件做不了结构,如果他能做,并能做成的话,他的东西就
不会选择投nchb,早就换cns去了。
楼主现在的问题只能拿已经解出的同源蛋白结构(如果有这个同源结构)作参考,由此
可能会少一点工作量,但相当数量的突变map还是要做的。
假设楼主已经发现了某些突变体(转运蛋白)可一改变化合物的量效,那下面你接着要
做化合物的修饰衍生物(类似于蛋白的突变),假设你的同一个突变体改变了化合物衍
生物的量效,但改变程度非常不一样,那你可以下99%的可信结论:这个化合物的修饰
部位距离突变蛋白的残基非常非常近(Double-Mutant Cycle Analysis的精髓即在此处
),换句话,你的化合物结合点就在这个氨基酸或其附近。如果与此同时,你这个氨基
酸 palys key role in 转运功能,那楼主你就发大了。 |
q******g 发帖数: 3858 | 12 我们的合作者已经拿到了纯化的蛋白。但transporter是膜蛋白,很难得到晶体,他们
做了一年还没有结果。
wangjingsioc说的ITC, SPR,Thermal shift,FA或许可以试试。合作者好像试过MTS
,但没有变化。
化合物衍生物是准备做。但还没有开始。
我曾经用MODEL来预测蛋白结构,将化合物docking,显示有一个binding pocket, 突变
了其中一个氨基酸,可以降低50%活性。准备做化合物和突变体的活性实验。但一个合
作者说,你需要把binding pocket所有的氨基酸都突变了,才有说服力。就没往下做,
而是直接交了。如果有个一个突变的结果,估计编辑这关或许能过去。 |
c*****u 发帖数: 357 | 13 像10楼说的一样,做些真正的binding study 和kinetics,才有说服力。 |