m*d
2 楼
观音姐姐说的
d*k
3 楼
att
★ 发自iPhone App: ChineseWeb 7.8
★ 发自iPhone App: ChineseWeb 7.8
m*e
4 楼
我们最近研究一个丝氨酸蛋白酶,它是以多聚体的形式发挥活性。我们突变了一个位于
两个亚基(protomer)界面的氨基酸,发现这个蛋白酶的活力提高了(Vmax提高了,Km
降低了),我们推测这个突变稳定了这个酶的活性构象,但不知道这个“稳定”可以用
什么样的实验具体证明。请有这方面经验的生化牛牛不吝赐教。多谢!
两个亚基(protomer)界面的氨基酸,发现这个蛋白酶的活力提高了(Vmax提高了,Km
降低了),我们推测这个突变稳定了这个酶的活性构象,但不知道这个“稳定”可以用
什么样的实验具体证明。请有这方面经验的生化牛牛不吝赐教。多谢!
c*o
6 楼
晕倒
y*u
7 楼
版主你出来干点啥吧,你的板斧都一个多月没上站了。难道是falling的马甲?
i*0
8 楼
something like a melting temperature? You can try isothermal titration
calorimetry. Add one subunit, and then add the other subunit, monitor the
heat release. If heat release increase with mutation, this means the complex
is more stable compared with WT. However, I doubt a single mutation will
make a great difference that could be detected by this method.
calorimetry. Add one subunit, and then add the other subunit, monitor the
heat release. If heat release increase with mutation, this means the complex
is more stable compared with WT. However, I doubt a single mutation will
make a great difference that could be detected by this method.
p*m
10 楼
太坏了 把我们的委比都扣下了
b*y
11 楼
LZ wants a method that can monitor whether one conformation (active) is
preferentially stabilized versus others (inactive ones). No easy method,
though.
You might have to solve the structures of both wt and mutant and hope there
is difference. NMR is good at revealing conformational changes but your
protein might be too big for it. If you can design a good single molecular
FRET experiment, you might find more than one mode of conformations. In your
mutant, you see one of the conformation is more populated than the rest.
That is all I can think of. none is easy.
preferentially stabilized versus others (inactive ones). No easy method,
though.
You might have to solve the structures of both wt and mutant and hope there
is difference. NMR is good at revealing conformational changes but your
protein might be too big for it. If you can design a good single molecular
FRET experiment, you might find more than one mode of conformations. In your
mutant, you see one of the conformation is more populated than the rest.
That is all I can think of. none is easy.
y*u
13 楼
顶一下,版主看到没啊?
k*0
14 楼
你是否认为蛋白质形成多具体是个动态过程,而突变稳定了多具体,因此提高了蛋白质
活性。
你可以利用dynamic light scattering观测多具体的形成和大小,然后加入变性剂看看
多聚体间亚基的蛋白质相互作用是否因为突变而更稳定了。
活性。
你可以利用dynamic light scattering观测多具体的形成和大小,然后加入变性剂看看
多聚体间亚基的蛋白质相互作用是否因为突变而更稳定了。
d*k
15 楼
att
★ 发自iPhone App: ChineseWeb 7.8
★ 发自iPhone App: ChineseWeb 7.8
y*u
17 楼
版主你出来干点啥吧,你的板斧都一个多月没上站了。难道是falling的马甲?
p*m
19 楼
太坏了 把我们的委比都扣下了
y*u
21 楼
顶一下,版主看到没啊?
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