Dear All,
Thank you all for the responses and suggestions.
Regards
Kavya
> Is the binding site accessible in the context of the crystal? Have you
tried to measure binding in the conditions of crystallization as a
control?
>
> Sent from my iPad
>
>> On 6 Jan 2016, at 09:29, Kavyashree Manjunath <[log in to unmask]>
wrote:
>>
>> Dear CCP4 users,
>>
>> I am trying to capture small molecule bound to the protein.
>> These small molecules are solubilised in 100% DMSO. And the
>> biochemical and biophysical assays shows micromolar affinity
>> (two or three digit) towards the protein.
>>
>> We have extensively tried soaking (max 15% DMSO), long period
>> soaking, growing protein crystals in the well precoated with
>> the ligand and cocrystallization (max 5% DMSO) experiments,
>> but have not succeeded to get any density for the ligand.
>>
>> The molar ratio that was used in the biochemical experiment
>> is ~ 1:1000 (protein:ligand) in order to observe any binding.
>> [one of the experiment used was microscale thermophoresis]. In
>> these experiments the protein is in nM and small molecule is in microM
quantities.
>>
>> For crystallization we are able to reach a maximum of 1:90 (in
>> cocrystallization). It is not possible to approach a higher molar ratio
because of the limitations of DMSO.
>>
>> So is there a possibility that we can capture the ligand in the
crystal? I have come across ligands that have been captured even though
>> they had mM binding affinities. So can anyone please suggest
>> possible ways I can try to capture the small molecule.
>>
>> Thank you
>> Regards
>> Kavya
>>
>>
>> --
>> This message has been scanned for viruses and
>> dangerous content by MailScanner, and is
>> believed to be clean.
>
--
This message has been scanned for viruses and
dangerous content by MailScanner, and is
believed to be clean.
|