Re: [ccp4bb] Contradictory result between ITC and cocrystal structure

[Petri Kursula]

Hi,
a likely scenario is that your mutant crystallises in the same 
conformation/packing as the wild-type protein, and this conformation is good 
for ligand binding. In solution, your mutant protein may be more flexible/open 
than the wild-type and affinity hence lower. We see this quite often. Various 
solution structure techniques will shed light on this.

Another possibility, assuming that you did ITC only at one temperature, is that 
you are “lucky” enough to be at the temperature where enthalpy for binding is 
zero for the mutant (but not wild-type). This you can find out by carrying out 
ITC at different temperatures.
Petri
 
Petri Kursula
--
Professor 
--
Department of Biomedicine
University of Bergen, Norway
http://www.uib.no/en/rg/petrikursula
petri.kurs...@uib.no
--
Faculty of Biochemistry and Molecular Medicine
Biocenter Oulu
University of Oulu, Finland
--





> On 22 Feb 2020, at 07:31, monika chandravanshi 
>  wrote:
> 
> Dear All,
> 
>   I have a situation, where a mutant protein does not exhibit any 
> heat change upon titration with cognate ligand in the ITC experiment. 
> However, it co-crystallizes with the respective cognate ligand. Also, the 
> cocrystal structure reveals the conservation of the hydrogen bonding networks 
> except for the mutated residues. I would like to know the possible reason for 
> the no heat change in the ITC experiment. 
> 
> Looking forward to hearing from you.
> 
> -- 
> -
> 
> With Kind Regards
> 
> Monika Chandravanshi
> PhD Scholar, 
> Department of Biosciences and Bioengineering
> Indian Institute of Technology Guwahati, Guwahati India
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 
> 



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Re: [ccp4bb] Contradictory result between ITC and cocrystal structure

[Srivastava, Dhiraj]

As other said, having co-crystal doesn’t mean you will have measurable 
affinity. Further no heat in ITC experiments doesn’t mean no binding. Which 
buffer you used during previous ITC experiment? If heat in the previous 
experiment (wild type) was due to heat of ionization and you removed the 
residue involved in protonation/deprotonation event, you will not see any heat.
If there is 10-20 fold decrease in affinity by mutation, you may not be able to 
see significant heat during ITC experiment as well.


Dhiraj



From: CCP4 bulletin board  on behalf of vipul panchal 

Sent: Saturday, February 22, 2020 3:25 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] Contradictory result between ITC and cocrystal structure

Hi Monika,

If the protein cocrystalize with ligand doesn't mean it interacts with protein.
You have provided insufficient information here. Does the ligand is bound to 
mutant protein with atleast 2 or 3 hydrogen bonds (how many residues are 
mutated?) ? If no that means, it is just cocrystalizing but not interacting 
with mutant protein.

Another possibility is, during cocrystalization ligand concentration is very 
high so as to force the interaction which otherwise is not possible as you 
observed with ITC. In other terms, affinity is too low to be detected by ITC.

Cheers,
Vipul

On Sat, 22 Feb, 2020, 12:01 PM monika chandravanshi, 
mailto:chandravanshi.monik...@gmail.com>> 
wrote:
Dear All,

  I have a situation, where a mutant protein does not exhibit any 
heat change upon titration with cognate ligand in the ITC experiment. However, 
it co-crystallizes with the respective cognate ligand. Also, the cocrystal 
structure reveals the conservation of the hydrogen bonding networks except for 
the mutated residues. I would like to know the possible reason for the no heat 
change in the ITC experiment.

Looking forward to hearing from you.

--
-

With Kind Regards

Monika Chandravanshi
PhD Scholar,
Department of Biosciences and Bioengineering
Indian Institute of Technology Guwahati, Guwahati India



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Re: [ccp4bb] Contradictory result between ITC and cocrystal structure

[Srivastava, Dhiraj]

As other said, having co-crystal doesn’t mean you will have measurable 
affinity. Further no heat in ITC experiments doesn’t mean no binding. Which 
buffer you used during previous ITC experiment? If heat in the previous 
experiment (wild type) was due to heat of ionization and you removed the 
residue involved in protonation/deprotonation event, you will not see any heat.
If there is 10-20 fold decrease in affinity by mutation, you may not be able to 
see significant heat during ITC experiment as well.


Dhiraj



From: CCP4 bulletin board  on behalf of vipul panchal 

Sent: Saturday, February 22, 2020 3:25 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] Contradictory result between ITC and cocrystal structure

Hi Monika,

If the protein cocrystalize with ligand doesn't mean it interacts with protein.
You have provided insufficient information here. Does the ligand is bound to 
mutant protein with atleast 2 or 3 hydrogen bonds (how many residues are 
mutated?) ? If no that means, it is just cocrystalizing but not interacting 
with mutant protein.

Another possibility is, during cocrystalization ligand concentration is very 
high so as to force the interaction which otherwise is not possible as you 
observed with ITC. In other terms, affinity is too low to be detected by ITC.

Cheers,
Vipul

On Sat, 22 Feb, 2020, 12:01 PM monika chandravanshi, 
mailto:chandravanshi.monik...@gmail.com>> 
wrote:
Dear All,

  I have a situation, where a mutant protein does not exhibit any 
heat change upon titration with cognate ligand in the ITC experiment. However, 
it co-crystallizes with the respective cognate ligand. Also, the cocrystal 
structure reveals the conservation of the hydrogen bonding networks except for 
the mutated residues. I would like to know the possible reason for the no heat 
change in the ITC experiment.

Looking forward to hearing from you.

--
-

With Kind Regards

Monika Chandravanshi
PhD Scholar,
Department of Biosciences and Bioengineering
Indian Institute of Technology Guwahati, Guwahati India



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Re: [ccp4bb] Contradictory result between ITC and cocrystal structure

[Patra, Dhabaleswar]

Dear Monika,

If your protein-ligand interaction is at millimolar, you need to increase 
injection volume in order to see heat change. Also, you can do the experiment 
at low temperature such as 10 degree Celsius. You can follow this paper where I 
have crystallized weak binding ligand and complemented with ITC.
https://pubmed.ncbi.nlm.nih.gov/24957055-structure-interactions-and-evolutionary-implications-of-a-domain-swapped-lectin-dimer-from-mycobacterium-smegmatis/?from_term=Dhabaleswar+Patra_pos=7
Structure, Interactions and Evolutionary Implications of a Domain-Swapped 
Lectin Dimer From Mycobacterium Smegmatis - 
PubMed<https://pubmed.ncbi.nlm.nih.gov/24957055-structure-interactions-and-evolutionary-implications-of-a-domain-swapped-lectin-dimer-from-mycobacterium-smegmatis/?from_term=Dhabaleswar+Patra_pos=7>
Crystal structure determination of the lectin domain of MSMEG_3662 from 
Mycobacterium smegmatis and its complexes with mannose and methyl-α-mannose, 
the first effort of its kind on a mycobacterial lectin, reveals a structure 
very similar to β-prism II fold lectins from plant sources, but with extens …
pubmed.ncbi.nlm.nih.gov
Hope this help.

Regards,
Dhabaleswar Patra
Purdue University, IN



From: CCP4 bulletin board  on behalf of vipul panchal 

Sent: Saturday, February 22, 2020 4:25 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] Contradictory result between ITC and cocrystal structure

Hi Monika,

If the protein cocrystalize with ligand doesn't mean it interacts with protein.
You have provided insufficient information here. Does the ligand is bound to 
mutant protein with atleast 2 or 3 hydrogen bonds (how many residues are 
mutated?) ? If no that means, it is just cocrystalizing but not interacting 
with mutant protein.

Another possibility is, during cocrystalization ligand concentration is very 
high so as to force the interaction which otherwise is not possible as you 
observed with ITC. In other terms, affinity is too low to be detected by ITC.

Cheers,
Vipul

On Sat, 22 Feb, 2020, 12:01 PM monika chandravanshi, 
mailto:chandravanshi.monik...@gmail.com>> 
wrote:
Dear All,

  I have a situation, where a mutant protein does not exhibit any 
heat change upon titration with cognate ligand in the ITC experiment. However, 
it co-crystallizes with the respective cognate ligand. Also, the cocrystal 
structure reveals the conservation of the hydrogen bonding networks except for 
the mutated residues. I would like to know the possible reason for the no heat 
change in the ITC experiment.

Looking forward to hearing from you.

--
-

With Kind Regards

Monika Chandravanshi
PhD Scholar,
Department of Biosciences and Bioengineering
Indian Institute of Technology Guwahati, Guwahati India



To unsubscribe from the CCP4BB list, click the following link:
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Re: [ccp4bb] Contradictory result between ITC and cocrystal structure

[vipul panchal]

Hi Monika,

If the protein cocrystalize with ligand doesn't mean it interacts with
protein.
You have provided insufficient information here. Does the ligand is bound
to mutant protein with atleast 2 or 3 hydrogen bonds (how many residues are
mutated?) ? If no that means, it is just cocrystalizing but not interacting
with mutant protein.

Another possibility is, during cocrystalization ligand concentration is
very high so as to force the interaction which otherwise is not possible as
you observed with ITC. In other terms, affinity is too low to be detected
by ITC.

Cheers,
Vipul

On Sat, 22 Feb, 2020, 12:01 PM monika chandravanshi, <
chandravanshi.monik...@gmail.com> wrote:

> Dear All,
>
>   I have a situation, where a mutant protein does not exhibit
> any heat change upon titration with cognate ligand in the ITC experiment.
> However, it co-crystallizes with the respective cognate ligand. Also, the
> cocrystal structure reveals the conservation of the hydrogen bonding
> networks except for the mutated residues. I would like to know the possible
> reason for the no heat change in the ITC experiment.
>
> Looking forward to hearing from you.
>
> --
> *-*
>
> *With Kind Regards*
>
> *Monika Chandravanshi*
> *PhD Scholar, *
> *Department of Biosciences and Bioengineering*
> *Indian Institute of Technology Guwahati, Guwahati India*
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>



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Re: [ccp4bb] Contradictory result between ITC and cocrystal structure

[Natesh Ramanathan]

Dear Monika,
Did you do the proper positive and -ve control for ITC.
It is impossible that there are no changes in thermodynamics.. when it
binds..
Either your detector is not sensing it OR your experiment is not running
properly..
HTH
Best wishes,
Natesh

On Sat, 22 Feb 2020 at 12:01, monika chandravanshi <
chandravanshi.monik...@gmail.com> wrote:

> Dear All,
>
>   I have a situation, where a mutant protein does not exhibit
> any heat change upon titration with cognate ligand in the ITC experiment.
> However, it co-crystallizes with the respective cognate ligand. Also, the
> cocrystal structure reveals the conservation of the hydrogen bonding
> networks except for the mutated residues. I would like to know the possible
> reason for the no heat change in the ITC experiment.
>
> Looking forward to hearing from you.
>
> --
> *-*
>
> *With Kind Regards*
>
> *Monika Chandravanshi*
> *PhD Scholar, *
> *Department of Biosciences and Bioengineering*
> *Indian Institute of Technology Guwahati, Guwahati India*
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>


-- 
--
"Live Simply and do Serious Things .. "
- Dorothy Mary Crowfoot Hodgkin OM, FRS

"In Science truth always wins"
- Max Ferdinand Perutz OM FRS
--
Dr. Ramanathan Natesh
Assistant Professor,
School of Biology,
Indian Institute of Science Education and Research Thiruvananthapuram
(IISER-TVM),
Maruthamala P.O., Vithura,
Thiruvananthapuram,  695551, Kerala, India

nat...@iisertvm.ac.in
http://www.researcherid.com/rid/C-4488-2008
ORCID: http://orcid.org/-0002-1145-5962
https://publons.com/author/1520837/ramanathan-natesh#profile
http://faculty.iisertvm.ac.in/natesh

Office Ph. 0091- 471-2778087



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[ccp4bb] Contradictory result between ITC and cocrystal structure

[monika chandravanshi]

Dear All,

  I have a situation, where a mutant protein does not exhibit
any heat change upon titration with cognate ligand in the ITC experiment.
However, it co-crystallizes with the respective cognate ligand. Also, the
cocrystal structure reveals the conservation of the hydrogen bonding
networks except for the mutated residues. I would like to know the possible
reason for the no heat change in the ITC experiment.

Looking forward to hearing from you.

-- 
*-*

*With Kind Regards*

*Monika Chandravanshi*
*PhD Scholar, *
*Department of Biosciences and Bioengineering*
*Indian Institute of Technology Guwahati, Guwahati India*



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