Hi Robert,
CCPN use the equations from the Vanderbilt website.
Use the 9th equation down form protein-ligand. The longest equation is for monomer-dimer binding.
Cheers,
Ken
Senior NMR Scientist
Drug Discovery Programme
CRUK Beatson Institute
Garscube Estate
Switchback Road
Bearsden
Scotland G61 1BD
Tel: 0141 330 6410
From: CcpNmr software mailing list [mailto:[log in to unmask]]
On Behalf Of Brian Smith
Sent: 08 May 2018 09:58
To: [log in to unmask]
Subject: Re: Kd from chemical shift analysis
Hi Robert,
Yes CCPNmr Analysis v2.x can hep you do this easily. Before you try fitting the data, evaluate whether you have data that will actually be worth fitting - do the chemical shifts titrate with increasing ligand concentration and follow what appears to be a saturation
process, i.e. do they initially shift a lot for small ligand additions, but then you reach a ligand concentration beyond which you see no further changes?
For the actual procedure, some of this is covered in the analysis beginners course tutorial part 2
https://www.ccpn.ac.uk/v2-software/software/tutorials/analysis-beginners/beginners-analysis-2
but not in much detail - partly because it's quite simple. In outline:
-
Organise your HSQCs in separate shiftLists so that it's easy to propagate assignments from one to another.
-
Set up an NMR Series for your different conditions (ligand concentrations).
-
Peak pick your starting HSQC and Initialize Root Resonances - you now have resonance and spin system identifiers for all the atoms and residues that you can use to track shifts.
-
Choose a few peaks that are easy to follow by eye and peak pick their peaks in your titration series, then propagate their assignments from the starting HSQC.
-
Use the Follow Shift Changes popup with Assign groups? unchecked and the appropriate Fitting Function (usually the longest one in the drop down list).
If everything works well, for your chosen peaks, you can get braver and pick more peaks and even try trusting Follow Shift Changes to propagate the assignments for you, but this can get messy, so be sure to Archive your project before attempting anything risky.
Dr. Brian O. Smith --------------------------- Brian Smith at glasgow ac uk
Institute of Molecular, Cell and Systems Biology & School of Life Sciences,
College of Medical, Veterinary & Life Sciences,
Joseph Black Building, University of Glasgow, Glasgow G12 8QQ, UK.
Tel: 0141 330 5167/6459/3089
----------------------------------------------------------------------
The University of Glasgow, charity number SC004401
From: CcpNmr
software mailing list [[log in to unmask]] on behalf of Hammond, Robert Glenn [[log in to unmask]]
Sent: 07 May 2018 23:16
To: [log in to unmask]
Subject: Kd from chemical shift analysis
Hello,
I am trying to calculate dissociation constant (Kd) of a ligand when it binds to my protein. I have found that CCPNmr can use binding models to do this but I am unsure on the protocol. I already have
my HSQC spectra overlaid in a window. Although, I do not have the backbone assigned, I want to get a grasp on how to pick peaks and make the calculation. If anyone has experience doing this, I would be grateful if you could clarify a few things for me.
Also, if anyone has any alternative suggestions, I am open to trying a different method. Thank you.
Regards,
Robert
Robert Hammond
PhD Candidate
Department of Chemistry
University of Alabama at Birmingham
(775)453-6605