Re: [ccp4bb] Off-topic: ligand enrichment
Yingjie Peng wrote: .. After I have solved my strucutre, I have found my target ligand bound at the potential binding site. Also, I have found that there are two more ligand molecules bound along the path from solvent to the binding site. I think this can enrich the ligand to binding site, enhancing the local concentration of the ligand, thus reducing the Km of the ligand. I'm not sure if this is anything like what you see, but sometimes more than one ligand molecule unexpectedly binds in the active site. e.g. http://www.rcsb.org/pdb/explore.do?structureId=1GX http://www.ncbi.nlm.nih.gov/pubmed/15102853?dopt=Abstract Cheers, Charlie -- Charlie Bond Professorial Fellow University of Western Australia School of Biomedical, Biomolecular and Chemical Sciences M310 35 Stirling Highway Crawley WA 6009 Australia charles.b...@uwa.edu.au +61 8 6488 4406
[ccp4bb] Off-topic: ligand enrichment
Dear guys, Sorry for the off-topic question. After I have solved my strucutre, I have found my target ligand bound at the potential binding site. Also, I have found that there are two more ligand molecules bound along the path from solvent to the binding site. I think this can enrich the ligand to binding site, enhancing the local concentration of the ligand, thus reducing the Km of the ligand. I am wondering if anybody can give some suggestions on how to solve this problem clearly. If there is any similar case, it will be better. Thank you in advance. Best wishes, Yingjie Yingjie PENG, Ph.D. student Structural Biology Group Shanghai Institute of Biochemistry and Cell Biology (SIBCB) Shanghai Institute of Biological Sciences (SIBS) Chinese Academy of Sciences (CAS) 320 Yue Yang Road, Shanghai 200031 P. R. China 86-21-54921117 Email: yjp...@sibs.ac.cn
Re: [ccp4bb] Off-topic: ligand enrichment
Yingjie Peng wrote: .. After I have solved my strucutre, I have found my target ligand bound at the potential binding site. Also, I have found that there are two more ligand molecules bound along the path from solvent to the binding site. I think this can enrich the ligand to binding site, enhancing the local concentration of the ligand, thus reducing the Km of the ligand. I've heard this kind of explanation for alternate binding sites before, but I am skeptical. To the extent that the bound ligands are in equilibrium with the bulk phase, the local activity of the ligand will be the same as in the bulk phase- i.e. the bound ligands don't count in figuring the effective concentration. If anything they lower the activity, competing with the active site if the concentration of ligand is not [enzyme]. There would be a local buffering effect, so if the enzyme is gated by a nerve impulse or absorption of a quantum of light so that it is usually inactive and turns on suddenly, the local binding sites could release their load in response to the local depletion faster than ligand could diffuse in from the bulk- Then during the next off period all the local binding sites recharge. This would be like the function of a bypass capacitor in a digital electronic circuit. But during steady-state turnover I don't see how the bound ligand could help any. It may be easy to get ligand in physiologically irrelevant low-affinity sites due to the high concentration of protein in crystallization experiments. The protein is a good fraction of 1 mM, whereas physiologically important binding sites are often in the nM to uM range. So if you add a 3-fold excess of your ligand, and one equivalent binds at the specific site, there will still be 100 uM or so free ligand, which may bind at low affinity non-specific sites. Whether this loosely bound ligand will be well-ordered enough to identify in the density is another question. Just my thoughts on the matter, Ed
Re: [ccp4bb] Off-topic: ligand enrichment
Dear Yingjie, I agree with Ed Berry that I do not believe that nearby binding sites influence the Km (~Kd) which depend on bound and unbound concentrations. However, there could be a strong kinetic effect, e.g. these secondary binding sites could act as stepping stones when the path to the primary binding site would otherwise be difficult to pass. The crystal structure of the potassium channel provides a beautiful example of this. Best regards, Herman From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Yingjie Peng Sent: Monday, February 16, 2009 11:09 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Off-topic: ligand enrichment Dear guys, Sorry for the off-topic question. After I have solved my strucutre, I have found my target ligand bound at the potential binding site. Also, I have found that there are two more ligand molecules bound along the path from solvent to the binding site. I think this can enrich the ligand to binding site, enhancing the local concentration of the ligand, thus reducing the Km of the ligand. I am wondering if anybody can give some suggestions on how to solve this problem clearly. If there is any similar case, it will be better. Thank you in advance. Best wishes, Yingjie Yingjie PENG, Ph.D. student Structural Biology Group Shanghai Institute of Biochemistry and Cell Biology (SIBCB) Shanghai Institute of Biological Sciences (SIBS) Chinese Academy of Sciences (CAS) 320 Yue Yang Road, Shanghai 200031 P. R. China 86-21-54921117 Email: yjp...@sibs.ac.cn
