Re: [ccp4bb] oof topic: pH effect on substrate analog
On Tue, 2012-10-30 at 16:12 +, Peter Hsu wrote: I'm wondering, since I lack activity at this pH point, would it lead to no binding of a substrate analog? Not necessarily. You should check pH dependence of the Km - it might be that lower activity is primarily due to reduction in kcat. Binding studies are always a good idea before trying to soak/cocrystallize. With that said, it's entirely possible that you don't see ligand in the electron density because the enzyme is stuck in a wrong conformation or binding site is blocked. I wonder if you can get resolve pH issue by cross-linking the crystals at which point you can use whichever soaking buffer pH you want (assuming no loss of diffraction) Cheers, Ed. -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
[ccp4bb] oof topic: pH effect on substrate analog
Hi all, I'm working on a protein that I recently got crystals of. My functional studies show that the protein has optimal activity at lower pHs, while losing 90% activity at about pH8. I've been trying to soak/cocrystallize a substrate analog (small molecule) into my crystals (grown at ~pH8) with no real luck. I'm wondering, since I lack activity at this pH point, would it lead to no binding of a substrate analog? Thanks for any insights Peter
Re: [ccp4bb] oof topic: pH effect on substrate analog
Dear Peter, You could check your putative binding site to see if there are charged groups which need to be protonated for your substrate analog to bind (or whether the substrate analog needs to be protonated). In order to draw physiologically relevant conclusions, you would need a crystal structure at a pH where the protein is active. Try if you can transfer your crystals to lower pH, perhaps by significantly increasing the precipitant concentration. Good luck! Herman -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Peter Hsu Sent: Tuesday, October 30, 2012 5:13 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] oof topic: pH effect on substrate analog Hi all, I'm working on a protein that I recently got crystals of. My functional studies show that the protein has optimal activity at lower pHs, while losing 90% activity at about pH8. I've been trying to soak/cocrystallize a substrate analog (small molecule) into my crystals (grown at ~pH8) with no real luck. I'm wondering, since I lack activity at this pH point, would it lead to no binding of a substrate analog? Thanks for any insights Peter
Re: [ccp4bb] oof topic: pH effect on substrate analog
Peter, I think it would depend if the substrate analog have ionizable groups? If the analog does not have ionizable groups, it is hard to imagine how the the titration of ionizable groups on the protein would impair the binding. Chitta - Original Message - From: Peter Hsu hsuu...@u.washington.edu To: CCP4BB@JISCMAIL.AC.UK Sent: Tuesday, October 30, 2012 12:12:58 PM Subject: [ccp4bb] oof topic: pH effect on substrate analog Hi all, I'm working on a protein that I recently got crystals of. My functional studies show that the protein has optimal activity at lower pHs, while losing 90% activity at about pH8. I've been trying to soak/cocrystallize a substrate analog (small molecule) into my crystals (grown at ~pH8) with no real luck. I'm wondering, since I lack activity at this pH point, would it lead to no binding of a substrate analog? Thanks for any insights Peter
Re: [ccp4bb] oof topic: pH effect on substrate analog
As for most questions in science, the answer is unfortunately it depends. A substrate analog may bind just peachy at a pH in which the enzyme is inactive, depending on what interactions are involved in stabilizing the analog compared to the substrate transition state. Such a complex may still be structurally informative. Changing the ionization states of active site groups may or may not have a significant effect on binding. One must consider not only electrostatic interactions, but also hydrogen bonding donor-acceptor interactions. Lowering pH, for example, may turn H-bonding acceptors into obligate donors, or in the case of metalloenzymes, make metal-bound water/hydroxide labile for exchange. In any event, I'd be willing to bet that a substrate analog structure at a non-optimal pH is more informative than no structure at the optimal pH! :) Cheers, ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 10/30/2012 1:39 PM, Chittaranjan Das wrote: Peter, I think it would depend if the substrate analog have ionizable groups? If the analog does not have ionizable groups, it is hard to imagine how the the titration of ionizable groups on the protein would impair the binding. Chitta - Original Message - From: Peter Hsu hsuu...@u.washington.edu To: CCP4BB@JISCMAIL.AC.UK Sent: Tuesday, October 30, 2012 12:12:58 PM Subject: [ccp4bb] oof topic: pH effect on substrate analog Hi all, I'm working on a protein that I recently got crystals of. My functional studies show that the protein has optimal activity at lower pHs, while losing 90% activity at about pH8. I've been trying to soak/cocrystallize a substrate analog (small molecule) into my crystals (grown at ~pH8) with no real luck. I'm wondering, since I lack activity at this pH point, would it lead to no binding of a substrate analog? Thanks for any insights Peter
