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-- == response #3 == >From mndoci_at_yahoo.com Wed May 30 14:11:42 2001 Date: Mon, 28 May 2001 08:52:41 -0700 (PDT) From: Deepak <mndoci_at_yahoo.com> To: Christian Pilger <cpilger_at_oc30.uni-paderborn.de> Subject: Re: CCL:titratable amino acids and counter ions Dear Dr. Pilger Those are very valid questions and I suspect different people will have different answers. The best solution is some experimental evidence pointing to the protonation states of such residues. In the absence of experimental evidence, I personally like to examine the local environment of each residue. Counterions are best used to neutralize a on-neutral protein. Althouth the isoelectric point gives you a decent handle on gross protein properties, it is the local pKa that governs the protonation state of titratable groups. In doubt, I have actually performed calculations using both both protonation states and examining the differences and trying to reconcile them with any known observations. There are of cousre, methods to try and calculate the protonation states but I do not trust them yet. Hope this helps Regards Deepak == response #4 == >From rvenable_at_gandalf.cber.nih.gov Wed May 30 14:11:50 2001 Date: Mon, 28 May 2001 13:45:12 -0400 From: Rick Venable <rvenable_at_gandalf.cber.nih.gov> To: Christian Pilger <cpilger_at_oc30.uni-paderborn.de> Subject: Re: CCL:titratable amino acids and counter ions On Mon, 28 May 2001, Christian Pilger wrote: > while preparing a protein structure as input for MD simulations, I > encountered the following points, which I would like to ask your advice: > > - How do you usually deal with titratable residues (GLU, ASP, LYS, ARG) ? > Are all of them simply taken in their ionized forms ? Would it be good > to try to rationalize the ionization states of these residues by > manually inspecting their environment ? Which methods/rules of thumb are > available for the estimation of these ionization states ? It might be worth considering (or calculating) burial, e.g. accessible surface area, for GLU, ASP, LYS, ARG; the rule of thumb for these 4 is that they are typically charged. HIS residues can be a bigger problem, esp. for enzymes, because of the tautomerism in addition to the charge state. People have used computational methods to try to estimate the protonation state, esp. Poisson-Boltzman methods. I know there are some freely available progs for this, but I don't have refs handy. > - In my case the protein has an experimentally determined isoelectric > point of roughly pH=4.7. Does this mean, that the protein will have a > negative net charge at pH=7 ? Then, is there a method to correlate the > isoelectric point with this net charge ? And, are MD calculations > reasonable, when the net charge of the system differs from zero ? Yes, the net charge will be negative. There can be problems with a non-zero net charge for particle-mesh Ewald methods. > - In case the net charge is neutralized by counter ions: how and where > should these be positioned ? Should one counter ion be placed directly > at each titratable residue (i.e. number of counter ions = number of > ioniziable residues) or should counter ions be placed (arbitrarily ?) > around the protein structure (i.e. number counter ions = net charge of > the whole system) ? I don't think there's complete agreement about the best wat to get a net zero charged system, or how to place the ions. I use a method for randomly replacing water molecules more than 5 or 6 A away from any non-water molecule. Two common neutralizing approaches are: (1) just enough counterions for net zero charge, or (2) e.g. 0.1 M salt added to (1) to better model ionic strength. =+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+= Rick Venable FDA/CBER/OVRR Biophysics Lab 1401 Rockville Pike HFM-419 Rockville, MD 20852-1448 U.S.A. (301) 496-1905 Rick_Venable_at_nih.gov ALT email: rvenable_at_speakeasy.org =+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+=+= == response #5 == >From mford_at_ccrc.uga.edu Wed May 30 14:11:57 2001 Date: Mon, 28 May 2001 13:57:53 -0400 From: Michael Ford <mford_at_ccrc.uga.edu> To: Christian Pilger <cpilger_at_oc30.uni-paderborn.de> Subject: Placing counter ions We have found that it may be best to simply place the ions near their corresponding charged group then run 500-100ps and take the avg coordinates for the ion. The CION program, part of AMBER, is really designed for DNA and my give you a "salty" system with a bunch of Na and Cl ions. Mike Ford == response #6 == >From kaynjay_at_igalaxy.net Wed May 30 14:12:03 2001 Date: Mon, 28 May 2001 14:28:19 -0700 From: Kenward Vaughan <kaynjay_at_igalaxy.net> To: Christian Pilger <cpilger_at_oc30.uni-paderborn.de> Subject: Re: titratable amino acids and counter ions On Mon, May 28, 2001 at 01:49:36PM +0200, Christian Pilger wrote: > Dear protein modelers, > > while preparing a protein structure as input for MD simulations, I > encountered the following points, which I would like to ask your advice: > > - How do you usually deal with titratable residues (GLU, ASP, LYS, ARG) ? > Are all of them simply taken in their ionized forms ? Would it be good > to try to rationalize the ionization states of these residues by > manually inspecting their environment ? Which methods/rules of thumb are > available for the estimation of these ionization states ? I have no experience with this in the field of MD,etc, but from a chemical perspective the side chains of residues (not the amino acids themselves) will behave like any other carboxylic acid or amine. With the vast bulk of those having pKa's and pKb's in the range of 4-5 (the amines being related to pOH), it is totally reasonable to assume they are >99% ionized at neutral pH (pOH). If surrounded by others of similar behavior (bases-bases and acids-acids) the expected effect of multiple ionizations becoming less and less likely makes sense, but as the strength of electrostatic attractions follow the inverse-square law I cannot guess how important this effect is. Unless there are several within say.. 5 or 6 angstroms, it may not make a lot of difference on the % ionization. But this is speculation on my part. > - In my case the protein has an experimentally determined isoelectric > point of roughly pH=4.7. Does this mean, that the protein will have a > negative net charge at pH=7 ? Then, is there a method to correlate the > isoelectric point with this net charge ? And, are MD calculations > reasonable, when the net charge of the system differs from zero ? Yes to the negative at pH 7 question. Don't know about the 2nd one. And an "I would certainly hope so!" to the 3rd. :-) > - In case the net charge is neutralized by counter ions: how and where > should these be positioned ? Should one counter ion be placed directly > at each titratable residue (i.e. number of counter ions = number of > ioniziable residues) or should counter ions be placed (arbitrarily ?) > around the protein structure (i.e. number counter ions = net charge of > the whole system) ? Beyond my experience, sorry. I would guess place them arbitrarily and let them settle into place. > Any comments are highly appreciated. ... Hope this helps! Kenward VaughanReceived on Wed May 30 2001 - 05:23:30 PDT