From cletner -A_T- remcure.bmb.wright.edu Wed Jul 6 09:01:36 1994 Received: from remcure.bmb.wright.edu for cletner%!at!%remcure.bmb.wright.edu by www.ccl.net (8.6.9/930601.1506) id IAA25468; Wed, 6 Jul 1994 08:28:26 -0400 Received: by remcure.bmb.wright.edu (920330.SGI/921111.SGI.AUTO) for CHEMISTRY-: at :-ccl.net id AA15154; Wed, 6 Jul 94 08:18:44 -0700 Date: Wed, 6 Jul 1994 07:58:32 -0700 (PDT) From: Charles Letner Subject: Protonation state summary To: Computational Chemistry List Message-Id: Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII Hello, I've gotten a number of usefull responses. As was expected the response turned to determining the pKa's of the AA's in the protein (I didn't specifically ask this question because I wanted to see if there where other answers not anticipated). Two other references from Bill Welsh's summary from May 23 are also usefull. Rob Setlik recommends: Bashford et al., (1993) Biochem 32, 8045. Yang et al., (1993) Protein 15, 252-265. Both of these are good papers. The Bashford et al. lists an ftp site ftp.scripps.edu in pub/electrostatics) to obtain the programs and data (Is this site possible a David Case piece of work, could be :) ). I've been there and there is an alpha version of a the program MEAD. I haven't unpacked this yet so I can't tell you much other than it exist. Thanks for the help. Untill next time, Chuck Charles Letner Wright State University Department of Biochemistry Dayton, OH 45435 e-mail: cletner -x- at -x- remcure.bmb.wright.edu ----------------- response summary ----------------------------------- From: "Dr. Walter J. Stevens" Quite a bit of work has been done on ionization states of amino acid side chains in proteins. See for example: M.K. Gilson, Proteins: Structure, Function and Genetics -15-, 266-282 (1993) J. Antosiewocz, J.A. McCammon, and M.K. Gilson J. Molec. Biol. -238-, 415-436 (1994) and references therein. This is a good review of the complexity of the problem you have addressed. -------------------------------------------------- Dr. Walter J. Stevens Center for Advanced Research in Biotechnology National Institute of Standards and Technology and University of Maryland Biotechnology Institute 9600 Gudelsky Drive Rockville, MD 20850 Phone: (301) 738-6264 FAX : (301) 738-6255 E-MAIL: walt /at\ibm1.carb.nist.gov -------------------------------------------------- From: "Jeffrey L. Nauss" I'm running into the same situation myself. The rule of thumb I am using is that if a protein is accessible to the solvent it is protonated as appropriate for the pH of the system under study. Fortunately, I am looking at systems at pH 7.0 or at low pH levels (3-4). Therefore, I feel I can still go for an all or nothing protonation for a given type of amino acid (i.e. all Glu will be protonated at the low pH). And, I do not have to worry about subtle variations in pKa due to local environments. As for salt bridges, the residues involved are charged. Residues at the active site present a different problem. There subtle pKa changes may have dramatic effects. Frankly, I am still a bit baffled as to how to handle them, especially one in my system of interest with a solvent accessible surface of only 1 Angstrom**2. I hope this rambling will be of some benefit to you. I am interested in your summary. Jeff Nauss **************************************************************************** * UU UU Jeffrey L. Nauss, PhD * * UU UU Director, Molecular Modeling Services * * UU UU Department of Chemistry * * UU UU CCCCCCC University of Cincinnati * * UU UU CCCCCCCC Cincinnati, OH 45221-0172 * * UUUU CC * * CC Telephone: 513-556-0148 Fax: 513-556-9239 * * CC * * CCCCCCCC e-mail: nauss #at# ucmod2.che.uc.edu * * CCCCCCC * **************************************************************************** From: "Jim Briggs, Ph.D., U of Houston, Chem. (713)743-3315" Chuck, I can't speak to your main question of the importance of having correct protonation states except that if you get one wrong in a critical area, you will likely see a large effect. I did want to respond to your secondary question of assigning protonation states to AAs in proteins. Using the approach that you suggest (i.e. the "null" model) should work most of the time. Assuming that residues that are on the surface are appropriately ionized depending on the pH and that internal residues are ionized if in "polar" environments (again, depending on pH). Obviously, you need to take a look at ALL histidines... Anyhow, there is an approach that was developed recently to predict ionization states depending on the pH, ionic strength, and electrostatic environment of each AA. The approach uses the electrostatics module of the UHBD (University of Houston Brownian Dynamics) program. The procedure and results are described in a recent paper: J. Antosiewicz, A. McCammon, M. Gilson J. Mol. Biol. 1994, 238, 415-436. In most cases, this approach does better than the "null" model as compared with experimentally determined ionization states. The UHBD manual can be downloaded for free from the anonymous ftp site: 129.7.8.16 under: pub/uhbd in DVI and PostScript forms. The code is available for a nominal fee to academic institutions and is distributed commercially through Molecular Simulations, Inc. I'm sure that a similar approach is available in conjunction with Barry Honig's DELPHI program... Jim Briggs JBriggs-: at :-uh.edu From: Soaring Bear Hi Chuck: I'm glad you posed that question because I've been wondering the same thing about drugs which contain amines which can equilibrate between both protonated & unprotenated states at physiological pH (as can Histidine). The grunt way of handling it would be to run the model each way seperatly but it would seem this is a common enough situation that modeling software ought to incorporate handling of this as a feature. I look foward to seeing the responses you get. bear * UU UU SOARING BEAR * * UU UU Computer Aided Drug Design * * UU UU A University of Arizona, New Pharmacy 404 * * UUUU AAA Tucson, AZ 85721 602-626-6133 * * AA AA where the sun shines * * AAAAAAA * * AA AA e-mail:bear: at :ellington.pharm.arizona.edu * From: Xiaoou Xiang Hi, Chuck: The only way to make certain about the protonation state is, of course, by getting hands wet with test tubes. The computational way to do that, not always reliable, is to locally minimize with the side chain under consideration in all protonation states, and then choose the one in which the side chain has the lowest energy of interaction with the local environment. In practice, one often only needs to ascertain the protonation state of residues in the region of interest in the protein, and assign arbitrary protonation states to other residues, assuming that the influence of (de)protonation does not propagate beyond 8-10 anstroems. Phil X. Xiang Brandeis University From: Paul Beroza People have been worried about this question, which usually falls under the topic, how do you determine an amino acid's pKa when it is bound to a protein? Here are a few references that I have at my fingertips. Paul (pberoza # - at - # ucsd.edu) --- Russel and Warshel, Journal of Molecular Biology, 185:389 (1985). "The Energetics of Ionized Groups in Bovine Pancreatic Trypsin Inhibitor" Bashford and Karplus, Biochemistry, 29: 10219 (1990), "pKa's of Ionizable Groups in Proteins: Atomic Detail from a Continuum Model" Beroza, et al., Proc. Natl. Acad. Sci., 88:5804 (1991), "Protonation of Interacting Residues in a Protein by a Monte Carlo Method: Application to Lysozyme and the Photosynthetic Reaction Center of Rhodobacter sphaeroides" Yang, et al., Proteins, 15:252 (1993), "On the Calculation of pKa's" in Proteins"