Protonation state summary

 	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 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,
 Charles Letner
 Wright State University
 Department of Biochemistry
 Dayton, OH 45435
 e-mail: cletner[ AT ]
 ----------------- response summary -----------------------------------
 From: "Dr. Walter J. Stevens" <walt[ AT ]>
 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 ]
 From: "Jeffrey L. Nauss" <nauss[ AT ]ucmod2.che.uc.EDU>
 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                           *
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 From: "Jim Briggs, Ph.D., U of Houston,
        Chem. (713)743-3315" <BRIGGS[ AT ]>
   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:  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 ]
 From: Soaring Bear<bear[ AT ]ellington.pharm.Arizona.EDU>
 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.
 * 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              *
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 *    AA     AA e-mail:bear[ AT ] *
 From: Xiaoou Xiang <xiang[ AT ]>
 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 <ppb[ AT ]>
 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.
 (pberoza[ AT ]
 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"