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. 1994 Sep;3(9):1602–1604. doi: 10.1002/pro.5560030925

Identification of a molecular switch that selects between two crystals forms of bovine pancreatic trypsin inhibitor.

W H Gallagher 1, K M Croker 1
PMCID: PMC2142951  PMID: 7530544

Abstract

Two crystals forms of bovine pancreatic trypsin inhibitor are produced between pH 8.39 and 10.13 when crystals are grown at room temperature from solutions of 1.5 M potassium phosphate. Lower pH values favor the form II crystals, whereas higher pH values favor the form III. The transition from one crystal form to the other occurs at pH 9.35. We examined the crystal lattice contacts in both crystal forms and identified an unusual interaction we believe explains these observations. Spanning the crystallographic 2-fold axis in form III crystals, the Lys 41 side-chain amino nitrogens from 2 symmetry-related molecules are only 2.72 A apart, implying they are hydrogen bonded to one another. In form II crystals, the Lys 41 side-chain amino group is protonated and forms a salt bridge with a solvent-derived phosphate group. For the Lys 41 side-chain amino groups to hydrogen bond in form III crystals, at least 1 member of the pair must be deprotonated. The transition that occurs at pH 9.35 marks the pKa for deprotonation. In solution, the pKa for the Lys 41 side chain is around 10.8. The pKa for one of the interacting Lys 41 side chains in form III crystals is therefore shifted downward by about 1.5 pH units. The energy for lowering the pKa value comes from the many additional intermolecular hydrogen bonds that are present in form III crystals: 19 compared to only 8 in form II crystals.

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Selected References

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  1. Artymiuk P. J., Blake C. C., Grace D. E., Oatley S. J., Phillips D. C., Sternberg M. J. Crystallographic studies of the dynamic properties of lysozyme. Nature. 1979 Aug 16;280(5723):563–568. doi: 10.1038/280563a0. [DOI] [PubMed] [Google Scholar]
  2. Gallagher W., Tao F., Woodward C. Comparison of hydrogen exchange rates for bovine pancreatic trypsin inhibitor in crystals and in solution. Biochemistry. 1992 May 19;31(19):4673–4680. doi: 10.1021/bi00134a020. [DOI] [PubMed] [Google Scholar]
  3. Gros P., Betzel C., Dauter Z., Wilson K. S., Hol W. G. Molecular dynamics refinement of a thermitase-eglin-c complex at 1.98 A resolution and comparison of two crystal forms that differ in calcium content. J Mol Biol. 1989 Nov 20;210(2):347–367. doi: 10.1016/0022-2836(89)90336-7. [DOI] [PubMed] [Google Scholar]
  4. Heinz D. W., Priestle J. P., Rahuel J., Wilson K. S., Grütter M. G. Refined crystal structures of subtilisin novo in complex with wild-type and two mutant eglins. Comparison with other serine proteinase inhibitor complexes. J Mol Biol. 1991 Jan 20;217(2):353–371. doi: 10.1016/0022-2836(91)90549-l. [DOI] [PubMed] [Google Scholar]
  5. Madden D. R., Saper M. A., Garrett T. P., Bjorkman P. J., Strominger J. L., Wiley D. C. Comparison of orthorhombic and monoclinic crystal structures of HLA-A2. Cold Spring Harb Symp Quant Biol. 1989;54(Pt 1):353–359. doi: 10.1101/sqb.1989.054.01.043. [DOI] [PubMed] [Google Scholar]
  6. Phillips G. N., Jr Comparison of the dynamics of myoglobin in different crystal forms. Biophys J. 1990 Feb;57(2):381–383. doi: 10.1016/S0006-3495(90)82540-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Sheriff S., Hendrickson W. A., Stenkamp R. E., Sieker L. C., Jensen L. H. Influence of solvent accessibility and intermolecular contacts on atomic mobilities in hemerythrins. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1104–1107. doi: 10.1073/pnas.82.4.1104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Sobek H., Hecht H. J., Aehle W., Schomburg D. X-ray structure determination and comparison of two crystal forms of a variant (Asn115Arg) of the alkaline protease from Bacillus alcalophilus refined at 1.85 A resolution. J Mol Biol. 1992 Nov 5;228(1):108–117. doi: 10.1016/0022-2836(92)90495-6. [DOI] [PubMed] [Google Scholar]
  9. Tong L., Wengler G., Rossmann M. G. Refined structure of Sindbis virus core protein and comparison with other chymotrypsin-like serine proteinase structures. J Mol Biol. 1993 Mar 5;230(1):228–247. doi: 10.1006/jmbi.1993.1139. [DOI] [PubMed] [Google Scholar]
  10. Walter J., Huber R. Pancreatic trypsin inhibitor. A new crystal form and its analysis. J Mol Biol. 1983 Jul 15;167(4):911–917. doi: 10.1016/s0022-2836(83)80120-x. [DOI] [PubMed] [Google Scholar]
  11. Wlodawer A., Nachman J., Gilliland G. L., Gallagher W., Woodward C. Structure of form III crystals of bovine pancreatic trypsin inhibitor. J Mol Biol. 1987 Dec 5;198(3):469–480. doi: 10.1016/0022-2836(87)90294-4. [DOI] [PubMed] [Google Scholar]
  12. Wlodawer A., Walter J., Huber R., Sjölin L. Structure of bovine pancreatic trypsin inhibitor. Results of joint neutron and X-ray refinement of crystal form II. J Mol Biol. 1984 Dec 5;180(2):301–329. doi: 10.1016/s0022-2836(84)80006-6. [DOI] [PubMed] [Google Scholar]
  13. Yang A. S., Gunner M. R., Sampogna R., Sharp K., Honig B. On the calculation of pKas in proteins. Proteins. 1993 Mar;15(3):252–265. doi: 10.1002/prot.340150304. [DOI] [PubMed] [Google Scholar]

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