Data from the García-Moreno Lab, Department of Biophysics, Johns
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1 Data from the García-Moreno Lab, Department of Biophysics, Johns Hopkins U. Note about numbering of sequence: Many of our experimental studies, and many of our crystal structures and on-going studies with NMR spectroscopy, were performed with hyperstable forms of staphylococcal nuclease. PHS nuclease is a variant with three substitutions (P117G, H124L, and S128A). Δ+PHS is PHS with two more substitutions (G50F, V51N) and with a deletion (residues 44 - 49). Please note that the sequence of Δ+PHS was numbered using the sequence of wild type nuclease. For this reason there is a gap between residues 44 and 49. Also note that the N and C termini of nuclease are usually not observed in crystal structures. Residues in this region of the protein are missing in all pdb files. Some crystals include an inhibitor: Some of the structures were obtained from crystals grown in the presence of Ca2+ and the inhibitor pdTp. Table I: Crystal structures of wild type staphylococcal nuclease (SNase) and hyperstable variants known as PHS and Δ+PHS. Accession code Protein Parent Protein Temp of Xray (K) Temp (K) and ionic strength (M) of pKa Authors NMR data pKa of interest Ref 1stn wt - 100 298, 0.01, 0.10, 1.0 Fox - All His 1,16, 17 1snc wt + Ca & pdTp - 100 - Lattman - - 2 3bdc Δ+PHS - 100 298, 0.01, 0.10, 1.0 GM Lab - All His, Asp and Glu 3 1ey8 PHS - 100 - Stites - - 4 2 Table II: Variants with internal ionizable groups for which pKa values and structures have been published (or are under review). Accession code Protein Parent Protein Temp of Xray (K) Temp (K) and ionic strength (M) of pKa Authors NMR data 1 pKa of interest2 Ref 2rks L38K PHS 100 298, 0.1 GM Lab Yes Lys-38 5 3d6c L38E PHS 100 298, 0.1 GM Lab Yes Glu-38 6 2snm V66K wt 100 298, 0.1 Lattman No Lys-66 7 no structure V66K PHS 298, 0.1 GM Lab No Lys-66 8 Available from GM V66K Δ+PHS 100, 298 298, 0.1 GM Lab Yes Lys-66 9 1u9r V66E PHS 298 298, 0.1 GM Lab Yes Glu-66 10 2oxp V66D PHS 100 298, 0.1 GM Lab Yes Asp-66 11 1tt2 I92K Δ+PHS 100 298, 0.1 Lattman Yes Lys-92 12 1tr5 I92E Δ+PHS 298 298, 0.1 Lattman Not yet Glu-92 12 1tqo I92E Δ+PHS 100 298, 0.1 Lattman Not yet Glu-92 12 2oeo I92D Δ+PHS 100 298, 0.1 Lattman Not yet Asp-92 12 3 Table III: Variants with internal ionizable groups for which structures have been released to the PDB in advance of publication, but for which pKa values have not been published. All pKa values were measured in variants made from the Δ+PHS background. Accession code Protein Parent Protein Temp of Xray (K) Temp (K) and ionic strength (M) of pKa Authors NMR data 1 pKa of interest2 Ref 3erq L25K Δ+PHS 100 298, 0.1 GM Lab Yes Lys-25 153 3evq L25E Δ+PHS 100 298, 0.1 GM Lab Not yet Glu-25 153 3eji L36K Δ+PHS 100 298, 0.1 GM Lab Yes Lys-36 153 3dmu T62K PHS 100 298, 0.1 GM Lab Yes Lys-62 153 2rbm I72K Δ+PHS 100 298, 0.1 GM Lab Yes Lys-72 153 3ero I72E Δ+PHS 100 298, 0.1 GM Lab Not yet Glu-72 153 3d8g I72R Δ+PHS 100 298, 0.1 GM Lab Not yet Arg-72 153 3dhq A90R Δ+PHS 100 298, 0.1 GM Lab Not yet Arg-90 153 3d4d Y91E Δ+PHS 100 298, 0.1 GM Lab Not yet Glu-91 153 3e5s L103K Δ+PHS 100 298, 0.1 GM Lab Yes Lys-103 153 3c1f V104K Δ+PHS 100 298, 0.1 GM Lab Yes Lys-104 153 3d4w A109R Δ+PHS 100 298, 0.1 GM Lab Not yet Arg-109 153 3c1e L125K PHS 100 298, 0.1 GM Lab Yes Lys-125 153 4 Table IV: Other internal sites that were substituted with Glu, Lys, Arg, Asp. Neither structures nor pKa values have been published. All pKa values were measured in variants made from the Δ+PHS background. Accession code Protein Parent Protein Tem p of Xray (K) Temp Authors (K) and ionic strength (M) of pKa NMR data1 pKa of interest2 Ref - G20D Δ+PHS 100 298, 0.1 GM Lab Not yet Asp-20 153 - G20E Δ+PHS 100 298, 0.1 GM Lab Not yet Glu-20 153 - G20K Δ+PHS 100 298, 0.1 GM Lab Yes Lys-20 153 - G20R Δ+PHS 100 298, 0.1 GM Lab Not yet Arg-20 153 - V23D Δ+PHS 100 298, 0.1 GM Lab Not yet Asp-23 153 - V23E Δ+PHS 100 298, 0.1 GM Lab Not yet Glu-23 153 - V23K Δ+PHS 100 298, 0.1 GM Lab Yes Lys-23 153 - V23R Δ+PHS 100 298, 0.1 GM Lab Not yet Arg-23 153 - L25D Δ+PHS 100 298, 0.1 GM Lab Not yet Asp-25 153 - L25R Δ+PHS 100 298, 0.1 GM Lab Not yet Arg-25 153 - F34D Δ+PHS 100 298, 0.1 GM Lab Not yet Asp-34 153 - F34E Δ+PHS 100 298, 0.1 GM Lab Not yet Glu-34 153 - F34K Δ+PHS 100 298, 0.1 GM Lab Yes Lys-34 153 - F34R Δ+PHS 100 298, 0.1 GM Lab Not yet Arg-34 153 5 - L36D Δ+PHS 100 298, 0.1 GM Lab Not yet Asp-36 153 - L36E Δ+PHS 100 298, 0.1 GM Lab Not yet Glu-36 153 - L36R Δ+PHS 100 298, 0.1 GM Lab Not yet Arg-36 153 - L37D Δ+PHS 100 298, 0.1 GM Lab Not yet Asp-37 153 - L37E Δ+PHS 100 298, 0.1 GM Lab Not yet Glu-37 153 - L37K Δ+PHS 100 298, 0.1 GM Lab Yes Lys-37 153 - L37R Δ+PHS 100 298, 0.1 GM Lab Not yet Arg-37 153 - L38D Δ+PHS 100 298, 0.1 GM Lab Not yet Asp-38 153 - L38R Δ+PHS 100 298, 0.1 GM Lab Not yet Arg-38 153 - V39D Δ+PHS 100 298, 0.1 GM Lab Not yet Asp-39 153 - V39E Δ+PHS 100 298, 0.1 GM Lab Not yet Glu-39 153 - V39K Δ+PHS 100 298, 0.1 GM Lab Yes Lys-39 153 - V39R Δ+PHS 100 298, 0.1 GM Lab Not yet Arg-39 153 - T41D Δ+PHS 100 298, 0.1 GM Lab Not yet Asp-41 153 - T41E Δ+PHS 100 298, 0.1 GM Lab Not yet Glu-41 153 - T41K Δ+PHS 100 298, 0.1 GM Lab Yes Lys-41 153 - T41R Δ+PHS 100 298, 0.1 GM Lab Not yet Arg-41 153 - A58D Δ+PHS 100 298, 0.1 GM Lab Not yet Asp-58 153 - A58E Δ+PHS 100 298, 0.1 GM Lab Not yet Glu-58 153 6 - A58K Δ+PHS 100 298, 0.1 GM Lab Yes Lys-58 153 - A58R Δ+PHS 100 298, 0.1 GM Lab Not yet Arg-58 153 - T62D Δ+PHS 100 298, 0.1 GM Lab Not yet Asp-62 153 - T62E Δ+PHS 100 298, 0.1 GM Lab Not yet Glu-62 153 - T62R Δ+PHS 100 298, 0.1 GM Lab Not yet Arg-62 153 - I72D Δ+PHS 100 298, 0.1 GM Lab Not yet Asp-72 153 - V74D Δ+PHS 100 298, 0.1 GM Lab Not yet Asp-74 153 - V74E Δ+PHS 100 298, 0.1 GM Lab Not yet Glu-74 153 - V74K Δ+PHS 100 298, 0.1 GM Lab Yes Lys-74 153 - V74R Δ+PHS 100 298, 0.1 GM Lab Not yet Arg-74 153 - A90D Δ+PHS 100 298, 0.1 GM Lab Not yet Asp-90 153 - A90E Δ+PHS 100 298, 0.1 GM Lab Not yet Glu-90 153 - A90K Δ+PHS 100 298, 0.1 GM Lab Yes Lys-90 153 - Y91D Δ+PHS 100 298, 0.1 GM Lab Not yet Asp-91 153 - Y91K Δ+PHS 100 298, 0.1 GM Lab Yes Lys-91 153 - Y91R Δ+PHS 100 298, 0.1 GM Lab Not yet Arg-91 153 - V99D Δ+PHS 100 298, 0.1 GM Lab Not yet Asp-99 153 - V99E Δ+PHS 100 298, 0.1 GM Lab Not yet Glu-99 153 - V99K Δ+PHS 100 298, 0.1 GM Lab Yes Lys-99 153 7 - V99R Δ+PHS 100 298, 0.1 GM Lab Not yet Arg-99 153 - N100D Δ+PHS 100 298, 0.1 GM Lab Not yet Asp-100 153 - N100E Δ+PHS 100 298, 0.1 GM Lab Not yet Glu-100 153 - N100K Δ+PHS 100 298, 0.1 GM Lab Yes Lys-100 153 - N100R Δ+PHS 100 298, 0.1 GM Lab Not yet Arg-100 153 - L103D Δ+PHS 100 298, 0.1 GM Lab Not yet Asp-103 153 - L103E Δ+PHS 100 298, 0.1 GM Lab Not yet Glu-103 153 - L103R Δ+PHS 100 298, 0.1 GM Lab Not yet Arg-103 153 - V104D Δ+PHS 100 298, 0.1 GM Lab Not yet Asp-104 153 - V104E Δ+PHS 100 298, 0.1 GM Lab Not yet Glu-104 153 - V104R Δ+PHS 100 298, 0.1 GM Lab Not yet Arg-104 153 - A109D Δ+PHS 100 298, 0.1 GM Lab Not yet Asp-109 153 - A109E Δ+PHS 100 298, 0.1 GM Lab Not yet Glu-109 153 - A109K Δ+PHS 100 298, 0.1 GM Lab Yes Lys-109 153 - N118D Δ+PHS 100 298, 0.1 GM Lab Not yet Asp-118 153 - N118E Δ+PHS 100 298, 0.1 GM Lab Not yet Glu-118 153 - N118K Δ+PHS 100 298, 0.1 GM Lab Yes Lys-118 153 - N118R Δ+PHS 100 298, 0.1 GM Lab Not yet Arg-118 153 - L125D Δ+PHS 100 298, 0.1 GM Lab Not yet Asp-125 153 8 - L125E Δ+PHS 100 298, 0.1 GM Lab Not yet Glu-125 153 - L125R Δ+PHS 100 298, 0.1 GM Lab Not yet Arg-125 153 - A132D Δ+PHS 100 298, 0.1 GM Lab Not yet Asp-132 153 - A132E Δ+PHS 100 298, 0.1 GM Lab Not yet Glu-132 153 - A132K Δ+PHS 100 298, 0.1 GM Lab Yes Lys-132 153 - A132R Δ+PHS 100 298, 0.1 GM Lab Not yet Arg-132 153 Table V: Other potentially relevant structures, especially to those interested in the problem of water penetration or electrostatic interactions through networks. Accession code Protein Parent Protein Tem p of Xray (K) Temp (K) and ionic strength (M) of pKa Authors NMR data1 pKa of interest2 Ref 2pw5 V66Y PHS 298 - GM Lab No - 13 2pw7 V66Y PHS 100 - GM Lab No - 13 2pzw V66N PHS 298 - GM Lab No - 13 2pzu V66N PHS 100 - GM Lab No - 13 2pyk V66Q PHS 298 - GM Lab No - 13 2pzt V66Q PHS 100 - GM Lab No - 13 9 2qdb E75Q NVIAGA 100 298, 0.1, 1.0 GM Lab Yes histidines 14 2rdf E75A NVIAGA 100 298, 0.1, 1.0 GM Lab Yes histidines 14 1 The pKa values of all the internal ionizable groups have been measured by analysis of the pH dependence of stability (ΔG). Some have also been measured with NMR spectroscopy. We have also began to gather NMR data to describe the structural consequences of ionization of the internal groups. None of the NMR data have been released although, data for some of the Lyscontaining variants is in preparation for submission. 2 The pKa values of surface Asp and Glu residues are being measured with NMR spectroscopy in some of the variants with internal Lys-residues. 3 This publication only describes stability at pH 5, 7, and 10. The pKa values of Glu, Lys, Arg and Asp at the 25 internal positions will likely appear, in that order, before July 2009. 1. Hynes, T. R. and Fox, R. O. (1991) Proteins: Structure, Function, Genetics, 10: 92-105. 2. P. J. Loll and E. E. Lattman (1989) Proteins: Structure, Function, Genetics 5: 183-201. 3. C. A. Castaneda, C. A. Fitch, A. Majumdar, V. Khangulov, J. L. Schlessman, B. GarciaMoreno (under review in Proteins) 4. J. Chen, Z. Lu, J. Sakon, W. E. Stites (2000) J. Mol. Biol. 303:125-130. 5. M. J. Harms, J. L. Schlessman, M. S. Chimenti, G. R. Se, A. Damjanovic, and B . GarciaMoreno (2008) Protein Science 17: 833-845. 6. M. J. Harms, C. A. Castaneda, J. L. Schlessman, G. R. Sue, and B. Garcia-Moreno (under review in JMB). 7. W. E. Stites, A. G. Gittis, E. E. Lattman, D. Shortle (1991) J. Mol. Biol. 221: 7-14 8. B. Garcia-Moreno, J. J. Dwyer, A. G. Gittis, E. E. Lattman, D. S. Spencer, W. E. Stites (1997) Biophys. Chem. 64: 211-224. 9. C. A. Fitch, D. A. Karp, K. K. Lee, W. E. Stites, E. E. Lattman, and B. Garcia-Moreno (2002) Biophys. J. 82: 3289-3304 10. J. J. Dwyer, A. G. Gittis, D. A. Karp, E. E. Lattman, D. S. Spencer, W. E. Stites, B. Garcia-Moreno (2000) Biophys. J. 79: 1610-1620 11. D. A. Karp, A. G. Gittis, M. R. Stahley, C. A. Fitch, W. E. Stites, B. Garcia-Moreno (2007) Biophys. J. 92: 2041-2053. 12. D. M. Nguyen, R. L. Reynald, A. G. Gittis and E. E. Lattman (2004) J. Mol. Biol. 341: 565-574. 13. J. L. Schlessman, C. Abe, A. Gittis, D. A. Karp, M. A. Dolan, and B. Garcia-Moreno (2008) Biophys. J. 94: 3208-33216 10 14. K. L. Baran, M. S. Chimenti, J. L. Schlessman, C. A. Fitch, K. J. Herbst, and B. GarciaMoreno J. Mol. Biol. 379: 1045-1062. 15. D. G. Isom, B. R. Cannon, C. A. Castaneda, A. Robinson, and B. Garcia-Moreno (2008) Proc. Natl. Acad. Sci. USA 105: 17784-17788 16. K. K. Lee, C. A. Fitch, J. T. J. Lecomte, and B. Garcia-Moreno (2002) Biochemistry 41, 5656-5667 17. K. K. Lee, C. A. Fitch, and B. Garcia-Moreno (2002) Protein Science 11:1004-1016
