title: Protonation-state determination in proteins using high-resolution X-ray crystallography: effects of resolution and completeness.
abstract: A bond-distance analysis has been undertaken to determine the protonation states of ionizable amino acids in trypsin, subtilisin and lysozyme. The diffraction resolutions were 1.2 Å for trypsin (97% complete, 12% H-atom visibility at 2.5σ), 1.26 Å for subtilisin (100% complete, 11% H-atom visibility at 2.5σ) and 0.65 Å for lysozyme (PDB entry 2vb1; 98% complete, 30% H-atom visibility at 3σ). These studies provide a wide diffraction resolution range for assessment. The bond-length e.s.d.s obtained are as small as 0.008 Å and thus provide an exceptional opportunity for bond-length analyses. The results indicate that useful information can be obtained from diffraction data at around 1.2-1.3 Å resolution and that minor increases in resolution can have significant effects on reducing the associated bond-length standard deviations. The protonation states in histidine residues were also considered; however, owing to the smaller differences between the protonated and deprotonated forms it is much more difficult to infer the protonation states of these residues. Not even the 0.65 Å resolution lysozyme structure provided the necessary accuracy to determine the protonation states of histidine.
authors: School of Chemistry, University of Manchester, Brunswick Street, Manchester M13 9PL, England. fisher@ill.fr
authors: S J
authors: SJ
authors: Fisher
authors: M P
authors: MP
authors: Blakeley
authors: M
authors: M
authors: Cianci
authors: S
authors: S
authors: McSweeney
authors: J R
authors: JR
authors: Helliwell
mesh_terms: D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001407:Bacillus / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D002352:Carrier Proteins / Q000737:chemistry; D002417:Cattle; D002645:Chickens; D018360:Crystallography, X-Ray / Q000379:methods; D006639:Histidine / Q000737:chemistry; D006801:Humans; D007477:Ions / Q000737:chemistry; D009113:Muramidase / Q000737:chemistry*; D011522:Protons; D013381:Subtilisins / Q000737:chemistry*; D014357:Trypsin / Q000737:chemistry*; D000099163:Myosin Binding Protein C
chemical_list: D000596:Amino Acids; D001426:Bacterial Proteins; D002352:Carrier Proteins; D006639:Histidine; D007477:Ions; D009113:Muramidase; D011522:Protons; D013381:Subtilisins; D014357:Trypsin; D000099163:Myosin Binding Protein C