Characterization of the pH Titration Shifts of Ribonuclease A by One- and Two-Dimensional Nuclear Magnetic Resonance Spectroscopy (original) (raw)
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FEBS Letters, 1994
Structures of a semisynthetic RNase have been obtained to a resolution of 2.0 8, at pH values of 5.2, 6.5, 7.5, and 8.8, respectively. The principle structural transformation occurring over this pH range is the conversion of the side chain of active site residue His-l 19 from one conformation k, = -43" to -57") at low pH to another k, = + 159" to + 168") at higher pH values. On the basis of this observation, a model is proposed that reconciles the disparate pK values for His-l 19 in the enzyme-substrate complex that have been deduced from kinetic studies and from proton NMR measurements in the presence of pseudosubstrates.
The determination of protonation states in proteins
Acta Crystallographica Section D Biological Crystallography, 2007
The protonation state of aspartic and glutamic acids in native (Mn, Ca) concanavalin A at 0.94, 1.20 and 0.92 Å resolutions. All the graphs that have error bars are derived from the full matrix inversion in SHELXL-97 and the bond lengths are derived from the CGLS refinement in SHELXL-97. In figure 1 the carboxyl group bond lengths (restrained) in aspartic acids for native (Mn,Ca) concanavalin A at 0.94Å resolution is shown (as reported by Deacon et al., 1997; PDB code 1nls). The result shows that Asp28 is clearly protonated with weaker indication of Asp82 and Asp136. After resolution truncation to 1.20 Å (figure 2) there remains protonation indication of Asp28, Asp82 and Asp136 although degraded compared with 0.94 Å. At 1.50 Å resolution (figure 3) Asp28 surprisingly still shows an indication of protonation in spite of the truncation to 1.50 Å. When the restraints were removed at 0.94 Å resolution (figure 4) the protonation indication for Asp28 and Asp82 is clear but that for Asp136 is marginal. The determined distances for Asp28 agree closely with the expected values from the Cambridge Structural Database (CSD) values but those of Asp82 deviate somewhat from the CSD values. At 1.20 resolution (figure 5) Asp28 protonation indication remains confident but Asp82, Asp136 and Asp151 behaviour show differences from the CSD. When the resolution is truncated to 1.50 Å (figure 6), Asp28 protonation indication remains clear but Asp16, Asp58, Asp80, Asp82, Asp136, Asp151 and Asp203 could equally well be assigned 'protonated', which is incorrect. Hence, by 1.50 Å there is no confidence in the evidence that Asp28 is protonated. Figure 7 shows the carboxyl group bond lengths in aspartic acids for native (Mn,Ca) concanavalin A at 1.20 Å resolution, reported by Parkin et al. (1996); PDB code 1jbc. The impact of restraints here is to largely force the distances to 1.25 Å but curiously Asp82 here is still correctly showing indication of the protonation. In figure 8 the carboxyl group bond lengths in aspartic acids for native (Mn,Ca) concanavalin A at 0.92 Å resolution is shown (as reported by Price, 1999). The impact of restraints here does not alter the fact that Asp28, Asp82 and Asp136 are protonated as expected. The estimated single and double bond distances are of course slightly in error being pulled by the 1.25 Å restraint.
Journal of Molecular Biology, 2009
The pKa values of internal ionizable groups are usually very different from the normal pKa values of ionizable groups in water. To examine the molecular determinants of pKa values of internal groups, we compared the properties of Lys, Asp, and Glu at internal position 38 in staphylococcal nuclease. Lys38 titrates with a normal or elevated pKa, whereas Asp38 and Glu38 titrate with elevated pKa values of 7.0 and 7.2, respectively. In the structure of the L38K variant, the buried amino group of the Lys38 side chain makes an ion pair with Glu122, whereas in the structure of the L38E variant, the buried carboxyl group of Glu38 interacts with two backbone amides and has several nearby carboxyl oxygen atoms. Previously, we showed that the pKa of Lys38 is normal owing to structural reorganization and water penetration concomitant with ionization of the Lys side chain. In contrast, the pKa values of Asp38 and Glu38 are perturbed significantly owing to an imbalance between favorable polar interactions and unfavorable contributions from dehydration and from Coulomb interactions with surface carboxylic groups. Their ionization is also coupled to subtle structural reorganization. These results illustrate the complex interplay between local polarity, Coulomb interactions, and structural reorganization as determinants of pKa values of internal groups in proteins. This study suggests that improvements to computational methods for pKa calculations will require explicit treatment of the conformational reorganization that can occur when internal groups ionize.
Prediction of proton chemical shifts in RNA. Their use in structure refinement and validation
Journal of biomolecular NMR, 2001
An analysis is presented of experimental versus calculated chemical shifts of the non-exchangeable protons for 28 RNA structures deposited in the Protein Data Bank, covering a wide range of structural building blocks. We have used existing models for ring-current and magnetic-anisotropy contributions to calculate the proton chemical shifts from the structures. Two different parameter sets were tried: (i) parameters derived by Ribas-Prado and Giessner-Prettre (GP set) [(1981) J. Mol. Struct., 76, 81-92.]; (ii) parameters derived by Case [(1995) J. Biomol. NMR, 6, 341-346]. Both sets lead to similar results. The detailed analysis was carried using the GP set. The root-mean-square-deviation between the predicted and observed chemical shifts of the complete database is 0.16 ppm with a Pearson correlation coefficient of 0.79. For protons in the usually well-defined A-helix environment these numbers are, 0.08 ppm and 0.96, respectively. As a result of this good correspondence, a reliable ...
Proton Transfers in Enzyme and Ribozyme Active Sites
We will briefly discuss two biochemical problems where proton transfer (PT) plays a key role. In the first , we apply, using QM/MM simulations, a nontraditional picture for PT (in which the reaction coordinate is dominated by the rearrangement of the environment) to the initial rate-limiting enzymatic proton abstraction step in triosephosphate isomerase (TIM). Key groups involved in the environmental rearrangement of this enzymatic reaction and their energetic costs are identified. The contribution of multiple Valence Bond states to electronic structure along the reaction path is also discussed. In the second topic, we explore in an electronic structure study the role of PT in peptide bond formation (PBF), which involves a nucleophilic attack of an NH2 on an electrophilic carbonyl carbon. This is a crucial chemical step in the elongation cycle of protein synthesis, in the peptidyl transferase center (PTC) of the large ribosomal subunit, and the risome is known to be a ribozyme. The ...
Journal of the American Chemical Society, 1976
The pH dependence of the 31P chemical shifts of 3'-, 5'-, and 2'-cytidine monophosphate and 3'-uridine monophosphate, both free in solution and when bound to bovine, pancreatic ribonuclease A has been determined by both chemical exchange and direct observation methods. The 3 1 P N M R titration data demonstrate that each nucleotide binds to the enzyme in the dianionic ionization state around neutral pH. Two ionizations are observed for the complex, with pK1 = 4.0-5.5 and pK2 = 5.9-6.7. The first pK is associated with ionization of the monoanionic inhibitor and the second with ionization of the protonated histidine12 residue which hydrogen bonds to the phosphate. Apparently, the 31P chemical shifts of the phosphate esters are only affected by the protonation state and not by the highly positive local environment of the enzyme. Thus, the chemical shift of 3'-CMP is shifted upfield by only 10 H z at neutral pH despite the proximity of protonated histidinells and lysine41 and partially protonated histidinelz. In addition, as long as the imidazole N remains the hydrogen-bonding donor, the phosphate 31P chemical shift is unaffected by a hydrogen-bonding interaction. This property provides a unique opportunity to calculate microscopic ionization constants and allows a detailed description of the ionic states involved in the binding process.