Analysing the pH-dependent properties of proteins using pKa calculations

Nielsen, Jens Erik

doi:10.1016/j.jmgm.2006.05.007

Cited by 38 publications

(25 citation statements)

References 45 publications

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“…CHARMM-27 all-atom force field with CMAP correction(25) was used for the protein and ions, the TIP3P force field was used for water molecules, whereas the carbohydrate Chain Solution Force Field (CSFF) was used for the oligosaccharide tail(26). The pKa of all the charged residues was calculated using the pKaTool software(27) to determine the protonation state of the amino acids in the protein at pH 7. NAMD 2.9 software package(28) was used to run the simulations.…”

Section: Methodsmentioning

confidence: 99%

Loop Electrostatics Asymmetry Modulates the Preexisting Conformational Equilibrium in Thrombin

Pozzi

Zerbetto²,

Acquasaliente³

et al. 2016

Biochemistry

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Thrombin exists as an ensemble of active (E) and inactive (E*) conformations that differ for their accessibility to the active site. Here we show that redistribution of the E*-E equilibrium can be achieved by perturbing the electrostatic properties of the enzyme. Removal of the negative charge of the catalytic Asp102 or Asp189 in the primary specificity site destabilizes the E form and causes a shift in the 215–217 segment that compromises substrate entrance. Solution studies and existing structures of D102N document stabilization of the E* form. A new high resolution structure of D189A also reveals the mutant in the collapsed E* form. These findings establish a new paradigm for the control of the E*-E equilibrium in the trypsin fold.

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Section: Methodsmentioning

confidence: 99%

Loop Electrostatics Asymmetry Modulates the Preexisting Conformational Equilibrium in Thrombin

Pozzi

Zerbetto²,

Acquasaliente³

et al. 2016

Biochemistry

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“…The majority of DpK a values are calculated equally well with both methods, but for a significant fraction there is a marked difference in the results. These cases fall into two general categories: (1) mutations where the intrinsic pK a value of the mutant is inappropriate for inducing a pK a shift in the target, and (2) mutations that interact strongly with the target residue and thus cause either a breakdown of typical Henderson-Hasselbalch titrational behavior or create a nonadditive titratable system (Nielsen 2006). Figure 3A shows the distance dependence of DpK a values for single mutations, and it is seen that a single mutation must be quite close (<13Å ) to a target residue to achieve a significant effect on the target pK a value.…”

Section: The Data Setmentioning

confidence: 99%

“…Such buffering effects can exist in artificially constructed systems (Nielsen 2006), but it is uncertain whether enzyme active sites display such effects. Table 1 compares the average DpK a values obtained for active site targets with those obtained for all targets, and it is clear that active site DpK a values are not buffered by their environment.…”

Section: Multiple Mutationsmentioning

confidence: 99%

Redesigning protein pK_avalues

Tynan-Connolly

Nielsen

2007

Protein Science

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The ability to re-engineer enzymatic pH-activity profiles is of importance for industrial applications of enzymes. We theoretically explore the feasibility of re-engineering enzymatic pH-activity profiles by changing active site pK a values using point mutations. We calculate the maximum achievable DpK a values for 141 target titratable groups in seven enzymes by introducing conservative net-charge altering point mutations. We examine the importance of the number of mutations introduced, their distance from the target titratable group, and the characteristics of the target group itself. The results show that multiple mutations at 10Å can change pK a values up to two units, but that the introduction of a requirement to keep other pK a values constant reduces the magnitude of the achievable DpK a . The algorithm presented shows a good correlation with existing experimental data and is available for download and via a web server at http://enzyme.ucd.ie/pKD.

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“…E50 and E62 are in close proximity in the crystal structure (Figure S3C), and thus these observations suggest that the ionization of E50 and E62 may be coupled [46]. To determine the pK a values of E50 and E62, we used the method of global fitting of titration events (GloFTE) developed by Nielsen and co-workers [38], [47], which allows an estimate of the titration profiles for individual titratable residues (Figure 3D). The pK a fold values of all Asp and Glu determined at 298 K and 333 K are summarized in Table 1.…”

Section: Resultsmentioning

confidence: 92%

Electrostatic Contribution of Surface Charge Residues to the Stability of a Thermophilic Protein: Benchmarking Experimental and Predicted pKa Values

et al. 2012

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Optimization of the surface charges is a promising strategy for increasing thermostability of proteins. Electrostatic contribution of ionizable groups to the protein stability can be estimated from the differences between the pKa values in the folded and unfolded states of a protein. Using this pKa-shift approach, we experimentally measured the electrostatic contribution of all aspartate and glutamate residues to the stability of a thermophilic ribosomal protein L30e from Thermococcus celer. The pKa values in the unfolded state were found to be similar to model compound pKas. The pKa values in both the folded and unfolded states obtained at 298 and 333 K were similar, suggesting that electrostatic contribution of ionizable groups to the protein stability were insensitive to temperature changes. The experimental pKa values for the L30e protein in the folded state were used as a benchmark to test the robustness of pKa prediction by various computational methods such as H++, MCCE, MEAD, pKD, PropKa, and UHBD. Although the predicted pKa values were affected by crystal contacts that may alter the side-chain conformation of surface charged residues, most computational methods performed well, with correlation coefficients between experimental and calculated pKa values ranging from 0.49 to 0.91 (p<0.01). The changes in protein stability derived from the experimental pKa-shift approach correlate well (r = 0.81) with those obtained from stability measurements of charge-to-alanine substituted variants of the L30e protein. Our results demonstrate that the knowledge of the pKa values in the folded state provides sufficient rationale for the redesign of protein surface charges leading to improved protein stability.

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Analysing the pH-dependent properties of proteins using pKa calculations

Cited by 38 publications

References 45 publications

Loop Electrostatics Asymmetry Modulates the Preexisting Conformational Equilibrium in Thrombin

Loop Electrostatics Asymmetry Modulates the Preexisting Conformational Equilibrium in Thrombin

Redesigning protein pK_avalues

Electrostatic Contribution of Surface Charge Residues to the Stability of a Thermophilic Protein: Benchmarking Experimental and Predicted pKa Values

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Analysing the pH-dependent properties of proteins using pKa calculations

Cited by 38 publications

References 45 publications

Loop Electrostatics Asymmetry Modulates the Preexisting Conformational Equilibrium in Thrombin

Loop Electrostatics Asymmetry Modulates the Preexisting Conformational Equilibrium in Thrombin

Redesigning protein pKavalues

Electrostatic Contribution of Surface Charge Residues to the Stability of a Thermophilic Protein: Benchmarking Experimental and Predicted pKa Values

Contact Info

Product

Resources

About

Redesigning protein pK_avalues