pH-Dependent Processes in Protein

Matthew, James B.; Gurd, Frank R. N.; García-Moreno, Bertrand; Flanagan, Margaret A.; March, Keith L.; Shire, Steven J.

doi:10.3109/10409238509085133

Cited by 202 publications

(183 citation statements)

References 169 publications

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“…The apparent pK a values of Glu residues at 23 of the 25 internal positions ( Fig. 2A and Table 1) are much higher than the normal pK a of 4.5 for Glu in water (19,20). The upward shifts in pK a are consistent with the ionizable moieties of the Glu residues being internal, at least partially removed from bulk water, and from the influence of the large number of basic residues on the surface of the protein.…”

Section: Resultssupporting

confidence: 55%

Charges in the hydrophobic interior of proteins

Isom

Castañeda

Cannon

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

214

322

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Charges are inherently incompatible with hydrophobic environments. Presumably for this reason, ionizable residues are usually excluded from the hydrophobic interior of proteins and are found instead at the surface, where they can interact with bulk water. Paradoxically, ionizable groups buried in the hydrophobic interior of proteins play essential roles, especially in biological energy transduction. To examine the unusual properties of internal ionizable groups we measured the pK a of glutamic acid residues at 25 internal positions in a stable form of staphylococcal nuclease. Two of 25 Glu residues titrated with normal pK a near 4.5; the other 23 titrated with elevated pK a values ranging from 5.2-9.4, with an average value of 7.7. Trp fluorescence and far-UV circular dichroism were used to monitor the effects of internal charges on conformation. These data demonstrate that although charges buried in proteins are indeed destabilizing, charged side chains can be buried readily in the hydrophobic core of stable proteins without the need for specialized structural adaptations to stabilize them, and without inducing any major conformational reorganization. The apparent dielectric effect experienced by the internal charges is considerably higher than the low dielectric constants of hydrophobic matter used to represent the protein interior in electrostatic continuum models of proteins. The high thermodynamic stability required for proteins to withstand the presence of buried charges suggests a pathway for the evolution of enzymes, and it underscores the need to mind thermodynamic stability in any strategy for engineering novel or altered enzymatic active sites in proteins.dielectric effect | electrostatics | hydration | pKa | bioenergetics T he transfer of an ion from water into a less polar and polarizable environment, such as the hydrophobic interior of a protein, is energetically unfavorable. Internal charges usually destabilize the folded states of proteins, which is primarily why charged groups are largely excluded from the hydrophobic interior and found instead at the protein-water interface, where they can interact with bulk water (1). Paradoxically, internal ionizable groups in proteins are essential for biological energy transduction. These type of ionizable groups are found in the active sites of enzymes (2), and are necessary for e − transfer and H þ transport in proteins such as ATPase (3) and cytochrome c oxidase (4), for ion homeostasis (5, 6), and for light-activated processes in proteins such as bacteriorhodopsin (7,8). The structural adaptations necessary for proteins to tolerate internal ionizable groups, and the factors that stabilize internal charges, are poorly understood. For this reason, our understanding of fundamental aspects of function and evolution of proteins is still limited, as is our ability to manipulate and design novel enzymes.To examine systematically the capacity of globular proteins to tolerate the presence of buried charges, we measured the pK a of 25 internal glutamic acid resid...

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Section: Resultssupporting

confidence: 55%

Charges in the hydrophobic interior of proteins

Isom

Castañeda

Cannon

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

214

322

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“…The value of the effective dielectric permittivity E is dependent on the environment of the charges and cannot be readily estimated by theoretical considerations [19]. Semi-empirical relationships for c as a function of distance are available, however, and the scaled dielectric permittivity function reported by Mehler and Eichele [18] has been shown to lead to reasonable agreements between calculated and experimentally observed electrostatic pKa shifts for ionizing groups in wateraccessible regions of proteins.…”

Section: Theorymentioning

confidence: 92%

Electrostatic effects of bound NADH and NAD⁺ on ionizing groups in liver alcohol dehydrogenase

Pettersson

Eklund

1987

European Journal of Biochemistry

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A simple point charge model has been used to estimate the effect of electrostatic interactions of bound NADH and NAD+ on ionizing groups in liver alcohol dehydrogenase. The results provide clear evidence that the much‐discussed effects of pH on coenzyme binding in the pH range 7–10 are predominantly of electrostatic origin and attributable to ionization of the water molecule which is bound at the active‐site zinc ion. Ionization of Lys‐228 is proposed to account for the proton‐dependent destabilization of the enzyme · NADH and enzyme · NAD+ complexes which has been reported to occur above a pKa of 10.4.

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“…The solution pK a (pK a,sol ) and E m (E m,sol ) are obtained from measurements in water. pK a,sol for amino acids are taken from studies of peptides (68,69). The heme propionic acids are treated as described previously with a pK a,sol of 4.9 (20).…”

Section: Structures Ofmentioning

confidence: 99%

Electrostatic Environment of Hemes in Proteins: pK_as of Hydroxyl Ligands

2006

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The pK a s of ferric aquo-heme and aquo-heme electrochemical midpoints (E m s) at pH 7 in sperm whale myoglobin, Aplysia myoblogin, hemoglobin I, heme oxygenase 1, horseradish peroxidase and cytochrome c oxidase were calculated with Multi-Conformation Continuum Electrostatics (MCCE). The pK a s span 3.3 pH units from 7.6 in heme oxygenase 1 to 10.9 in peroxidase, and the E m s range from -250 mV in peroxidase to 125 mV in Aplysia myoglobin. Proteins with higher in situ ferric aquo-heme pK a s tend to have lower E m s. Both changes arise from the protein stabilizing a positively charged heme. However, compared with values in solution, the protein shifts the aquo-heme E m s more than the pK a s. Thus, the protein has a larger effective dielectric constant for the protonation reaction, showing that electron and proton transfers are coupled to different conformational changes that are captured in the MCCE analysis. The calculations reveal a breakdown in the classical continuum electrostatic analysis of pairwise interactions. Comparisons with DFT calculations show that Coulomb's law overestimates the large unfavorable interactions between the ferric water-heme and positively charged groups facing the heme plane by as much as 60%. If interactions with Cu B in cytochrome c oxidase and Arg 38 in horseradish peroxidase are not corrected, the pK a calculations are in error by as much as 6 pH units. With DFT corrected interactions calculated pK a s and E m s differ from measured values by less than 1 pH unit or 35 mV, respectively. The in situ aquo-heme pK a is important for the function of cytochrome c oxidase since it helps to control the stoichiometry of proton uptake coupled to electron transfer [Song, Michonova-Alexova, and Gunner ( . Heme oxygenase is an essential protein in heme metabolism, using three O 2 s and seven electrons to degrade heme to biliverdin (3). Peroxidases reduce toxic hydrogen peroxide to water and then carry out one electron oxidization of a wide range of substrates (4,5). Cytochrome c oxidase is the terminal electron acceptor in the respiratory chain (6-9). Here Heme a 3 forms a binuclear center with a copper complex (Cu B ) that reduces O 2 to water. The protein uses the released chemical energy to pump protons across the membrane. Other five-coordinate heme proteins carry out NO storage and transport (10), steroid synthesis (11, 12), and O 2 , CO, and NO sensing (13,14).Hemes can carry out many biological functions because the protein, especially the active site residues, modulates the ligand binding specificity and resultant chemistry. The diversity of in situ heme functions highlights important interactions of proteins with their cofactors and substrates. Extensive studies have explored how ligand geometry (15-17) and the protein scaffolding (18) affect in situ heme properties (1). The range of redox free energy and pK a s of protein-bound hemes and their ligands show the importance of electrostatic interactions (19,20). For example, sixcoordinate bis-His-hemes have E m s ranging f...

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pH-Dependent Processes in Protein

Cited by 202 publications

References 169 publications

Charges in the hydrophobic interior of proteins

Charges in the hydrophobic interior of proteins

Electrostatic effects of bound NADH and NAD⁺ on ionizing groups in liver alcohol dehydrogenase

Electrostatic Environment of Hemes in Proteins: pK_as of Hydroxyl Ligands

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pH-Dependent Processes in Protein

Cited by 202 publications

References 169 publications

Charges in the hydrophobic interior of proteins

Charges in the hydrophobic interior of proteins

Electrostatic effects of bound NADH and NAD+ on ionizing groups in liver alcohol dehydrogenase

Electrostatic Environment of Hemes in Proteins: pKas of Hydroxyl Ligands

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Product

Resources

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Electrostatic effects of bound NADH and NAD⁺ on ionizing groups in liver alcohol dehydrogenase

Electrostatic Environment of Hemes in Proteins: pK_as of Hydroxyl Ligands