Influence of charge and size of terminal amino-acid residues on local conformational states and shape of alanine-based peptides

We present results of conformational studies by Circular dichroism and NMR spectroscopy, differential scanning calorimetry, and molecular dynamics, of three alanine-based peptides: Ac-KK-(A)(7)-KK-NH(2) (KAK), Ac-OO-(A)(7)-DD-NH(2) (OAD), and Ac-KK-(A)(7)-EE-NH(2) (KAE), where A, K, O, D, and E, denote alanine, lysine, ornithine, aspartic acid, and glutamic acid residues, respectively. For OAD and KAE, canonical MD simulations with time-averaged NMR-derived restraints demonstrate the presence of an ensemble of structures with a variety of conformational states (polyproline II, alpha-helical, alpha', and extended, turn); for KAK the conformational states are predominantly polyproline II and extended. The OAD peptide exhibits a bent shape with its ends close to each other, whereas KAK and KAE are more extended. The bent shape was also observed in our earlier study of the Ac-XX-(A)(7)-OO-NH(2) (XAO) peptide, where X denotes the diaminobutyric acid residue; therefore, the shape seems to depend on the size of the charged side chains at the ends of the alanine sequence and not on their kind. This suggests that the bent shape of the alanine sequence is formed to enable screening of this nonpolar sequence from the solvent by sufficiently short charged side chains. As in our previous study of the XAO peptide, no long polyproline II segments were observed.     

Interplay of charge distribution and conformation in peptides: comparison of theory and experiment

We assessed the correlation between charge distribution and conformation of flexible peptides by comparing the theoretically calculated potentiometric-titration curves of two model peptides, Ac-Lys5-NHMe (a model of poly-L-lysine) and Ac-Lys-Ala11-Lys-Gly2-Tyr-NH2 (P1) in water and methanol, with the experimental curves. The calculation procedure consisted of three steps: (i) global conformational search of the peptide under study using the electrostatically driven Monte Carlo (EDMC) method with the empirical conformational energy program for peptides (ECEPP)/3 force field plus the surface-hydration (SRFOPT) or the generalized Born surface area (GBSA) solvation model as well as a molecular dynamics method with the assisted model building and energy refinement (AMBER)99/GBSA force field; (ii) reevaluation of the energy in the pH range considered by using the modified Poisson-Boltzmann approach and taking into account all possible protonation microstates of each conformation, and (iii) calculation of the average degree of protonation of the peptide at a given pH value by Boltzmann averaging over conformations. For Ac-Lys5-NHMe, the computed titration curve agrees qualitatively with the experimental curve of poly-L-lysine in 95% methanol. The experimental titration curves of peptide P1 in water and methanol indicate a remarkable downshift of the first pK(a) value compared to the values for reference compounds (n-butylamine and phenol, respectively), suggesting the presence of a hydrogen bond between the tyrosine hydroxyl oxygen and the H(epsilon) proton of a protonated lysine side chain. The theoretical titration curves agree well with the experimental curves, if conformations with such hydrogen bonds constitute a significant part of the ensemble; otherwise, the theory predicts too small a downward pH shift.     

Effects of pH-induced variations of the charge of the transmembrane alpha-helical peptide Ac-K2(LA)12K2-amide on the organization and dynamics of the host dimyristoylphosphatidylcholine bilayer membrane

The effects of the transmembrane alpha-helical peptide Ac-K(2)(LA)(12)K(2)-amide ((LA)(12)) on the phase transition and dynamics of saturated dimyristoylphosphatidylcholine (DMPC) membranes were investigated at different pH using conventional and saturation-recovery EPR observations of phosphatidylcholine spin labels. At a peptide-to-DMPC ratio of 1/10, the main phase-transition temperature of the DMPC bilayer is decreased by 4.0 degrees C when measured at pH 7.0, by 1.6 degrees C when measured at pH 9.5, and not affected when measured at pH 11.5. This reversible pH effect is due to the subsequent neutralization of the positive charges of lysine side chains at both ends of (LA)(12). Apparent pK(a)s of the lysine side chain amino groups of (LA)(12) in DMPC bilayer are 8.6 and approximately 10.9, as compared with the pK(a) value of 10.5 for these groups when lysine is dissolved in water. Saturation-recovery curves as a function of oxygen concentration using phosphatidylcholine spin labels in DMPC bilayer containing (LA)(12) are always mono-exponential when measured at pH 7.0 and 9.5. This observation is consistent with the hypothesis that the lipid exchange rates among the bulk, boundary, and (LA)(12)-rich regions are faster than 0.5 micros, the electron spin-lattice relaxation time in the presence of molecular oxygen, suggesting that stable oligomers of (LA)(12) do not form. Neutralization of one lysine side chain positive charge on each end of the peptide significantly decreases the ordering effect of (LA)(12) on the lipid hydrocarbon chains, while its effect on the reorientational motion of terminal groups of lipid hydrocarbon chains is rather moderate. It does not affect the local diffusion-solubility product of oxygen measured in the DMPC-(LA)(12) membrane interior.     

Effects of external pH on substrate binding and on the inward chloride translocation rate constant of band 3

To test the hypothesis that amino acid residues in band 3 with titratable positive charges play a role in the binding of anions to the outside-facing transport site, we measured the effects of changing external pH (pH(O)) on the dissociation constant for binding of external iodide to the transport site, K(O)(I). K(O)(I) increased with increasing pH(O), and a significant increase was seen even at pH(O) values as low as 9.9. The dependence of K(O)(I) on pH(O) can be explained by a model with one titratable site with pK 9.5 +/- 0.2 (probably lysine), which increases anion affinity for the external transport site when it is in the positively charged form. A more complex model, analogous to one recently proposed by Bjerrum (1992), with two titratable sites, one with pK 9.3 +/- 0.3 (probably lysine) and another with pK > 11 (probably arginine), gives a slightly better fit to the data. Thus, titratable positively charged residues seem to be functionally important for the binding of substrate anions to the outward-facing anion transport site. In addition, analysis of Dixon plot slopes for L inhibition of Cl- exchange at different pH 0 values, coupled with the assumption that pH(O) has parallel effects on external I- and Cl- binding, indicates that k', the rate-constant for inward translocation of the complex of Cl- with the extracellular transport site, decreases with increasing pH(O). The data are compatible with a model in which titration of the pK 9.3 residue decreases k to 14 +/- 10% of its value at neutral pH(O). This result, however, together with Bjerrum's (1992) observation that the maximum flux J(M)) increases 1.6- fold when this residue is deprotonated, makes quantitative predictions that raise significant questions about the adequacy of the two titratable site ping-pong model or the assumptions used in analyzing the data.     

Acid-base catalysis in the yeast alcohol dehydrogenase reaction.

The effect of pH on steady state kinetic parameters for the yeast alcohol dehydrogenase-catalyzed reduction of aldehydes and oxidation of alcohols has been studied. The oxidation of p-CH3 benzyl alcohol-1,1-h2 and -1,1-d2 by NAD+ was found to be characterized by large deuterium isotope effects (kH/kD = 4.1 plus or minus 0.1) between pH 7.5 and 9.5, indicating a rate-limiting hydride trahsfer step in this pH range; a plot of kCAT versus pH could be fit to a theoretical titration curve, pK = 8.25, where kCAT increases with increasing pH. The Michaelis constnat for p-CH3 benzyl alcohol was independent of pH. The reduction of p-CH3 benzaldehyde by NADH and reduced nicotinamide adenine dinucleotide with deuterium in the 4-A position (NADD) cound not be studied below pH 8.5 due to substrate inhibition; however, between pH 8.5 and 9.5, kCAT was found to decrease with increasing pH and to be characterized by significant isotope effects (kH/kD = 3.3 plus or minus 0.3). In the case of acetaldehyde reduction by NADH and NADD, isotope effects were found to be small and exxentially invariant (kH/kD = 2.O plus or minus 0.4) between pH 7.2 and 9.5, suggesting a partially rate-limiting hydride transger step for this substrate; a plot of kCAT/K'b (where K'b is the Michaelis constant for acetaldehyde) versus pH could be fit to a titration curve, pK = 8.25. The titration curve for acetaldehyde reduction has the same pK but is opposite in direction to that observed for p-CH3 benzyl alcohol oxidation. The data presented in this paper indicate a dependence on different enzyme forms for aldehyde reduction and alcohol oxidation and are consistent with a single active site side chain, pK = 8.25, which functions in acid-base catalysis of the hydride transfer step.     

Chemical mechanism of saccharopine dehydrogenase (NAD+, L-lysine-forming) as deduced from initial rate pH studies

The variation of kinetic parameters with pH has been determined so as to gain insight into the chemical mechanism of the saccharopine dehydrogenase (NAD+,L-lysine-forming)-catalyzed reaction. In the direction of reductive condensation of lysine and alpha-ketoglutarate (reverse reaction), the V/K profile for lysine shows a group with a pK of 6.3 must be unprotonated and a group with a pK of 8.0 must be protonated for activity. Similar pK's are obtained in the pKi profile for ornithine, which acts as a linear competitive inhibitor with respect to lysine. Temperature and solvent perturbation studies show that these groups are probably histidines. The V/K profile for alpha-ketoglutarate reveals a single group with pK = 8.4 (probably lysine) that must be protonated. It is proposed that one of the histidines is involved in the binding of the epsilon-amino group of the substrate lysine and the positively charged lysine residue hydrogen bonds to the carbonyl oxygen of alpha-ketoglutarate. In the direction of saccharopine cleavage, the V/K profile for saccharopine shows that two groups with pK values of 6.0 and 7.1, possibly a histidine and lysine, must be unprotonated for its reaction with the enzyme X NAD+ complex. The log V-pH plots for the forward and reverse reactions both show sigmoidal curves. At low pH, the activity is lower for the forward reaction, and is higher for the reverse reaction. The ionization of a single group appears to be responsible for the change in activity. A tentative scheme for the chemical reaction is presented.     

Total weak acid concentration and effective dissociation constant of nonvolatile buffers in human plasma.

The strong ion approach provides a quantitative physicochemical method for describing the mechanism for an acid-base disturbance. The approach requires species-specific values for the total concentration of plasma nonvolatile buffers (A(tot)) and the effective dissociation constant for plasma nonvolatile buffers (K(a)), but these values have not been determined for human plasma. Accordingly, the purpose of this study was to calculate accurate A(tot) and K(a) values using data obtained from in vitro strong ion titration and CO(2) tonometry. The calculated values for A(tot) (24.1 mmol/l) and K(a) (1.05 x 10(-7)) were significantly (P < 0.05) different from the experimentally determined values for horse plasma and differed from the empirically assumed values for human plasma (A(tot) = 19.0 meq/l and K(a) = 3.0 x 10(-7)). The derivatives of pH with respect to the three independent variables [strong ion difference (SID), PCO(2), and A(tot)] of the strong ion approach were calculated as follows: dpH/dSID(+) = [1 + 10(pK(a)-pH)](2)/(2.303 x [SPCO(2)10(pH-pK'(1)[1 + 10(pK(a)-pH](2) + A(tot)10(pK(a)-PH]]; dpH/dPCO(2) = S10(-pK'(1)/[2.303[A(tot)10(pH)(10(pH + 10(pK(a))(-2) - SID(+)10(-pH)]], dpH/dA(tot) = -1/[2.303[SPCO(2)10(pH-pK'(1) + SID(+)10(pK(a)-pH)]], where S is solubility of CO(2) in plasma. The derivatives provide a useful method for calculating the effect of independent changes in SID(+), PCO(2), and A(tot) on plasma pH. The calculated values for A(tot) and K(a) should facilitate application of the strong ion approach to acid-base disturbances in humans.     

Like-charged residues at the ends of oligoalanine sequences might induce a chain reversal

We have examined the effect of like-charged residues on the conformation of an oligoalanine sequence. This was facilitated by circular dichroism (CD) and NMR spectroscopic and differential scanning calorimetric (DSC) measurements, and molecular dynamics calculations of the following three alanine-based peptides: Ac-K-(A)(5) -K-NH(2) (KAK5), Ac-K-(A)(4) -K-NH(2) (KAK4), Ac-K-(A)(3) -K-NH(2) (KAK3), where A and K denote alanine and lysine residues, respectively. Our earlier studies suggested that the presence of like-charged residues at the end of a short polypeptide chain composed of nonpolar residues can induce a chain reversal. For all three peptides, canonical molecular dynamics simulations with NMR-derived restraints demonstrate the presence of ensembles of structures with a tendency to form a chain reversal. The KAK3 peptide exhibits a bent shape with its ends close to each other, while KAK4 and KAK5 are more extended. In the KAK5 peptide, the lysine residues do not have any influence on each other and are very mobile. Nevertheless, the tendency to form a more or less pronounced chain reversal is observed and it seems to be stable in all three peptides. This chain reversal seems to be caused by screening of the nonpolar core from the solvent by the hydrated charged residues.     

pH studies on the chemical mechanism of rabbit muscle pyruvate kinase. 2. Physiological substrates and phosphoenol-alpha-ketobutyrate

pH profiles have been determined for the reactions catalyzed by pyruvate kinase between pyruvate and MgATP and between phosphoenolpyruvate and MgADP. V, V/KMgATP, and V/Kpyruvate all decrease below a pK of 8.3 and above one of 9.2. The group with pK = 8.3 is probably a lysine that removes the proton from pyruvate during enolization, while the pK of 9.2 is that of water coordinated to enzyme-bound Mg2+. The fact that this pK shows in all three pH profiles shows that pyruvate forms a predominantly second sphere complex and cannot replace hydroxide to form the inner sphere complex that results in enolization and subsequent phosphorylation. On the basis of the displacement of the pK of the acid-base catalytic group in its V/K profile, phosphoenolpyruvate is a sticky substrate, reacting to give pyruvate approximately 5 times faster than it dissociates. The V/K profile for the slow substrate phosphoenol-alpha-ketobutyrate shows the pK of 8.3 for the acid-base catalytic group in its correct position, but this group must be protonated so that it can donate a proton to the intermediate enolate following phosphoryl transfer. The secondary phosphate pK of the substrate is seen in this V/K profile as well as in the pKi profile for phosphoglycolate (but not in those for glycolate O-sulfate or oxalate), showing a preference for the trianion for binding. The chemical mechanism with the natural substrates thus appears to involve phosphoryl transfer between MgADP and a Mg2+-bound enolate with metal coordination of the enolate serving to make it a good leaving group.     

Binding of cationic pentapeptides with modified side chain lengths to negatively charged lipid membranes: Complex interplay of electrostatic and hydrophobic interactions

Basic amino acids play a key role in the binding of membrane associated proteins to negatively charged membranes. However, side chains of basic amino acids like lysine do not only provide a positive charge, but also a flexible hydrocarbon spacer that enables hydrophobic interactions. We studied the influence of hydrophobic contributions to the binding by varying the side chain length of pentapeptides with ammonium groups starting with lysine to lysine analogs with shorter side chains, namely ornithine (Orn), α,γ-diaminobutyric acid (Dab) and α, β-diaminopropionic acid (Dap). The binding to negatively charged phosphatidylglycerol (PG) membranes was investigated by calorimetry, FT-infrared spectroscopy (FT-IR) and monolayer techniques. The binding was influenced by counteracting and sometimes compensating contributions. The influence of the bound peptides on the lipid phase behavior depends on the length of the peptide side chains. Isothermal titration calorimetry (ITC) experiments showed exothermic and endothermic effects compensating to a different extent as a function of side chain length. The increase in lipid phase transition temperature was more significant for peptides with shorter side chains. FTIR-spectroscopy revealed changes in hydration of the lipid bilayer interface after peptide binding. Using monolayer techniques, the contributions of electrostatic and hydrophobic effects could clearly be observed. Peptides with short side chains induced a pronounced decrease in surface pressure of PG monolayers whereas peptides with additional hydrophobic interactions decreased the surface pressure much less or even lead to an increase, indicating insertion of the hydrophobic part of the side chain into the lipid monolayer.     

Folding simulations of alanine-based peptides with lysine residues.

The folding of short alanine-based peptides with different numbers of lysine residues is simulated at constant temperature (274 K) using the rigid-element Monte Carlo method. The solvent-referenced potential has prevented the multiple-minima problem in helix folding. From various initial structures, the peptides with three lysine residues fold into helix-dominated conformations with the calculated average helicity in the range of 60-80%. The peptide with six lysine residues shows only 8-14% helicity. These results agree well with experimental observations. The intramolecular electrostatic interaction of the charged lysine side chains and their electrostatic hydration destabilize the helical conformations of the peptide with six lysine residues, whereas these effects on the peptides with three lysine residues are small. The simulations provide insight into the helix-folding mechanism, including the beta-bend intermediate in helix initiation, the (i, i + 3) hydrogen bonds, the asymmetrical helix propagation, and the asymmetrical helicities in the N- and C-terminal regions. These findings are consistent with previous studies.     

Effects of Ca(2+)-activated calmodulin on neuronal nitric oxide synthase reductase activity and binding of substrates: pH dependence of kinetic parameters.

The pH dependence of basal and calmodulin- (CaM-) stimulated neuronal nitric oxide synthase (nNOS) reduction of 2,6-dichloroindophenol (DCIP) and cytochrome c(3+) was investigated. The wave-shaped log V versus pH profile revealed that optimal DCIP reduction occurred when a group, pK(a) of 7.6-7.8, was ionized. The (V/K)(NADPH) and (V/K)(DCIP) versus pH profiles increased with the protonation of a group with a pK(a) of 6.5 or 5.9 and the ionization of two groups with the same pK(a) of 7.5 or 7.0, respectively. (V/K)(DCIP) decreased with the ionization of a group, pK(a) of 9.0. Similar V, (V/K)(NADPH), and (V/K)(DCIP) versus pH profiles for DCIP reduction were obtained with and without CaM, indicating that CaM does not influence ionizable groups involved in catalysis or substrate binding. In contrast, CaM affected the pH dependence of cytochrome c(3+) reduction. The wave-shaped log V versus pH profile for basal cytochrome c(3+) reduction revealed that ionization of a group, pK(a) of 8.6, increased catalysis. Log V for CaM-stimulated cytochrome c(3+) reduction displayed a bell-shaped pH dependence with the protonation of a group with a pK(a) of 6.4 and the ionization of a group with a pK(a) of 9.3, resulting in a loss of activity. The log(V/K)(cytc) versus pH profiles with and without CaM were bell-shaped with the ionization of a group at pK(a) of 7.1 or 7.6 (CaM) or pK(a) of 9.4 or 9.6 (CaM), increasing and decreasing (V/K)(cytc). These results suggest that CaM may change the nature of the rate-limiting catalytic steps or ionizable groups involved in cytochrome c(3+) reduction.     

Synthesis and acid ionization constants of cyclic cystine peptides H-Cys-(Gly)n-Cys-OH (n = 0-4)

Cyclic peptide disulfides of the general formula H-Cys-(Gly)n-Cys-OH (n = 0-4) were synthesized from the corresponding peptide derivatives [Boc-Cys(Trt)(Gly)-n-Cys(Trt)-OBut] by oxidation with iodine in methanol and by subsequent removal of the terminal groups with trifluoroacetic acid. Acid ionization constants of the obtained peptides were determined by potentiometric titration in aqueous KCl (0.1 mol/L) medium. All compounds have two dissociable hydrogens, corresponding to carboxyl (pK1 = 2.35-2.84) and to terminal amino group (pK2 = 5.61-6.93); pK1 values show first an upward and then a downward trend with the increase in ring size; the opposite is true for pK2 values. These trends could be tentatively attributed to the intramolecular salt bridge (-COO- ----NH+3-) formation.     

Investigation of substrate specificity of creatine kinase using chromium (III) and cobalt(III) complexes of adenosine 5'-diphosphate

The specificity for substrate binding to creatine kinase for metal-nucleotide complexes of the type Cr-(H2O)4-n(NH3)nADP (where n = 0, 3, or 4) and Co-(H2O)4-m(NH3)mADP (for m = 3 or 4) has been investigated over the pH range 5.5-7.8 with the delta-alpha, beta-bidentate diastereoisomers. These inert nucleotide complexes acted as competitive inhibitors vs. MgADP over this range. In addition, the pH dependence of the V, V/K, and Km values for MgADP has been determined. Metal-nucleotide binding to the enzyme is strongest below an approximate pK of 6.45 but again becomes pH independent above pH 7. This pK is not associated with the metal-nucleotide complex. Instead, we conclude that the pK of the acid-base catalyst (thought to be histidine) is about 6.45 in the absence of nucleotide but is raised to 7.2 in its presence. This perturbation of the pK may result from a protein conformational change that allows a hydrogen bond to form between the phosphorylated nitrogen of phosphocreatine and the acid-base catalyst. The pK of the water in Cr(H2O)(NH3)3ADP has been determined to be 6.6, and by comparison of the binding affinity of this complex with that of Cr(NH3)4ADP or Cr(H2O)4ADP, it can be deduced that the hydroxo species binds more strongly than the aquo complex. In general, chromium nucleotides are bound more strongly than cobalt complexes, and binding affinity increases as water replaces ammonia in the first coordination sphere of the metal. Both trends are a result of stronger hydrogen-bond interactions between the metal complex and protein.     

Unraveling the catalytic mechanism of nitrile hydratases

To elucidate a detailed catalytic mechanism for nitrile hydratases (NHases), the pH and temperature dependence of the kinetic constants k(cat) and K(m) for the cobalt-type NHase from Pseudonocardia thermophila JCM 3095 (PtNHase) were examined. PtNHase was found to exhibit a bell-shaped curve for plots of relative activity versus pH at pH 3.2-11 and was found to display maximal activity between pH 7.2 and 7.8. Fits of these data provided pK(E)(S1) and pK(E)(S2) values of 5.9 +/- 0.1 and 9.2 +/- 0.1 (k(cat)' = 130 +/- 1 s(-1)), respectively, and pK(E)(1) and pK(E)(2) values of 5.8 +/- 0.1 and 9.1 +/- 0.1 (k(cat)'/K(m)' = (6.5 +/- 0.1) x 10(3) s(-1) mm(-1)), respectively. Proton inventory studies indicated that two protons are transferred in the rate-limiting step of the reaction at pH 7.6. Because PtNHase is stable at 60 degrees C, an Arrhenius plot was constructed by plotting ln(k(cat)) versus 1/T, providing E(a) = 23.0 +/- 1.2 kJ/mol. The thermal stability of PtNHase also allowed DeltaH(0) ionization values to be determined, thus helping to identify the ionizing groups exhibiting the pK(E)(S1) and pK(E)(S2) values. Based on DeltaH(0)(ion) data, pK(E)(S1) is assigned to betaTyr(68), whereas pK(E)(S2) is assigned to betaArg(52), betaArg(157), or alphaSer(112) (NHases are alpha(2)beta(2)-heterotetramers). A combination of these data with those previously reported for NHases and synthetic model complexes, along with sequence comparisons of both iron- and cobalt-type NHases, allowed a novel catalytic mechanism for NHases to be proposed.     

Self-association of glutamic acid-rich fusion peptide analogs of influenza hemagglutinin in the membrane-mimic environments: effects of positional difference of glutamic acids on side chain ionization constant and intra- and inter-peptide interactions deduced from NMR and gel electrophoresis measurements

Two glutamic acid-rich fusion peptide analogs of influenza hemagglutinin were synthesized to study the organization of the charged peptides in the membranous media. Fluorescence and gel electrophoresis experiments suggested a loose association between the monomers in the vesicles. A model was built which showed that a positional difference of 3, 7 and 4, 8 results in the exposure of Glu3 and Glu7 side chains to the apolar lipidic core. Supportive results include: first, pK(a) values of two pH units higher than reference value in aqueous medium for Glu3 and Glu7 CgammaH, whereas the deviation of pK(a) from the reference value for Glu4 and Glu8 CgammaH is substantially smaller; second, Hill coefficients of titration shift of these protons indicate anti-cooperativity for Glu3 and Glu7 side chain protons but less so for Glu4 and Glu8, implying a strong electrostatic interaction between Glu3 and Glu7 possibly resulting from their localization in an apolar environment; third, positive and larger titration shift for NH of Glu3 is observed compared to that of Glu4, suggesting stronger hydrogen bond between the NH and the carboxylic group of Glu3 than that of Glu4, consistent with higher degree of exposure to hydrophobic medium for the side chain of Glu3.     

pK values of histidine residues in ribonuclease Sa: effect of salt and net charge

The primary goal of this study was to gain a better understanding of the effect of environment and ionic strength on the pK values of histidine residues in proteins. The salt-dependence of pK values for two histidine residues in ribonuclease Sa (RNase Sa) (pI=3.5) and a variant in which five acidic amino acids have been changed to lysine (5K) (pI=10.2) was measured and compared to pK values of model histidine-containing peptides. The pK of His53 is elevated by two pH units (pK=8.61) in RNase Sa and by nearly one pH unit (pK=7.39) in 5K at low salt relative to the pK of histidine in the model peptides (pK=6.6). The pK for His53 remains elevated in 1.5M NaCl (pK=7.89). The elevated pK for His53 is a result of screenable electrostatic interactions, particularly with Glu74, and a non-screenable hydrogen bond interaction with water. The pK of His85 in RNase Sa and 5K is slightly below the model pK at low salt and merges with this value at 1.5M NaCl. The pK of His85 reflects mainly effects of long-range Coulombic interactions that are screenable by salt. The tautomeric states of the neutral histidine residues are changed by charge reversal. The histidine pK values in RNase Sa are always higher than the pK values in the 5K variant. These results emphasize that the net charge of the protein influences the pK values of the histidine residues. Structure-based pK calculations capture the salt-dependence relatively well but are unable to predict absolute histidine pK values.     

Metal binding by melanins: studies of colloidal dihydroxyindole-melanin, and its complexation by Cu(II) and Zn(II) ions

Melanins are colloidal pigments known to have a high affinity for metal ions. In this work, the nature of the metal-binding sites are determined and the binding affinities are quantified. Initial potentiometric titrations have been performed on synthetic dihydroxyindole (DHI) melanin solutions to determine the chemical speciation of quinole/quinone subunits. Two types of acidic functionalities are assignable: catechol groups, with pK(a) between 9 and 13, and quinone imines (QI), with pK(a) of 6.3. The presence of the quinone-imine tautomer has, to our knowledge, never been assessed in polymeric melanins. Melanin solutions obtained from N-methylated DHI lack the pK(a) 6.3 buffer, consistent with its inability to form the quinone-imine tautomer. EPR spectroscopy of the DHI-melanin samples demonstrates that the semiquinone radical is in too low a concentration to contribute to the bulk binding of metals. Changes in the titration curves after addition of Cu(II) and Zn(II) ions were analyzed to obtain the binding constants and stoichiometry of the metal-melanin complexes, using the BEST7 program. UV-Vis spectra at neutral and high pH are used to identify absorbances due to Cu-bound quinone imine and catechol groups. The derived binding constants were used to determine speciation of the Cu(II) and Zn(II) ions coordinated to the quinone imine and catechol groups at various pH. The mixed complexes, Zn(QI)(Cat)(-) and Cu(QI)(Cat)(-) are shown to dominate at physiological pH.     

Simulating proteins at constant pH: An approach combining molecular dynamics and Monte Carlo simulation

For the structure and function of proteins, the pH of the solution is one of the determining parameters. Current molecular dynamics (MD) simulations account for the solution pH only in a limited way by keeping each titratable site in a chosen protonation state. We present an algorithm that generates trajectories at a Boltzmann distributed ensemble of protonation states by a combination of MD and Monte Carlo (MC) simulation. The algorithm is useful for pH-dependent structural studies and to investigate in detail the titration behavior of proteins. The method is tested on the acidic residues of the protein hen egg white lysozyme. It is shown that small structural changes may have a big effect on the pK(A) values of titratable residues.     

Interhelical ion pairing in coiled coils: solution structure of a heterodimeric leucine zipper and determination of pKa values of Glu side chains

Residues of opposite charge often populate heptad positions g (heptad i on chain 1) and e' (heptad i + 1 on chain 2) in dimeric coiled coils and may stabilize the dimer by formation of interchain ion pairs. To investigate the contribution to stability of such electrostatic interactions we have designed a disulfide-linked heterodimeric zipper (AB zipper) consisting of the acidic chain Ac-E-VAQLEKE-VAQAEAE-NYQLEQE-VAQLEHE-CG-NH(2) and the basic chain Ac-E-VQALKKR-VQALKAR-NYAAKQK-VQALRHK-CG-NH(2) in which all e and g positions are occupied by either E or K/R to form a maximum of seven interhelical salt bridges. Temperature-induced denaturation experiments monitored by circular dichroism reveal a stable coiled coil conformation below 50 degrees C and in the pH range 1.2-8.0. Stability is highest at pH approximately 4.0 [DeltaG(U) (37 degrees C) = 5.18 +/- 0.51 kcal mol(-)(1)]. The solution structure of the AB zipper at pH 5.65 has been elucidated on the basis of homonuclear (1)H NMR data collected at 800 MHz [heavy atom rmsd's for the ensemble of 50 calculated structures are 0.47 +/- 0.13 A (backbone) and 0.95 +/- 0.16 A (all)]. Both chains of the AB zipper are almost entirely in alpha-helical conformation and form a superhelix with a left-handed twist. Overhauser connectivities reveal close contacts between g position residues (heptad i on chain 1) and residues d/f (heptad i on chain 1), residues a/d (heptad i + 1 on chain 1), and residue a' (heptad i + 1 on chain 2). Residues in position e (heptad i on chain 1) are in contact with residues a/b/d/f (heptad i on chain 1) and residue d' (heptad i on chain 2). These connectivities hint at a relatively defined alignment of the side chains across the helix interface. Partial H-bond formation between the functional groups of residues g and e'(+1) is observed in the calculated structures. NMR pH titration experiments disclose pK(a) values for Glu delta-carboxylate groups: 4.14 +/- 0.02 (E(1)), 4.82 +/- 0.07 (E(6)), 4.52 +/- 0.01 (E(8)), 4.37 +/- 0.03 (E(13)), 4.11 +/- 0.02 (E(15)), 4.41 +/- 0.07 (E(20)), 4.82 +/- 0.03 (E(22)), 4.65 +/- 0.04 (E(27)), 4.63 +/- 0.03 (E(29)), 4.22 +/- 0.02 (E(1)(')). By comparison with pK(a) of Glu in unfolded peptides ( approximately 4. 3 +/- 0.1), our pK(a) data suggest marginal or even unfavorable contribution of charged Glu to the stability of the AB zipper. The electrostatic energy gained from interhelical ion pairs is likely to be surpassed by hydrophobic energy terms upon protonation of Glu, due to increased hydrophobicity of uncharged Glu and, thus, better packing against apolar residues at the chain interface.