Salt bridges destabilize a leucine zipper designed for maximized ion pairing between helices

Interhelical salt bridges are common in leucine zippers and are thought to stabilize the coiled coil conformation. Here we present a detailed thermodynamic investigation of the designed, disulfide-linked leucine zipper AB(SS) whose high-resolution NMR structure shows six interhelical ion pairs between heptad positions g of one helix and e' of the other helix but no ion pairing within single helices. The average pK(a) value of the Glu side chain carboxyl groups of AB(SS) is slightly higher than the pK(a) of a freely accessible Glu in an unfolded peptide [Marti, D. N., Jelesarov, I., and Bosshard, H. R. (2000) Biochemistry 39, 12804-12818]. This indicates that the salt bridges are destabilizing, a prediction we now have confirmed by determining the pH +/- stability profile of AB(SS). Circular dichroism-monitored unfolding by urea and by heating and differential scanning calorimetry show that the coiled coil conformation is approximately 5 kJ/mol more stable when salt bridges are broken by protonation of the carboxyl side chains. Using guanidinium chloride as the denaturant, the increase in the free energy of unfolding on protonation of the carboxyl side chains is larger, approximately 17 kJ/mol. The discrepancy between urea and guanidinium chloride unfolding can be ascribed to the ionic nature of guanidinium chloride, which screens charge-charge interactions. This work demonstrates the difficulty of predicting the energetic contribution of salt bridges from structural data alone even in a case where the ion pairs are seen in high-resolution NMR structures. The reason is that the contribution to stability results from a fine balance between energetically favorable Coulombic attractions and unfavorable desolvation of charges and conformational constraints of the residues involved in ion pairing. The apparent discrepancy between the results presented here and mutational studies indicating stabilization by salt bridges is discussed and resolved. An explanation is proposed for why interhelical salt bridges are frequently found in natural coiled coils despite evidence that they do not directly contribute to stability.     

Inverse electrostatic effect: electrostatic repulsion in the unfolded state stabilizes a leucine zipper

The pH-dependent stability of a protein is strongly affected by electrostatic interactions between ionizable residues in the folded as well as unfolded state. Here we characterize the individual contributions of charged Glu and His residues to stability and determine the NMR structure of the designed, heterodimeric leucine zipper AB consisting of an acidic A chain and a basic B chain. Thermodynamic parameters are compared with those of the homologous leucine zipper AB(SS) in which the A and B chains are disulfide-linked. NMR structures of AB based on (1)H NMR data collected at 600 MHz converge, and formation of the same six interchain salt bridges found previously in disulfide-linked AB(SS) [Marti, D. N., and Bosshard, H. R. (2003) J. Mol. Biol. 330, 621-637] is indicated. While the structures of AB and AB(SS) are very similar, their pH-dependent relative stabilities are strikingly different. The stability of AB peaks at pH approximately 4.5 and is higher at pH 8 than at pH 2. In contrast, AB(SS) is most stable at acidic pH where no interhelical salt bridges are formed. The different energetic contributions of charged Glu and His residues to stability of the two coiled coil structures were evaluated from pK(a) shifts induced by folding. The six charged Glu residues involved in salt bridges stabilize leucine zipper AB by 4.5 kJ/mol yet destabilize disulfide-linked AB(SS) by -1.1 kJ/mol. Two non-ion-paired Glu charges destabilize AB by only -1.8 kJ/mol but AB(SS) by -5.6 kJ/mol. The higher relative stability of AB at neutral pH is not caused by more favorable electrostatic interactions in the folded leucine zipper. It is due mainly to unfavorable electrostatic interactions in the unfolded A and B chains and may therefore be called an inverse electrostatic effect. This study illustrates the importance of residual interactions in the unfolded state and how the energetics of the unfolded state affect the stability of the folded protein.     

Electrostatic interactions in leucine zippers: thermodynamic analysis of the contributions of Glu and His residues and the effect of mutating salt bridges

Electrostatic interactions play a complex role in stabilizing proteins. Here, we present a rigorous thermodynamic analysis of the contribution of individual Glu and His residues to the relative pH-dependent stability of the designed disulfide-linked leucine zipper AB(SS). The contribution of an ionized side-chain to the pH-dependent stability is related to the shift of the pK(a) induced by folding of the coiled coil structure. pK(a)(F) values of ten Glu and two His side-chains in folded AB(SS) and the corresponding pK(a)(U) values in unfolded peptides with partial sequences of AB(SS) were determined by 1H NMR spectroscopy: of four Glu residues not involved in ion pairing, two are destabilizing (-5.6 kJ mol(-1)) and two are interacting with the positive alpha-helix dipoles and are thus stabilizing (+3.8 kJ mol(-1)) in charged form. The two His residues positioned in the C-terminal moiety of AB(SS) interact with the negative alpha-helix dipoles resulting in net stabilization of the coiled coil conformation carrying charged His (-2.6 kJ mol(-1)). Of the six Glu residues involved in inter-helical salt bridges, three are destabilizing and three are stabilizing in charged form, the net contribution of salt-bridged Glu side-chains being destabilizing (-1.1 kJ mol(-1)). The sum of the individual contributions of protonated Glu and His to the higher stability of AB(SS) at acidic pH (-5.4 kJ mol(-1)) agrees with the difference in stability determined by thermal unfolding at pH 8 and pH 2 (-5.3 kJ mol(-1)). To confirm salt bridge formation, the positive charge of the basic partner residue of one stabilizing and one destabilizing Glu was removed by isosteric mutations (Lys-->norleucine, Arg-->norvaline). Both mutations destabilize the coiled coil conformation at neutral pH and increase the pK(a) of the formerly ion-paired Glu side-chain, verifying the formation of a salt bridge even in the case where a charged side-chain is destabilizing. Because removing charges by a double mutation cycle mainly discloses the immediate charge-charge effect, mutational analysis tends to overestimate the overall energetic contribution of salt bridges to protein stability.     

Protein destabilization by electrostatic repulsions in the two-stranded alpha-helical coiled-coil/leucine zipper.

The destabilizing effect of electrostatic repulsions on protein stability has been studied by using synthetic two-stranded alpha-helical coiled-coils as a model system. The native coiled-coil consists of two identical 35-residue polypeptide chains with a heptad repeat QgVaGbAcLdQeKf and a Cys residue at position 2 to allow formation of an interchain disulfide bridge. This peptide, designed to contain no intrahelical or interhelical electrostatic interactions, forms a stable coiled-coil structure at 20 degrees C in benign medium (50 mM KCl, 25 mM PO4, pH 7) with a [urea]1/2 value of 6.1 M. Four mutant coiled-coils were designed to contain one or two Glu substitutions for Gln per polypeptide chain. The resulting coiled-coils contained potential i to i' + 5 Glu-Glu interchain repulsions (denoted as peptide E2(15,20)), i to i' + 2 Glu-Glu interchain repulsions (denoted E2(20,22)), or no interchain ionic interactions (denoted E2(13,22) and E1(20)). The stabilities of the coiled-coils were determined by measuring the ellipticities at 222 nm as a function of urea or guanidine hydrochloride concentration at 20 degrees C in the presence and absence of an interchain disulfide bridge. At pH 7, in the presence of urea, the stabilities of E2(13,22) and E2(20,22) were identical suggesting that the potential i to i' + 2 interchain Glu-Glu repulsion in the E2(20,22) coiled-coil does not occur. In contrast, the mutant E2(15,20) is substantially less stable than E2(13,22) or E2(15,20) by 0.9 kcal/mol due to the presence of two i to i' + 5 interchain Glu-Glu repulsions, which destabilize the coiled-coil by 0.45 kcal/mol each. At pH 3 the coiled-coils were found to increase in stability as the number of Glu substitutions were increased. This, combined with reversed-phase HPLC results at pH 7 and pH 2, supports the conclusion that the protonated Glu side chains present at low pH are significantly more hydrophobic than Gln side chains which are in turn more hydrophobic than the ionized Glu side chains present at neutral pH. The protonated Glu residues increase the hydrophobicity of the coiled-coil interface leading to higher coiled-coil stability. The guanidine hydrochloride results at pH 7 show similar stabilities between the native and mutant coiled-coils indicating that guanidine hydrochloride masks electrostatic repulsions due to its ionic nature and that Glu and Gln in the e and g positions of the heptad repeat have very similar effects on coiled-coil stability in the presence of GdnHCl.     

Oligomerization of the {gamma}-aminobutyric acid transporter-1 is driven by an interplay of polar and hydrophobic interactions in transmembrane helix II

The available evidence indicates that members of the neurotransmitter:sodium symporter family form constitutive oligomers. Their second transmembrane helix (TM2) contains a leucine heptad repeat proposed to be involved in oligomerization. In artificial transmembrane segments, interhelical interactions are stabilized by polar residues. We searched for these hydrogen bond donors in TM2 by mutating the five polar residues in TM2 of the gamma-aminobutyric acid transporter-1 (GAT1). We tested the ability of the resulting mutants to oligomerize by fluorescence microscopy, Foerster resonance energy transfer, and beta-lactamase fragment complementation. Of all generated mutants, only Y86A- (but not Y86F-), E101A-, E101Q-, and E101D-GAT1 were judged by these criteria to be deficient in oligomerization and were retained intracellularly. The observations are consistent with a model where the leucine heptad repeat in TM2 drives a homophilic association that is stabilized by Tyr(86) and Glu(101); Tyr(86) participates in hydrophobic stacking. Glu(101) is in the a-position of the leucine heptad repeat (where positions 1-7 are denoted a-g, and each leucine is in the central d-position). Thus, Glu(101) is in the position predicted for the hydrogen bond donor (i.e. sandwiched between Leu(97) and Leu(104), which are one helical turn above and below Glu(101)). These key residues, namely Tyr(86) and Glu(101), are conserved in related transporters from archaeae to humans; they are therefore likely to support oligomeric assembly in transporter orthologs and possibly other proteins with multiple transmembrane segments.     

Catalytic mechanism of Zn2+-dependent polyol dehydrogenases: kinetic comparison of sheep liver sorbitol dehydrogenase with wild-type and Glu154-->Cys forms of yeast xylitol dehydrogenase

Co-ordination of catalytic Zn2+ in sorbitol/xylitol dehydrogenases of the medium-chain dehydrogenase/reductase superfamily involves direct or water-mediated interactions from a glutamic acid residue, which substitutes a homologous cysteine ligand in alcohol dehydrogenases of the yeast and liver type. Glu154 of xylitol dehydrogenase from the yeast Galactocandida mastotermitis (termed GmXDH) was mutated to a cysteine residue (E154C) to revert this replacement. In spite of their variable Zn2+ content (0.10-0.40 atom/subunit), purified preparations of E154C exhibited a constant catalytic Zn2+ centre activity (kcat) of 1.19+/-0.03 s(-1) and did not require exogenous Zn2+ for activity or stability. E154C retained 0.019+/-0.003% and 0.74+/-0.03% of wild-type catalytic efficiency (kcat/K(sorbitol)=7800+/-700 M(-1) x s(-1)) and kcat (=161+/-4 s(-1)) for NAD+-dependent oxidation of sorbitol at 25 degrees C respectively. The pH profile of kcat/K(sorbitol) for E154C decreased below an apparent pK of 9.1+/-0.3, reflecting a shift in pK by about +1.7-1.9 pH units compared with the corresponding pH profiles for GmXDH and sheep liver sorbitol dehydrogenase (termed slSDH). The difference in pK for profiles determined in 1H2O and 2H2O solvent was similar and unusually small for all three enzymes (approximately +0.2 log units), suggesting that the observed pK in the binary enzyme-NAD+ complexes could be due to Zn2+-bound water. Under conditions eliminating their different pH-dependences, wild-type and mutant GmXDH displayed similar primary and solvent deuterium kinetic isotope effects of 1.7+/-0.2 (E154C, 1.7+/-0.1) and 1.9+/-0.3 (E154C, 2.4+/-0.2) on kcat/K(sorbitol) respectively. Transient kinetic studies of NAD+ reduction and proton release during sorbitol oxidation by slSDH at pH 8.2 show that two protons are lost with a rate constant of 687+/-12 s(-1) in the pre-steady state, which features a turnover of 0.9+/-0.1 enzyme equivalents as NADH was produced with a rate constant of 409+/-3 s(-1). The results support an auxiliary participation of Glu154 in catalysis, and possible mechanisms of proton transfer in sorbitol/xylitol dehydrogenases are discussed.     

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.     

Side-chain protonation and mobility in the sarcoplasmic reticulum Ca2+-ATPase: implications for proton countertransport and Ca2+ release

Protonation of acidic residues in the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA 1a) was studied by multiconformation continuum electrostatic calculations in the Ca(2+)-bound state Ca(2)E1, in the Ca(2+)-free state E2(TG) with bound thapsigargin, and in the E2P (ADP-insensitive phosphoenzyme) analog state with MgF(4)(2-) E2(TG+MgF(4)(2-)). Around physiological pH, all acidic Ca(2+) ligands (Glu(309), Glu(771), Asp(800), and Glu(908)) were unprotonated in Ca(2)E1; in E2(TG) and E2(TG+MgF(4)(2-)) Glu(771), Asp(800), and Glu(908) were protonated. Glu(771) and Glu(908) had calculated pK(a) values larger than 14 in E2(TG) and E2(TG+MgF(4)(2-)), whereas Asp(800) titrated with calculated pK(a) values near 7.5. Glu(309) had very different pK(a) values in the Ca(2+)-free states: 8.4 in E2(TG+MgF(4)(2-)) and 4.7 in E2(TG) because of a different local backbone conformation. This indicates that Glu(309) can switch between a high and a low pK(a) mode, depending on the local backbone conformation. Protonated Glu(309) occupied predominantly two main, very differently orientated side-chain conformations in E2(TG+MgF(4)(2-)): one oriented inward toward the other Ca(2+) ligands and one oriented outward toward a protein channel that seems to be in contact with the cytoplasm. Upon deprotonation, Glu(309) adopted completely the outwardly orientated side-chain conformation. The contact of Glu(309) with the cytoplasm in E2(TG+MgF(4)(2-)) makes this residue unlikely to bind lumenal protons. Instead it might serve as a proton shuttle between Ca(2+)-binding site I and the cytoplasm. Glu(771), Asp(800), and Glu(908) are proposed to take part in proton countertransport.     

Complementation of buried lysine and surface polar residues in a designed heterodimeric coiled coil

The coiled coil is an attractive target for protein design. The helices of coiled coils are characterized by a heptad repeat of residues denoted a to g. Residues at positions a and d form the interhelical interface and are usually hydrophobic. An established strategy to confer structural uniqueness to two-stranded coiled coils is the use of buried polar Asn residues at position a, which imparts dimerization and conformational specificity at the expense of stability. Here we show that polar interactions involving buried position-a Lys residues that can interact favorably only with surface e' or g' Glu residues also impart structural uniqueness to a designed heterodimeric coiled coil with the nativelike properties of sigmoidal thermal and urea-induced unfolding transitions, slow hydrogen exchange and lack of ANS binding. The position-a Lys residues do not, however, confer a single preference for helix orientation, likely reflecting the ability of Lys at position a to from favorable interactions with g' or e' Glu residues in the parallel and antiparallel orientations, respectively. The Lys-Glu polar interaction is less destabilizing than the Asn-Asn a-->a' interaction, presumably reflecting a higher desolvation penalty associated with the completely buried polar position-a groups. Our results extend the range of approaches for two-stranded coiled-coil design and illustrate the role of complementing polar groups associated with buried and surface positions of proteins in protein folding and design.     

Specific effects of chloride on the photocycle of E194Q and E204Q mutants of bacteriorhodopsin as measured by FTIR spectroscopy

Low-temperature Fourier transform infrared spectroscopy has been used to study mutants of Glu194 and Glu204, two amino acids that are involved in proton release to the extracellular side of bacteriorhodopsin. Difference spectra of films of E194Q, E204Q, E194Q/E204Q, E9Q/E194Q/E204Q, and E9Q/E74Q/E194Q/E204Q at 243, 277, and 293 K and several pH values were obtained by continuous illumination. A specific effect of Cl(-) ions was found for the mutants, promoting a N-like intermediate at alkaline pH and an O' intermediate at neutral or acid pH. The apparent pK(a) of Asp85 in the M intermediate was found to be decreased for E194Q in the presence of Cl(-) (pK(a) of 7.6), but it was unchanged for E204Q, as compared to wild-type. In the absence of Cl(-) (i.e., in the presence of SO(4)(2)(-)), mutation of Glu194 or of Glu204 produces M- (or M(N), M(G))-like intermediates under all of the conditions examined. The absence of N, O, and O' intermediates suggests a long-range effect of the mutation. Furthermore, it is suggested that Cl(-) acts by reaching the interior of the protein, rather than producing surface effects. The effect of low water content was also examined, in the presence of Cl(-). Similar spectra corresponding to the M(1) intermediate were found for dry samples of both mutants, indicating that the effects of the mutations or of Cl(-) ions are confined to the second part of the photocycle. The water O-H stretching data further confirms altered photocycles and the effect of Cl(-) on the accumulation of the N intermediate.     

Stabilization of a fibronectin type III domain by the removal of unfavorable electrostatic interactions on the protein surface

It is generally considered that electrostatic interactions on the protein surface, such as ion pairs, contribute little to protein stability, although they may play important roles in conformational specificity. We found that the tenth fibronectin type III domain of human fibronectin (FNfn10) is more stable at acidic pH than neutral pH, with an apparent midpoint of transition near pH 4. Determination of pK(a)'s for all the side chain carboxyl groups of Asp and Glu residues revealed that Asp 23 and Glu 9 have an upshifted pK(a). These residues and Asp 7 form a negatively charged patch on the surface of FNfn10, with Asp 7 centrally located between Asp 23 and Glu 9, suggesting repulsive electrostatic interactions among these residues at neutral pH. Mutant proteins, D7N and D7K, in which Asp 7 was replaced with Asn and Lys, respectively, exhibited a modest but significant increase in stability at neutral pH, compared to the wild type, and they no longer showed pH dependence of stability. The pK(a)'s of Asp 23 and Glu 9 in these mutant proteins shifted closer to their respective unperturbed values, indicating that the unfavorable electrostatic interactions have been reduced in the mutant proteins. Interestingly, the wild-type and mutant proteins were all stabilized to a similar degree by the addition of 1 M sodium chloride at both neutral and acidic pH, suggesting that the repulsive interactions between the carboxyl groups cannot be effectively shielded by 1 M sodium chloride. These results indicate that repulsive interactions between like charges on the protein surface can destabilize a protein, and protein stability can be significantly improved by relieving these interactions.     

Reverse protonation is the key to general acid-base catalysis in enolase

The pH dependence of enolase catalysis was studied to understand how enolase is able to utilize both general acid and general base catalysis in each direction of the reaction at near-neutral pHs. Wild-type enolase from yeast was assayed in the dehydration reaction (2-phospho-D-glycerate --> phosphoenolpyruvate + H(2)O) at different pHs. E211Q, a site-specific variant of enolase that catalyzes the exchange of the alpha-proton of 2-phospho-D-glycerate but not the complete dehydration, was assayed in a (1)H/(2)H exchange reaction at different pDs. Additionally, crystal structures of E211Q and E168Q were obtained at 2.0 and 1.8 A resolution, respectively. Analysis of the pH profile of k(cat)/K(Mg) for wild-type enolase yielded macroscopic pK(a) estimates of 7.4 +/- 0.3 and 9.0 +/- 0.3, while the results of the pD profile of the exchange reaction of E211Q led to a pK(a) estimate of 9.5 +/- 0.1. These values permit estimates of the four microscopic pK(a)s that describe the four relevant protonation states of the acid/base catalytic groups in the active site. The analysis indicates that the dehydration reaction is catalyzed by a small fraction of enzyme that is reverse-protonated (i.e., Lys345-NH(2), Glu211-COOH), whereas the hydration reaction is catalyzed by a larger fraction of the enzyme that is typically protonated (i.e., Lys345-NH(3)(+), Glu211-COO(-)). These two forms of the enzyme coexist in a constant, pH-independent ratio. The structures of E211Q and E168Q both show virtually identical folds and active-site architectures (as compared to wild-type enolase) and thus provide additional support to the conclusions reported herein. Other enzymes that require both general acid and general base catalysis likely require reverse protonation of catalytic groups in one direction of the reaction.     

Thermodynamic analysis of ionizable groups involved in the catalytic mechanism of human matrix metalloproteinase 7 (MMP-7)

Human matrix metalloproteinase 7 (MMP-7) exhibits a broad bell-shaped pH-dependence with the acidic and alkaline pK(e) (pK(e1) and pK(e2)) values of about 4 and 10. In this study, we estimated the ionizable groups involved in its catalytic mechanism by thermodynamic analysis. pK(a) of side chains of L-Asp, L-Glu, L-His, L-Cys, L-Tyr, L-Lys, and L-Arg at 25-45°C were determined by the pH titration of amino-acid solutions, from which their enthalpy changes, ?H°, of deprotonation were calculated. pK(e1) and pK(e2) of MMP-7 at 15-45°C were determined in the hydrolysis of (7-methoxycoumarin-4-yl)acetyl-L-Pro-L-Leu-Gly-L-Leu-[N(3)-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]-L-Ala-L-Arg-NH(2), from which ?H(o) for pK(e1) and pK(e2) was calculated. The ?H(o) for pK(e1) (-20.6±6.1kJmol(-1)) was similar to that for L-Glu (-23.6±5.8kJmol(-1)), and the ?H(o) for pK(e2) (89.9±4.0kJmol(-1)) was similar to those for L-Arg (87.6±5.5kJmol(-1)) and L-Lys (70.4±4.4kJmol(-1)). The mutation of the active-site residue Glu198 into Ala completely abolished the activity, suggesting that Glu198 is the ionizable group for pK(e1). On the other hand, no arginine or lysine residues are found in the active site of MMP-7. We proposed a possibility that a protein-bound water is the ionizable group for pK(e2).     

Oxidation of the non-heme iron complex in photosystem II

In photosystem II (PSII), the redox properties of the non-heme iron complex (Fe complex) are sensitive to the redox state of quinones (Q(A/)(B)), which may relate to the electron/proton transfer. We calculated the redox potentials for one-electron oxidation of the Fe complex in PSII [E(m)(Fe)] based on the reference value E(m)(Fe) = +400 mV at pH 7 in the Q(A)(0)Q(B)(0) state, considering the protein environment in atomic detail and the associated changes in protonation pattern. Our model yields the pH dependence of E(m)(Fe) with -60 mV/pH as observed in experimental redox titration. We observed significant deprotonation at D1-Glu244 in the hydrophilic loop region upon Fe complex oxidation. The calculated pK(a) value for D1-Glu244 depends on the Fe complex redox state, yielding a pK(a) of 7.5 and 5.5 for Fe(2+) and Fe(3+), respectively. To account for the pH dependence of E(m)(Fe), a model involving not only D1-Glu244 but also the other titratable residues (five Glu in the D-de loops and six basic residues near the Fe complex) seems to be needed, implying the existence of a network of residues serving as an internal proton reservoir. Reduction of Q(A/B) yields +302 mV and +268 mV for E(m)(Fe) in the Q(A)(-)Q(B)(0) and Q(A)(0)Q(B)(-) states, respectively. Upon formation of the Q(A)(0)Q(B)(-) state, D1-His252 becomes protonated. Forming Fe(3+)Q(B)H(2) by a proton-coupled electron transfer process from the initial state Fe(2+)Q(B)(-) results in deprotonation of D1-His252. The two EPR signals observed at g = 1.82 and g = 1.9 in the Fe(2+)Q(A)(-) state of PSII may be attributed to D1-His252 with variable and fixed protonation, respectively.     

Dissecting the electrostatic interactions and pH-dependent activity of a family 11 glycosidase

Previous studies of the low molecular mass family 11 xylanase from Bacillus circulans show that the ionization state of the nucleophile (Glu78, pK(a) 4.6) and the acid/base catalyst (Glu172, pK(a) 6.7) gives rise to its pH-dependent activity profile. Inspection of the crystal structure of BCX reveals that Glu78 and Glu172 are in very similar environments and are surrounded by several chemically equivalent and highly conserved active site residues. Hence, there are no obvious reasons why their apparent pK(a) values are different. To address this question, a mutagenic approach was implemented to determine what features establish the pK(a) values (measured directly by (13)C NMR and indirectly by pH-dependent activity profiles) of these two catalytic carboxylic acids. Analysis of several BCX variants indicates that the ionized form of Glu78 is preferentially stabilized over that of Glu172 in part by stronger hydrogen bonds contributed by two well-ordered residues, namely, Tyr69 and Gln127. In addition, theoretical pK(a) calculations show that Glu78 has a lower pK(a) value than Glu172 due to a smaller desolvation energy and more favorable background interactions with permanent partial charges and ionizable groups within the protein. The pK(a) value of Glu172 is in turn elevated due to electrostatic repulsion from the negatively charged glutamate at position 78. The results also indicate that all of the conserved active site residues act concertedly in establishing the pK(a) values of Glu78 and Glu172, with no particular residue being singly more important than any of the others. In general, residues that contribute positive charges and hydrogen bonds serve to lower the pK(a) values of Glu78 and Glu172. The degree to which a hydrogen bond lowers a pK(a) value is largely dependent on the length of the hydrogen bond (shorter bonds lower pK(a) values more) and the chemical nature of the donor (COOH > OH > CONH(2)). In contrast, neighboring carboxyl groups can either lower or raise the pK(a) values of the catalytic glutamic acids depending upon the electrostatic linkage of the ionization constants of the residues involved in the interaction. While the pH optimum of BCX can be shifted from -1.1 to +0.6 pH units by mutating neighboring residues within the active site, activity is usually compromised due to the loss of important ground and/or transition state interactions. These results suggest that the pH optima of an enzyme might be best engineered by making strategic amino acid substitutions, at positions outside of the "core" active site, that electrostatically influence catalytic residues without perturbing their immediate structural environment.     

The pH-dependence of amide chemical shift of Asp/Glu reflects its pKa in intrinsically disordered proteins with only local interactions

Detailed knowledge of the pH-dependence of ionizable residues in both folded and unfolded states of proteins is essential to understand the role of electrostatics in protein folding and stability. The reassembly of E. coli Thioredoxin (Trx) by complementation of its two disordered fragments (1-37/38-108) provides a folded heterodimer in equilibrium with its unfolded state which, based on circular dichroism and NMR spectroscopy, consists of two unfolded monomers. To gain insight into the role of electrostatics in protein folding and stability, we compared the pH-dependence of the carboxylate sidechain chemical shift of each Asp/Glu against that of its backbone amide chemical shift in the unfolded heterodimer. We monitored via C(CO)NH experiments four Asp and four Glu in fragments 38 to 108 (C37) of Trx in the pH range from 2.0 to 7.0 and compared them with results from (1)H(15)N HSQC experiments [Pujato et al., Biophys. J., 89 (2005) 3293-3302]. The (1)H(15)N HSQC analysis indicates two segments with quite distinct behavior: (A) a segment from Ala57 to Ala108 in which ionizable residues have up to three contiguous neighbors with pH-dependent backbone amide shifts, and (B) a segment of fifteen contiguous pH-dependent backbone amide shifts (Leu42 to Val56) in which two Asp and two Glu are implicated in medium range interactions. In all cases, the titration curves are simple modified sigmoidals from which a pH-midpoint (pH(m)) can be obtained by fitting. In segment A, the pH(m) of a given backbone amide of Asp/Glu mirrors within 0.15 pH-units that of its carboxylate sidechain (i.e., the pK(a)). In contrast, segment B shows significant differences with absolute values of 0.46 and 0.74 pH-units for Asp and Glu, respectively. The dispersion in the pH(m) of the backbone amide of Asp/Glu is also different in the two segments. Segment A shows a dispersion of 0.31 and 0.17 pH-units for Asp and Glu, respectively. Segment B shows a substantially larger dispersion (0.50 and 1.08 pH-units for Asp and Glu, respectively). In both segments, the dispersion in the pH(m) of its backbone amide is larger than in the pK(a) of the carboxylate sidechain (the latter is only 0.17 and 0.52 pH-units for Asp and Glu, respectively). Our results indicate that the pH(m) of the backbone amide chemical shift of Asp/Glu in a disordered polypeptide segment is a good predictor of its pK(a) whenever there are none or few neighboring backbone amides with similar pH-dependence.     

Determination of pK(a) values of carboxyl groups in the N-terminal domain of rat CD2: anomalous pK(a) of a glutamate on the ligand-binding surface

The ligand-binding surface of the T-lymphocyte glycoprotein CD2 has an unusually high proportion of charged residues, and ionic interactions are thought to play a significant role in defining the ligand specificity and binding affinity of CD2 with the structurally homologous ligands CD48 (in rodents) and CD58 (in humans). The determination of the electrostatic properties of these proteins can therefore contribute to our understanding of structure-activity relationships for these adhesion complexes that underpin T-cell adhesion to antigen-presenting cells. In this study, we investigated the pH titration behavior of the carboxyl groups of the N-terminal domain of rat CD2 (CD2d1) using the chemical shifts of backbone amide nitrogen-15 ((15)N) and proton NMR resonances, and carboxyl carbon-13 ((13)C) signals. The analysis revealed the presence of a glutamate (Glu41) on the binding surface of rat CD2 with an unusually elevated acidity constant (pK(a) = 6.73) for CD2d1 samples at 1.2 mM concentration. pH titration of CD2d1 at low protein concentration (0.1 mM) resulted in a slight decrease of the measured pK(a) of Glu41 to 6.36. The ionization of Glu41 exhibited reciprocal interactions with a second glutamate (Glu29) in a neighboring location, with both residues demonstrating characteristic biphasic titration behavior of the carboxyl (13)C resonances. Measurements at pH 5.5 of the two-bond deuterium isotope shift for the (13)C carboxyl resonances for Glu41 and Glu29 [(2)DeltaC(delta)(O(epsilon)D) = 0.2 and 0.1 ppm, respectively] were consistent with the assignment of the anomalous pK(a) to Glu41, under the strong influence of Glu29. The characterization of single site mutations of CD2d1 residues Glu41 and Glu29 to glutamine confirmed the anomalous pK(a) for Glu41, and indicated that electrostatic interaction with the Glu29 side chain is a significant contributing influence for this behavior in the wild-type protein. The implications of these observations are discussed with respect to recent structural and functional analyses of the interaction of rat CD2 with CD48. In particular, CD2 Glu41 must be a candidate residue to explain the previously reported strong pH dependence of binding of these two proteins in vitro.     

Catalytic mechanism of SGAP, a double-zinc aminopeptidase from Streptomyces griseus

The catalytic mechanism underlying the aminopeptidase from Streptomyces griseus (SGAP) was investigated. pH-dependent activity profiles revealed the enthalpy of ionization for the hydrolysis of leucine-para-nitroanilide by SGAP. The value obtained (30 +/- 5 kJ.mol(-1)) is typical of a zinc-bound water molecule, suggesting that the zinc-bound water/hydroxide molecule acts as the reaction nucleophile. Fluoride was found to act as a pure noncompetitive inhibitor of SGAP at pH values of 5.9-8 with a K(i) of 11.4 mM at pH 8.0, indicating that the fluoride ion interacts equally with the free enzyme as with the enzyme-substrate complex. pH-dependent pK(i) experiments resulted in a pK(a) value of 7.0, suggesting a single deprotonation step of the catalytic water molecule to an hydroxide ion. The number of proton transfers during the catalytic pathway was determined by monitoring the solvent isotope effect on SGAP and its general acid-base mutant SGAP(E131D) at different pHs. The results indicate that a single proton transfer is involved in catalysis at pH 8.0, whereas two proton transfers are implicated at pH 6.5. The role of Glu131 in binding and catalysis was assessed by determining the catalytic constants (K(m), k(cat)) over a temperature range of 293-329 degrees K for both SGAP and the E131D mutant. For the binding step, the measured and calculated thermodynamic parameters for the reaction (free energy, enthalpy and entropy) for both SGAP and the E131D mutant were similar. By contrast, the E131D point mutation resulted in a four orders of magnitude decrease in k(cat), corresponding to an increase of 9 kJ.mol(-1) in the activation energy for the E131D mutant, emphasizing the crucial role of Glu131 in catalysis.     

The effect of net charge on the solubility, activity, and stability of ribonuclease Sa

The net charge and isoelectric pH (pI) of a protein depend on the content of ionizable groups and their pK values. Ribonuclease Sa (RNase Sa) is an acidic protein with a pI = 3.5 that contains no Lys residues. By replacing Asp and Glu residues on the surface of RNase Sa with Lys residues, we have created a 3K variant (D1K, D17K, E41K) with a pI = 6.4 and a 5K variant (3K + D25K, E74K) with a pI = 10.2. We show that pI values estimated using pK values based on model compound data can be in error by >1 pH unit, and suggest how the estimation can be improved. For RNase Sa and the 3K and 5K variants, the solubility, activity, and stability have been measured as a function of pH. We find that the pH of minimum solubility varies with the pI of the protein, but that the pH of maximum activity and the pH of maximum stability do not.