Chemically accurate protein structures: Validation of protein NMR structures by comparison of measured and predicted pK a values

A new method is presented for evaluating the quality of protein structures obtained by NMR. This method exploits the dependence between measurable chemical properties of a protein, namely pK _a values of acidic residues, and protein structure. The accurate and fast empirical computational method employed by the PROPKA program () allows the user to test the ability of a given structure to reproduce known pK _a values, which in turn can be used as a criterion for the selection of more accurate structures. We demonstrate the feasibility of this novel idea for a series of proteins for which both␣NMR and X-ray structures, as well as pK _a values of all ionizable residues, have been determined. For the 17 NMR ensembles used in this study, this criterion is shown effective in the elimination of a large number of NMR structure ensemble members.     

Factors influencing the energetics of electron and proton transfers in proteins. What can be learned from calculations

A protein structure should provide the information needed to understand its observed properties. Significant progress has been made in developing accurate calculations of acid/base and oxidation/reduction reactions in proteins. Current methods and their strengths and weaknesses are discussed. The distribution and calculated ionization states in a survey of proteins is described, showing that a significant minority of acidic and basic residues are buried in the protein and that most of these remain ionized. The electrochemistry of heme and quinones are considered. Proton transfers in bacteriorhodopsin and coupled electron and proton transfers in photosynthetic reaction centers, 5-coordinate heme binding proteins and cytochrome c oxidase are highlighted as systems where calculations have provided insight into the reaction mechanism.     

No direct linkage between proton pumping and photosynthate unloading from seed coats of Phaseolus vulgaris L.

The energization of the active sucrose release from bean seed-coat halves was investigated. For this purpose, seed coat tissues adjacent to the apoplastic space were exposed to a variety of treatments and proton and photosynthate release were measured. Fusicoccin (10^–5 moll^–1) stimulated proton pump activities. Orthovanadate (2×10^–4 moll^–1) and abscisic acid (10^–5 moll^–1) diminished the proton extrusion evoked by fusicoccin. Fusicoccin inhibited sucrose release, whereas orthovanadate and abscisic acid stimulated it. Addition of 100 mmoll^–1 K⁺ had a promotory effect on photosynthate unloading, fading away with time. This extra unloading was linearly related to an enhanced proton loss. It was concluded that the photosynthate unloading apparently is not a proton/sucrose antiport and that a pump-leak system for photosynthate release is unlikely. A tentative model for photosynthate/proton symport not directly linked to proton pumping is presented as the mechanism of unloading.Abbreviations ABA abscisic acid - CCCP carbonyl cyanide m-chlorophenyl hydrazone - DTE diethioerythritol - FC fusicoccin - MES 2-(N-morpholino) ethanesulfonic acid monohydrate - NEM n-ethylmaleimide - PCMBS p-chloromercuriphenylsulfonic acid - TRIS 2-amino-2-(hydroxymethyl) propane-1,3 diol - VAN sodium orthovanadate     

Effects of geometric isomerism in dinuclear platinum antitumor complexes on aquation reactions in the presence of perchlorate, acetate and phosphate

The aquation and subsequent reactions of the dinuclear Pt antitumor complexes [{trans-PtCl(NH(3))(2)}(2)(mu-NH(2)(CH(2))(6)NH(2))](2+) (1,1/t,t) and [{cis-PtCl(NH(3))(2)}(2)(mu-NH(2)(CH(2))(6)NH(2))](2+) (1,1/c,c) in 15 mM perchlorate, acetate or phosphate solutions were followed at 298 K by [(1)H,(15)N] HSQC 2D NMR spectroscopy. Rate and equilibrium constants for the initial reversible aquation and the subsequent reversible reaction with phosphate or acetate are reported. The rate constant for the first aquation step is two-fold lower for 1,1/c,c than 1,1/t,t but the anation rate constants are similar so that the equilibrium lies further towards the chloro form for the 1,1/c,c compound. A pK (a) value of 6.01+/-0.03 was determined for the diaquated species [{cis-Pt(NH(3))(2)(H(2)O)}(2)(mu-NH(2)(CH(2))(6)NH(2))](4+) (1,1/c,c-3) which is 0.4 units higher than that of the 1,1/t,t compound. The rate constants for the binding of acetate and phosphate to 1,1/t,t are similar, but the rate constant for the reverse reaction is close to ten-fold higher in the case of phosphate so that equilibrium conditions are attained more rapidly (12 h compared with 64 h). On the other hand, for 1,1/c,c the rate constants for the forward and reverse reactions with acetate and phosphate are quite similar so that equilibrium conditions are reached very slowly (80-100 h) and a greater proportion of phosphate-bound species are present. The reduced lability of the bound phosphate for 1,1/c,c is attributed to the formation of a macrochelate phosphate-bridged species which was characterized by (31)P NMR and ESI-MS. The speciation profiles of 1,1/t,t and 1,1/c,c under physiological conditions are explored.     

Physicochemical properties of hydroxylated polychlorinated biphenyls aid in predicting their interactions with rat sulfotransferase 1A1 (rSULT1A1)

Hydroxylated metabolites of polychlorinated biphenyls (OHPCBs) interact with rat sulfotransferase 1A1 (rSULT1A1) as substrates and inhibitors. Previous studies have shown that there are complex and incompletely understood structure–activity relationships governing the interaction of rSULT1A1 with these molecules. Furthermore, modification of the enzyme with glutathione disulfide (GSSG) results in the conversion of some OHPCBs from inhibitors to substrates. We have now examined estimated values for the acid-dissociation constant (K_a) and the octanol–water distribution coefficient (D), as well as experimentally determined dissociation constants for enzyme complexes, to assist in the prediction of interactions of OHPCBs with rSULT1A1. Under reducing conditions, initial velocities for rSULT1A1-catalyzed sulfation exhibited a positive correlation with pK_a and a negative correlation with log D of the OHPCBs. IC₅₀ values of inhibitory OHPCBs decreased with decreasing pK_a values for both the glutathione (GSH)-pretreated and GSSG-pretreated forms of rSULT1A1. Comparison of GSH- and GSSG-pretreated forms of rSULT1A1 with respect to binding of OHPCB in the presence and absence of adenosine 3′,5′-diphosphate (PAP) revealed that the dissociation constants with the two redox states of the enzyme were similar for each OHPCB. Thus, pK_a and log D values are useful in predicting the binding of OHPCBs to the two redox forms of rSULT1A1 as well as the rates of sulfation of those OHPCBs that are substrates. However, the differences in substrate specificity for OHPCBs that are seen with changes in redox status of the enzyme are not directly related to specific structural effects of individual OHPCBs within inhibitory enzyme–PAP–OHPCB complexes.     

Molecular modeling studies of the DCCD-treated cytochrome bc1 complex: predicted conformational changes and inhibition of proton translocation

Dicyclohexylcarbodiimide (DCCD) binds covalently to an acidic amino acid located in the cd loop connecting membrane-spanning helices C and D of cytochrome b resulting in an inhibition of proton translocation in the cytochrome bc ₁ complex with minimal effects on the steady state rate of electron transfer. Single turnover studies performed with the yeast cytochrome bc ₁ complex indicated that the initial phase of cytochrome b reduction was inhibited 25–45% in the DCCD-treated cytochrome bc ₁ complex, while the rate of cytochrome c ₁ reduction was unaffected. Simulations by molecular modeling predict that binding of DCCD to glutamate 163 located in the cd2 loop of cytochrome b of chicken liver mitochondria results in major conformational changes in the protein. The conformation of the cd loop and the end of helix C appeared twisted with a concomitant rearrangement of the amino acid residues of both cd1 and cd2 loops. The predicted rearrangement of the amino acid residues of the cd loop results in disruptions of the hydrogen bonds predicted to form between amino acid residues of the cd and ef loops. Simultaneously, two new hydrogen bonds are predicted to form between glutamate 272 and two residues, aspartate 253 and tyrosine 272. Formation of these new hydrogen bonds would restrict the rotation and protonation of glutamate 272, which may be necessary for the release of the second electrogenic proton obtained during ubiquinol oxidation in the bc₁ complex.     

Electrostatic control of proton pumping in cytochrome c oxidase

As part of the mitochondrial respiratory chain, cytochrome c oxidase utilizes the energy produced by the reduction of O₂ to water to fuel vectorial proton transport. The mechanism coupling proton pumping to redox chemistry is unknown. Recent advances have provided evidence that each of the four observable transitions in the complex catalytic cycle consists of a similar sequence of events. However, the physico-chemical basis underlying this recurring sequence has not been identified. We identify this recurring pattern based on a comprehensive model of the catalytic cycle derived from the analysis of oxygen chemistry and available experimental evidence. The catalytic cycle involves the periodic repetition of a sequence of three states differing in the spatial distribution of charge in the active site: [0|1], [1|0], and [1|1], where the total charge of heme a and the binuclear center appears on the left and on the right, respectively. This sequence recurs four times per turnover despite differences in the redox chemistry. This model leads to a simple, robust, and reproducible sequence of electron and proton transfer steps and rationalizes the pumping mechanism in terms of electrostatic coupling of proton translocation to redox chemistry. Continuum electrostatic calculations support the proposed mechanism and suggest an electrostatic origin for the decoupled and inactive phenotypes of ionic mutants in the principal proton-uptake pathway.     

Carbon-13 NMR studies of the lysine side chains of calmodulin and its proteolytic fragments

ThepH-titration and dynamic behaviour of the seven lysine side chains in bovine calmodulin were studied by carbon-13 NMR. The amino groups of the calcium saturated protein and its proteolytic fragments TR₁C(1–75) and TR₂C (78–148) were dimethylated with carbon-13 labeled formaldehyde; this modification did not alter the protein's structure or its ability to activate the enzyme cyclic nucleotide phosphodiesterase. Tentative assignments for 5 out of the 7 dimethyl lysine resonances could be obtained by comparing spectra of the fully and partially modified protein, with those of the proteolytic fragments. ThepK_a values measured for calcium saturated calmodulin ranged between 9.5 (Lys 75) and 10.2 (Lys 13); two residues (Lys 94 and Lys 13) showed a biphasic titration curve suggesting their possible involvement in ion-pairs. The dynamic behavior of the lysine side chains was deduced from spin lattice relaxation measurements. All side chains were flexible and this was not influenced by the removal of calcium, or the addition of the calmodulin antagonist trifluoperazine. The latter data suggest that the lysine side chains are not directly involved in calmodulin's target binding sites.     

Towards the mechanism of proton pumping by the haem-copper oxidases

The haem-copper oxidases comprise a large family of enzymes that is widespread among aerobic organisms. These remarkable membrane-bound proteins catalyse the respiratory reduction of dioxygen to water, and conserve free energy from this reaction by operating as proton pumps. The mechanism of redox-dependent proton translocation has been elusive despite the availability of high resolution crystal structures from several oxidases. Here, we discuss some recent as well as some older results that may shed light on this mechanism. We conclude that proton-pumping is initiated by vectorial proton transfer from a conserved glutamic acid (Glu242 in the bovine enzyme) to a proton acceptor above the haem groups, and that this primary event is mechanistically coupled to electron transfer from haem a to the binuclear haem a₃/Cu_B centre. Subsequently, Glu242 is reprotonated from the negatively charged side of the membrane. Next this proton is transferred to the binuclear site to complete the chemistry, Glu242 is reprotonated once more, and the “prepumped” proton is ejected on the opposite side of the membrane. The different kinetics of electron-coupled proton transfer in different steps of the catalytic cycle may be related to differences in the driving force due to different E_m values of the electron acceptor in the binuclear site.     

Carbanions from decarboxylation of orotate analogs: stability and mechanistic implications

The pKa's of the 6-CH groups of 1,3-dimethyluracil, N-methyl-2-pyridone, and N-methyl-4-pyridone were determined through their reactions with bases derived from carbon acids with known pKa and the reactions of their corresponding carbanions with the carbon acids. No correlation between the stability of the carbanions and the rate of decarboxylation of corresponding carboxylic acids was found.     

Roles of N-terminal pyroglutamate in maintaining structural integrity and pKa values of catalytic histidine residues in bullfrog ribonuclease 3

Many proteins and bioactive peptides contain an N-terminal pyroglutamate residue (Pyr1). This residue reduces the susceptibility of the protein to aminopeptidases and often has important functional roles. The antitumor ribonuclease RC-RNase 3 (RNase 3) from oocytes of Rana catesbeiana (bullfrog) is one such protein. We have produced recombinant RNase 3 containing the N-terminal Pyr1 (pRNase 3) and found it to be indistinguishable from the native RNase 3 by mass spectrometry and a variety of other biochemical and immunological criteria. We demonstrated by NMR analysis that the Pyr1 of pRNase 3 forms hydrogen bonds with Lys9 and Ile96 and stabilizes the N-terminal alpha-helix in a rigid conformation. In contrast, the N-terminal alpha-helix becomes flexible and the pKa values of the catalytic residues His10 and His97 altered when Pyr1 formation is blocked by an extra methionine at the N terminus in the recombinant mqRNase 3. Thus, our results provide a mechanistic explanation on the essential role of Pyr1 in maintaining the structural integrity, especially at the N-terminal alpha-helix, and in providing the proper environment for the ionization of His10 and His97 residues for catalysis and cytotoxicity against HeLa cells.     

Geometrical deformation and failure behavior of C60 fullerene dimer under applied external electric field

By the quantum-molecular dynamics (QMD) technique based on the Roothaan–Hall equation and the Newton motion law, geometrical deformation and failure behavior of C₆₀ fullerene dimer (2C₆₀) as well as single C₆₀ fullerene under applied external electric field are simulated. Further, the effects of the electric field direction on the electric field-induced deformation, polarization-charge distribution and dipole moment of the fullerene molecules are discussed systemically. It is found that the geometrical configuration and failure behavior of the 2C₆₀ molecule are sensitive to the electric field direction, that when the electric field direction is parallel to the bridging C–C bonds of the 2C₆₀ molecule the 2C₆₀ fails easily, and that when the electric field direction is perpendicular to the 2C₆₀ fails difficultly and has the same polarization and failure mechanism as the single C₆₀.     

Protonation state of Asp30 exerts crucial influence over surface loop rearrangements responsible for NO release in nitrophorin 4

pK(a) values of ionizable residues were calculated for the crystal structures describing the pH and NO binding dependant conformations of nitrophorin 4, a pH sensitive NO carrier heme protein. Comparison of resultant H-bonding patterns allowed the identification of the amino acids that take part in signaling pH change. We carried out MD simulations to show that the protonation state of Asp30, buried in the closed conformation, is crucial for maintaining the tight packed conformation of the closed form of the complex - presenting a model for the functional decrease of NO binding affinity of nitrophorins at physiological pH.     

Relevance of phenylalanine 216 in the affinity of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> phosphoenolpyruvate carboxykinase for Mn(II)

Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase catalyzes the reversible formation of oxaloacetate and adenosine triphosphate from PEP, adenosine diphosphate and carbon dioxide, and uses Mn²⁺ as the activating metal ion. Comparison with the crystalline structure of homologous Escherichia coli PEP carboxykinase [Tari et al. (1997) Nature Struct. Biol. 4, 990–994] shows that Lys²¹³ is one of the ligands to Mn²⁺ at the enzyme active site. Coordination of Mn²⁺ to a lysyl residue is not common and suggests a low pK _a value for the ε-NH₂ group of Lys²¹³. In this work, we evaluate the role of neighboring Phe²¹⁶ in contributing to provide a low polarity microenvironment suitable to keep the ε-NH₂ of Lys²¹³ in the unprotonated form. Mutation Phe216Tyr shows that the introduction of a hydroxyl group in the lateral chain of the residue produces a substantial loss in the enzyme affinity for Mn²⁺, suggesting an increase of the pK _a of Lys²¹³. In agreement with this interpretation, theoretical calculations indicate an alkaline shift of 2.8 pH units in the pK _a of the ε-amino group of Lys²¹³ upon Phe216Tyr mutation.     

Ion exchange properties of plant root cell walls

Acid-base properties and the swelling capacity of wheat, lupin and pea root cell walls were investigated. Roots of seedlings and green plants of different age were analysed by the potentiometric method. The ion exchange capacity (S _i) and the swelling coefficient (K ^cw) of root cell walls were estimated at various pH values (from 2 to 12) and at different ionic strength (between 0.3 and 1000 mM). To analyse the polysigmoid titration curves pH_i = f (S _i), the Gregor's equation was employed. It was shown that the Gregor's model fits well the experimental data. The total number of the cation exchange (S _t ^cat) and the anion exchange (S _t ^an) groups were determined in the root cell walls. The number of the functional group of each type (S ^j) was estimated, and the corresponding values of pK _a ^j were calculated. It was shown that for all types of cation exchangeable groups arranged in the cell wall structure the acid properties are enhanced by the increasing concentration of electrolyte. For each ionogenic group the coefficients of Helfferich's equation [pK _a ^j = f (C ^K+)] were determined. It was found that the swelling of root cell walls changes with pH, C ^K+ and strongly depends on plant species. Within the experimental pH and C ^K+ range the swelling coefficient changes as follows: lupin > pea > wheat. The obtained results show that for the plant species under investigation the differences in the swelling coefficients originate from (a) the differences in the cross-linking degrees of polymeric chains arranged in the cell wall structure, (b) the differences in the number of carboxyl groups and (c) the differences in the total number of functional groups. Based on the estimated swelling coefficients in water it could be inferred that for wheat the cross-linking degree of the polymeric chains in the root cell walls is higher than those for lupin or pea. It has been emphasized that the calculated parameters (S ^j, pK _a ^j, K ^cw), the equation {pK _a ^j = f (C^K+)} and the dependencies {K ^cw = f (C^K+, pH)} allow to estimate quantitatively the changes in the ion exchange capacity of the root cell walls in response to the changes in an ionic composition of an outer solution. The results of these estimations allow to suggest that (a) the root apoplast is a compartment where the accumulation of cations takes place during the first stage of cation uptake from an outer medium, and (b) the accumulation degree is defined by pH and ionic composition of an outer solution. On the basis of the literature review and the results of the present experimental study it was proposed that the changes in the cell wall swelling in response to variances of environmental or experimental conditions could lead to a change of the water flow through a root apoplast. It has been supported that there is direct relationship between the swelling of root cell walls and the water flow within the plant root apoplast.     

Structure,redox, p<Emphasis Type="Italic">K</Emphasis><Subscript>a</Subscript>, spin. A golden tetrad for understanding metalloenzyme energetics and reaction pathways

After a review of the current status of density functional theory (DFT) for spin-polarized and spin-coupled systems, we focus on the resting states and intermediates of redox-active metalloenzymes and electron transfer proteins, showing how comparisons of DFT-calculated spectroscopic parameters with experiment and evaluation of related energies and geometries provide important information. The topics we examine include (1) models for the active-site structure of methane monooxygenase intermediate Q and ribonucleotide reductase intermediate X; (2) the coupling of electron transfer to proton transfer in manganese superoxide dismutase, with implications for reaction kinetics; (3) redox, pK(a), and electronic structure issues in the Rieske iron-sulfur protein, including their connection to coupled electron/proton transfer, and an analysis of how partial electron delocalization strongly alters the electron paramagnetic resonance spectrum; (4) the connection between protein-induced structural distortion and the electronic structure of oxidized high-potential 4Fe4S proteins with implications for cluster reactivity; (5) an analysis of cluster assembly and central-atom insertion into the FeMo cofactor center of nitrogenase based on DFT structural and redox potential calculations.     

Synthesis of N-acridinyl-N'-alkylguanidines: dramatic influence of amine to guanidine replacement on the physicochemical properties

Transformation of aminoacridines into N-acridinyl-N′-alkylguanidines is described. The chosen procedure allows introduction of pendent substituents (exemplified by N,N-dimethylaminopropyl chain) into key acridinyl thioureas, thus opening the way to structural diversity. Spectroscopic study and pK_a determination show that the presence of the strongly basic guanidine has a dramatic influence on the ionization of the acridine nucleus by lowering the pk_a value down to 4.49.     

The suitability of different DHB isomers as matrices for the MALDI-TOF MS analysis of phospholipids: which isomer for what purpose?

Although the analysis of large biomolecules is the prime application of matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS), there is also increasing interest in lipid analysis. Since lipids possess relatively small molecular weights, matrix signals should be as small as possible to avoid overlap with lipid peaks. Although 2,5-dihydroxybenzoic acid (DHB) is an established MALDI matrix, the question whether just this isomer is ideal for lipid analysis was not yet addressed. UV absorptions of all six DHB isomers were determined and their laser desorption spectra recorded. In addition, all isomers were used as matrices to record positive and negative ion mass spectra of selected phospholipids (phosphatidylcholine and -serine): In the order 2,5-, 2,6-, 2,3- and 2,4-DHB, the quality of the positive ion lipid spectra decreases. This correlates well with the decreasing acidity of the applied DHB isomers. The 3,4- and 3,5- isomers give only very weak positive ion signals especially of acidic lipids. In contrast, the most suitable matrices in the negative ion mode are 2,5-, 2,4- and 3,5-DHB. 2,6-DHB does not provide any signal in the negative ion mode due to its marked acidity. Finally, differences in the crystallization behavior of the pure matrix and the matrix/lipid co-crystals were also monitored by atomic force microscopy (AFM): 2,5-DHB gave the smallest crystals and the skinniest layer. It is concluded that basically all DHB isomers can be used as MALDI matrices but the 2,5-isomer represents the most versatile compound. Dedicated to Prof. Dr. Klaus Arnold on the occasion of his 65th birthday.     

Investigation of protonatable residues in Rhodothermus marinus caa 3 haem-copper oxygen reductase: comparison with Paracoccus denitrificans aa 3 haem-copper oxygen reductase

The Rhodothermus marinus caa ₃ haem-copper oxygen reductase contains all the residues of the so-called D- and K-proton channels, with the notable exception of the helix VI glutamate residue (Glu278^I in Paracoccus denitrificans aa ₃), being nevertheless a true oxygen reductase reducing O₂ to water, and an efficient proton pump. Instead, in the same helix, but one turn below, it has a tyrosine residue (Tyr256^I, R. marinus caa ₃ numbering), whose hydroxyl group occupies the same spatial position as the carboxylate group of Glu278^I, as deduced by comparative modelling techniques. Therefore, we proposed previously that this tyrosine residue could play an important role in the proton pathway. In this article we further study this hypothesis, by investigating the equilibrium thermodynamics of protonation in R. marinus caa ₃, using theoretical methodologies based on the structural model previously obtained. Control calculations are also performed for the P. denitrificans aa ₃ oxygen reductase. In both oxygen reductases we find several residues that are proton active (i.e., that display partial protonation) at physiological pH, some of them being redox sensitive (i.e., sensitive to the protein redox state). However, the caa ₃ Tyr256^I is not proton active at physiological pH, in contrast to the aa ₃ Glu278^I which is both proton active at physiological pH and shows a high redox sensitivity. In R. marinus caa ₃ we do not find any other residues in the same protein zone that can have this property. Therefore, there are no putative D-channel residues that are proton active in this oxidase. The protonatable residues of the K-channel are much more functionally conserved in both oxygen reductases than the same type of residues in the D-channel. Two (Tyr262^I and Lys336^I, caa ₃ numbering) out of three protonatable K-channel residues are proton active and redox sensitive in both proteins.     

The role of intramolecular hydrogen bonding on nucleobase acidification following metal coordination: possible implications of an “indirect” role of metals in acid–base catalysis of nucleic acids

The acidifying effect of Pt^II on nucleobase –NH and –NH₂ groups depends both on the site of metal coordination and on the efficiency of stabilization of the deprotonated nucleobase via intracomplex hydrogen bonding. Weakly acidic nucleobase protons with pK _a values between 9 and 17 can be acidified by a single Pt^II to have pK _a values which are well within the physiological pH range. This could open the possibility of an acid–base catalysis occurring at pH 7, with the metal–nucleobase entity functioning either as an acid or a base. Examples of Pt^II complexes studied here include, among others, mixed nucleobase systems of 1-methylcytosine and 1,9-dimethyladenine as well as a complex of the rare iminooxo tautomer of 1-methylcytosine having the metal bonded at N4.