Hydrogen-bonded Salt Bridges Research Articles

Design Glutamate Dehydrogenase for Nonaqueous System by Motifs Reassembly and Interaction Network Analysis.

Glutamate dehydrogenases (GDH) serve as the key regulated enzyme that links protein and carbohydrate metabolism. Combined with motif reassembly and mutation, novel GDHs were designed. Motif reassembly of thermophilic GDH and malate dehydrogenase aims to overcome stability and activity tradeoff in nonaqueous systems. Structural compatibility and dynamic cooperation of the designed AaDHs were studied by molecular dynamics simulation. Furthermore, multipoint mutations improved its catalytic activity for unnatural substrates. Amino acid interaction network analysis indicated that the high density of hydrogen-bonded salt bridges is beneficial to the stability. Finally, the experimental verification determines the kinetics of AaDHs in a nonaqueous system. The activity of Aa05 was increased by 1.78-fold with ionic liquid [EMIM]BF4. This study presents the strategy of a combination of rigid motif assembly and mutations of active sites for robust dehydrogenases with high activity in the nonaqueous system, which overcomes the activity-stability tradeoff effect.

Oxidation of 2-mercaptopyridine N-oxide upon iodine agent: structural and FT-IR studies on charge-assisted hydrogen bonds CAHB(+) and I\u2026I halogen interactions in 2,2\u2032-dithiobis(pyridine N-oxide) ionic cocrystal

2-Mercaptopyridine N-oxide (I) undergoes spontaneous dimerization to the disulfide form due to reaction with iodine acting as an oxidizing reagent. As a result, a di-N-oxide disulfide derivative of pyridine is obtained. During the process of crystallization, one of N-oxide groups undergoes protonation and a cation form of disulfide moiety cocrystallizes with I3− counterion forming a salt structure. Therefore, in the crystalline state, the 2,2′-dithiobis(pyridine N-oxide) molecule exists in a not observed previously form of monocation. Interestingly, the protonated N-oxide group does not form hydrogen-bonded salt bridges (of the CAHB(±) type with I3− anions) but prefers to be involved in intermolecular interactions with the unprotonated N-oxide group of the adjacent molecule This results in formation of intermolecular CAHB(+) hydrogen bridges finally linking molecules into infinite chains. The crystal structure is also stabilized by other various noncovalent interactions, including iodine...iodine and sulfur...iodine contacts.

Controlling Second Coordination Sphere Effects in Luminescent Ruthenium Complexes by Means of External Pressure.

Two luminescent heteroleptic RuII complexes with a 2,2'-biimidazole (biimH2 ) ligand form doubly hydrogen-bonded salt bridges to 4-sulfobenzoate anions in single crystals. The structure of one of these cation-anion adducts shows that the biimH2 ligand is deprotonated. Its 3 MLCT luminescence band does not shift significantly under the influence of an external hydrostatic pressure, a behavior typical for these electronic transitions. In contrast, hydrostatic pressure on the other crystalline cation-anion adduct induces a shift of proton density from the peripheral N-H groups of biimH2 towards benzoate, leading to a pronounced redshift of the 3 MLCT luminescence band. Such a significant and pressure-tunable influence from an interaction in the second coordination sphere is unprecedented in artificial small-molecule-based systems.

Contextual Role of a Salt Bridge in the Phage P22 Coat Protein I-Domain

The I-domain is a genetic insertion in the phage P22 coat protein that chaperones its folding and stability. Of 11 acidic residues in the I-domain, seven participate in stabilizing electrostatic interactions with basic residues across elements of secondary structure, fastening the β-barrel fold. A hydrogen-bonded salt bridge between Asp-302 and His-305 is particularly interesting as Asp-302 is the site of a temperature-sensitive-folding mutation. The pKa of His-305 is raised to 9.0, indicating the salt bridge stabilizes the I-domain by ∼4 kcal/mol. Consistently, urea denaturation experiments indicate the stability of the WT I-domain decreases by 4 kcal/mol between neutral and basic pH. The mutants D302A and H305A remove the pH dependence of stability. The D302A substitution destabilizes the I-domain by 4 kcal/mol, whereas H305A had smaller effects, on the order of 1-2 kcal/mol. The destabilizing effects of D302A are perpetuated in the full-length coat protein as shown by a higher sensitivity to protease digestion, decreased procapsid assembly rates, and impaired phage production in vivo By contrast, the mutants have only minor effects on capsid expansion or stability in vitro The effects of the Asp-302-His-305 salt bridge are thus complex and context-dependent. Substitutions that abolish the salt bridge destabilize coat protein monomers and impair capsid self-assembly, but once capsids are formed the effects of the substitutions are overcome by new quaternary interactions between subunits.

Symmetric allosteric mechanism of hexameric Escherichia coli arginine repressor exploits competition between L-arginine ligands and resident arginine residues.

An elegantly simple and probably ancient molecular mechanism of allostery is described for the Escherichia coli arginine repressor ArgR, the master feedback regulator of transcription in L-arginine metabolism. Molecular dynamics simulations with ArgRC, the hexameric domain that binds L-arginine with negative cooperativity, reveal that conserved arginine and aspartate residues in each ligand-binding pocket promote rotational oscillation of apoArgRC trimers by engagement and release of hydrogen-bonded salt bridges. Binding of exogenous L-arginine displaces resident arginine residues and arrests oscillation, shifting the equilibrium quaternary ensemble and promoting motions that maintain the configurational entropy of the system. A single L-arg ligand is necessary and sufficient to arrest oscillation, and enables formation of a cooperative hydrogen-bond network at the subunit interface. The results are used to construct a free-energy reaction coordinate that accounts for the negative cooperativity and distinctive thermodynamic signature of L-arginine binding detected by calorimetry. The symmetry of the hexamer is maintained as each ligand binds, despite the conceptual asymmetry of partially-liganded states. The results thus offer the first opportunity to describe in structural and thermodynamic terms the symmetric relaxed state predicted by the concerted allostery model of Monod, Wyman, and Changeux, revealing that this state is achieved by exploiting the dynamics of the assembly and the distributed nature of its cohesive free energy. The ArgR example reveals that symmetry can be maintained even when binding sites fill sequentially due to negative cooperativity, which was not anticipated by the Monod, Wyman, and Changeux model. The molecular mechanism identified here neither specifies nor requires a pathway for transmission of the allosteric signal through the protein, and it suggests the possibility that binding of free amino acids was an early innovation in the evolution of allostery.

Selective molecular recognition of arginine by anionic salt bridge formation with bis-phosphate crown ethers: implications for gas phase peptide acidity from adduct dissociation

Arginine forms a stable noncovalent anionic salt bridge complex with DP (a crown ether which contains two endocyclic dialkylhydrogenphosphate esters). Abundant adduct formation with DP is observed for complexes with arginine, YAKR, HPPGFSPFR, AAKRKAA, RR, RPPGFSPFR, RYLGYL, RGDS, and YGGFMRGL in electrospray ionization mass spectrometry (ESI-MS) experiments. DFT calculations predict a hydrogen bonded salt bridge structure with a protonated guanidinium flanked by two deprotonated phosphates to be the lowest energy structure. Dissociation of DP/peptide adducts reveals that, in general, the relative gas phase acidity of a peptide is dependent on peptide length, with longer peptides being more acidic. In particular, peptides that are six residues or more in length can stabilize the deprotonated C-terminus by extensive hydrogen bonding with the peptide backbone. Dissociation of DP/peptide complexes often yields the deprotonated peptide, allowing for the facile formation of anionic peptides that otherwise would be difficult to generate in high abundance. Although DP has a preference for binding to arginine residues in peptides, DP is also observed to form less abundant complexes with peptides containing multiple lysines. Lys-Xxx-Lys and Lys-Lys sequences form low abundance anionic adducts with DP. For example, KKKK exclusively forms a double adduct with one net negative charge on the complex.

Synthesis and conformational features of human salivary mucin C-terminal derived peptide epitope carrying Thomsen-Friedenreich antigen: implications for its role in self-association.

The conformational features of a chemically synthesized 23-residue glycopeptide construct (II) carrying Gal-beta-(1,3)-alpha-GalNAc and its deglycosylated counterpart (I; Gal: galactose; GalNAc: N-acetyl galactosamine) derived from the C-terminal domain of human salivary mucin (MUC7) were investigated using CD spectroscopy as well as molecular dynamic simulation studies. The corresponding deglycosylated peptide (I) was essentially used to compare and study the influence of the sugar moiety on peptide backbone conformation. CD measurements in aqueous medium revealed that the apopeptide (I) contains significant populations of beta-strand conformation while the glycopeptide (II) possess, partly, helical structure. This transition in the secondary structure upon glycosylation from beta-strand to helical conformation clearly demonstrates that the carbohydrate moiety exerts significant influence on the peptide backbone. On the other hand, upon titrating structure stabilizing organic cosolvent, trifluoroethanol (TFE), both the peptides showed pronounced helical structure. However, the propensity for helical structure formation is less pronounced in glycopeptide compared to apopeptide suggesting that the bulky carbohydrate moiety possibly posing steric hindrance to the formation of TFE-induced secondary structure in II. Energy-minimized molecular model for the glycopeptide revealed that the preferred helix conformation in aqueous medium appears to be stabilized by the hydrogen-bonded salt bridge like interaction between carbohydrate --OH and Lys-10 side--N(+)H(3) group. Size exclusion chromatographic analysis of both (glyco)peptides I and II showed an apparent Kd of 2.3 and 0.52 microM, respectively, indicating that glycopeptide (II) has greater tendency for self-association. Due to high amphipathic character as well as due to the presence of a leucine zipper motif ( approximately LLYMKNLL approximately ), which is known to increase the stability at the coiled-coil interface via hydrophobic interactions, we propose therefore that, this domain could be one of the key elements involved in the self-association of intact MUC7 in vivo. Profound conformational effects governed by glycosylation exemplified herein could have implications in determining structure-function relationships of mucin glycoproteins.

Structure of tick anticoagulant peptide at 1.6 A resolution complexed with bovine pancreatic trypsin inhibitor.

The structure of tick anticoagulant peptide (TAP) has been determined by X-ray crystallography at 1.6 A resolution complexed with bovine pancreatic trypsin inhibitor (BPTI). The TAP-BPTI crystals are tetragonal, a = b = 46.87, c = 50.35 A, space group P41, four complexes per unit cell. The TAP molecules are highly dipolar and form an intermolecular helical array along the c-axis with a diameter of about 45 A. Individual TAP units interact in a head-to-tail fashion, the positive end of one molecule associating with the distal negative end of another, and vice versa. The BPTI molecules have a uniformly distributed positively charged surface that interacts extensively through 14 hydrogen bonds and two hydrogen bonded salt bridges with the helical groove around the helical TAP chains. Comparing the structure of TAP in TAP-BPTI with TAP bound to factor Xa(Xa) suggests a massive reorganization in the N-terminal tetrapeptide and the first disulfide loop of TAP (Cys5T-Cys15T) upon binding to Xa. The Tyr1(T)OH atom of TAP moves 14.2 A to interact with Asp189 of the S1 specificity site, Arg3(T)CZ moves 5.0 A with the guanidinium group forming a cation-pi-electron complex in the S4 subsite of Xa, while Lys7(T)NZ differs in position by 10.6 A in TAP-BPTI and TAP-Xa, all of which indicates a different pre-Xa-bound conformation for the N-terminal of TAP in its native state. In contrast to TAP, the BPTI structure of TAP-BPTI is practically the same as all those of previously determined structures of BPTI, only arginine and lysine side-chain conformations showing significant differences.

Binding of biotin to streptavidin stabilizes intersubunit salt bridges between Asp61 and His87 at low pH

The remarkable stability of the streptavidin tetramer towards subunit dissociation becomes even greater upon binding of biotin. At two equivalent extensive monomer-monomer interfaces, monomers tightly associate into dimers that in turn associate into the tetramer at a less extensive dimer-dimer interface. To probe the structural basis for the enhancement of the stability of streptavidin by biotin, the crystal structures of apostreptavidin and its complexes with biotin and other small molecule and cyclic peptide ligands were determined and compared at resolutions as high as 1.36 Å over a range of pH values from as low as 1.39. At low pH dramatic changes occur in the conformation and intersubunit hydrogen bonds involving the loop comprising Asp61 to Ser69. The hydrogen-bonded salt bridge between Asp61 O δ2 and His87 N δ1, observed at higher pH, is replaced with a strong hydrogen bond between Asp61 O δ1and Asn85 O δ1. Through crystallography at multiple pH values, the pH where this conformational change occurs, and thus the p K a of Asp61, was determined in crystals of space group I222 and/or I4 122 of apostreptavidin and complexes. A range in p K a values for Asp61 was observed in these structures, the lowest being 1.78 ± 0.19 for I222 streptavidin-biotin in 2.9 M (NH 4) 2SO 4. At low pH the decrease in p K a of Asp61 and preservation of the intersubunit Asp61 O δ2 -N δ1 His87 hydrogen-bonded salt bridge in streptavidin-biotin versus apostreptavidin or streptavidin-peptide complexes is associated with an ordering of the flexible flap comprising residues Ala46 to Glu51, that in turn orders the Arg84 side-chain of a neighboring loop through resulting hydrogen bonds. Ordering of Arg84 in close proximity to the strong intersubunit interface appears to stabilize the conformation associated with the Asp61 O δ2 -N δ1 His87 hydrogen-bonded salt bridge. Thus, in addition to the established role of biotin in tetramer stabilization by direct mediation of intersubunit interactions at the weak interface through contact with Trp120, biotin may enhance tetramer stability at the strong interface more indirectly by ordering loop residues.

Characterization of the apparent negative co-operativity induced in Escherichia coli aspartate aminotransferase by the replacement of Asp222 with alanine. Evidence for an extremely slow conformational change.

The strictly conserved active site residue, Asp222, which forms a hydrogen-bonded salt bridge with the pyridine nitrogen atom of the pyridoxal 5' phosphate (PLP) co-factor of aspartate aminotransferase (AATase), was replaced with alanine (D222A) in the Escherichia coli enzyme. The D222A mutant exhibits non-hyberbolic saturation behavior with amino acid substrates which appear as apparent negative cooperativity in steady-state kinetic analyses. Single turnover progress curves for D222A are well described by the sum of two exponentials, contrasting with the monophasic kinetics of the wild-type enzyme. An active/inactive heterodimer containing the D222A mutation retains this biphasic kinetic response, proving that the observed cooperativity is not the result of induced allostery. The anomalous behavior is explained by a hysteretic kinetic model involving two slowly interconverting enzyme forms, only one of which is catalytically competent. The slow functional transition between the two forms has a half-life of approximately 10 mins. Preincubation of the mutant with the dicarboxylic inhibitor maleate shifts the equilibrium population of the enzyme towards the catalytically active form, suggesting that the slow transition is related to the domain closure known to occur upon association of this inhibitor with the wild-type enzyme. The importance of Asp222 in the chemical steps of transamination is confirmed by the approximately 10(5)-fold decrease in catalytic competence in the D222A mutant, and by the large primary C alpha-deuterium kinetic isotope effect (6.7 versus 2.2 for the wild-type). The transamination activity of the D222A mutant is enhanced 4- to 20-fold by reconstitution with the co-factor analog N-methylpyridoxal-5'-phosphate (N-MePLP), and the C alpha-proton abstraction step is less rate determining, as evidenced by the decrease in the primary kinetic isotope effect from 6.7 to 2.3. These results suggest that the conserved interaction between the protonated pyridine nitrogen of PLP and the negatively charged carboxylate of Asp222 is important not only for efficient C alpha-proton abstraction, but also for conformational transitions concomitant with the transamination process.

Estimating the contribution of engineered surface electrostatic interactions to protein stability by using double-mutant cycles

Coulombic interactions between charges on the surface of proteins contribute to stability. It is difficult, however, to estimate their importance by protein engineering methods because mutation of one residue in an ion pair alters the energetics of many interactions in addition to the coulombic energy between the two components. We have estimated the interaction energy between two charged residues, Asp-12 and Arg-16, in an alpha-helix on the surface of a barnase mutant by invoking a double-mutant cycle involving wild-type enzyme (Asp-12, Thr-16), the single mutants Thr----Arg-16 and Asp----Ala-12, and the double mutant Asp----Ala-12, Thr----Arg-16. The changes in free energy of unfolding of the single mutants are not additive because of the coulombic interaction energy. Additivity is restored at high concentrations of salt that shield electrostatic interactions. The geometry of the ion pair in the mutant was assumed to be the same as that in the highly homologous ribonuclease from Bacillus intermedius, binase, which has Asp-12 and Arg-16 in the native enzyme. The ion pair does not form a hydrogen-bonded salt bridge, but the charges are separated by 5-6 A. The mutant barnase containing the ion pair Asp-12/Arg-16 is more stable than wild type by 0.5 kcal/mol, but only a part of the increased stability is attributable to the electrostatic interaction. We present a formal analysis of how double-mutant cycles can be used to measure the energetics of pairwise interactions.