Transient Hydrogen Bonds Research Articles

Beyond Induced-Fit Receptor-Ligand Interactions: Structural Changes that Can Significantly Extend Bond Lifetimes

While the lifetime of conventional receptor-ligand interactions is shortened by tensile mechanical force, some recently discovered interactions, termed catch bonds, can be strengthened by force. Motivated by the search for the underpinning structural mechanisms, we here explore the structural dynamics of the binding site of the bacterial adhesive protein FimH by molecular dynamics and steered molecular dynamics. While the crystal structure of only one FimH conformation has been reported so far, we describe two distinctively different conformations of the mannose-bound FimH binding site. Force-induced dissociation was slowed when the mannose ring rotated such that additional force-bearing hydrogen bonds formed with the base of the FimH binding pocket. The lifetime of the complex was further enhanced significantly by rigidifying this base. We finally show how even sub-angstrom spatial alterations of the hydrogen bonding pattern within the base can lead to significantly decreased bond lifetimes.

Raman-Assisted Crystallography Reveals End-On Peroxide Intermediates in a Nonheme Iron Enzyme

Iron-peroxide intermediates are central in the reaction cycle of many iron-containing biomolecules. We trapped iron(III)-(hydro)peroxo species in crystals of superoxide reductase (SOR), a nonheme mononuclear iron enzyme that scavenges superoxide radicals. X-ray diffraction data at 1.95 angstrom resolution and Raman spectra recorded in crystallo revealed iron-(hydro)peroxo intermediates with the (hydro)peroxo group bound end-on. The dynamic SOR active site promotes the formation of transient hydrogen bond networks, which presumably assist the cleavage of the iron-oxygen bond in order to release the reaction product, hydrogen peroxide.

Early Time Hydrogen-Bonding Dynamics of Photoexcited Coumarin 102 in Hydrogen-Donating Solvents: Theoretical Study

To study the early time hydrogen-bonding dynamics of chromophore in hydrogen-donating solvents upon photoexcitation, the infrared spectra of the hydrogen-bonded solute-solvent complexes in electronically excited states have been calculated using the time-dependent density functional theory (TDDFT) method. The hydrogen-bonding dynamics in electronically excited states can be widely monitored by the spectral shifts of some characteristic vibrational modes involved in the formation of hydrogen bonds. In this study, we have demonstrated that the intermolecular hydrogen bonds between coumarin 102 (C102) and hydrogen-donating solvents are strengthened in the early time of photoexcitation to the electronically excited state by theoretically monitoring the stretching modes of C=O and H-O groups. This is significantly contrasted with the ultrafast hydrogen bond cleavage taking place within a 200-fs time scale upon electronic excitation, proposed in many femtosecond time-resolved vibrational spectroscopy experiments. The transient hydrogen bond strengthening behaviors in excited states of chromophores in hydrogen-donating solvents, which we have demonstrated here for the first time, may take place widely in many other systems in solution and are very important to explain the fluorescence-quenching phenomena associated with some radiationless deactivation processes, for example, the ultrafast solute-solvent intermolecular electron transfer and the internal conversion process from the fluorescent state to the ground state.

Hyaluronan: the absence of amide-carboxylate hydrogen bonds and the chain conformation in aqueous solution are incompatible with stable secondary and tertiary structure models.

Contradictory descriptions for the aqueous solution conformation of the glycosaminoglycan hyaluronan (HA) exist in the literature. According to hydrodynamic and simulation data, HA molecules are stiffened by a rapidly interchanging network of transient hydrogen bonds at the local level and do not significantly associate at the global level. In marked contrast, models derived from NMR data suggest that the secondary structure involves persistent hydrogen bonds and that strong associations between chains can occur to form vast stable tertiary structures. These models require an extended 2-fold helical conformation of the HA chain and specific hydrogen bonds between amide and carboxylate groups. To test these descriptions, we have used 15N-labelled oligosaccharides and high-field NMR to measure pertinent properties of the acetamido group. The amide proton chemical shift perturbation and carboxylate group pK(a) value are inconsistent with a highly populated hydrogen bond between the amide and carboxylate groups. Amide proton temperature coefficients and chemical exchange rates confirm this conclusion. Comparison of oligomer properties with polymeric HA indicates that there is no discernible difference in amide proton environment between the centre of octasaccharides and the polymer, inconsistent with the formation of tertiary structures. A [1H-1H-15N] NOESY-HSQC (heteronuclear single-quantum correlation) spectrum recorded on an HA octasaccharide revealed that amide groups in the centre are in a trans orientation and that the average solution conformation is not an extended 2-fold helix. Therefore the two key aspects of the secondary and tertiary structure models are unlikely to be correct. Rather, these new NMR data agree with descriptions from hydrodynamic and simulations data.

Molecular Dynamics Simulation Study of Superhydrated Perdeuterated Natrolite Using a New Interaction Potential Model

To test a new interaction potential, molecular dynamics simulations of zeolite natrolite were performed for the structures under ambient conditions hydrated by perdeuterated water and at high pressure (1.87 GPa) in the superhydrated phase, which were recently studied by neutron diffraction. The experimental structures were reproduced with reasonable accuracy, and the hydrogen bond features are discussed. As in ordinary natrolite, a flip motion of water molecules around the HOH bisector is found, which, together with translational oscillations, gives rise to transient hydrogen bonds between water molecules, which do not appear from experimental equilibrium coordinates. The dynamics of water molecules can explain some problems encountered in refining the experimental structure. Vibrational spectra of natrolite containing perdeuterated water, which are not yet measured, were simulated, and their qualitative trend is discussed.

Hyaluronan: The Local Solution Conformation Determined by NMR and Computer Modeling is Close to a Contracted Left-handed 4-Fold Helix

The polysaccharide hyaluronan (HA) is a ubiquitous component of the vertebrate extracellular matrix with diverse physiological roles from space-filling to acting as a scaffold for other macromolecules. The molecular interactions responsible for these solution properties have been the subject of much debate and, primarily due to the lack of residue-specific experimental data, no consensus model for the three-dimensional conformation nor dynamics of HA in solution has emerged. Here, the solution conformation of HA is investigated using molecular dynamics (MD) simulations and high-field nuclear magnetic resonance (NMR). In contrast to previous studies, MD simulations incorporated explicit water molecules and sodium ions, while NMR experiments utilized (15)N-enriched oligosaccharides to allow residue-specific information to be obtained. The resultant average conformation is predicted to be almost a contracted left-handed 4-fold helix; i.e. similar to that observed for sodium hyaluronate fibers by X-ray diffraction, but with the acetamido side-chain trans to H(2). The glycosidic linkages and acetamido side-chains are predicted to have standard deviation rotations of 13 degrees and 18 degrees around their mean conformations in free solution, respectively, and are not observed to be stabilized by strong intramolecular hydrogen bonds as X-ray fiber diffraction refinements describe for the solid-state. Rather, weak and transient hydrogen bonds that are in rapid interchange with solvent molecules are predicted. These predictions are quantitatively consistent with demanding residue-specific NMR data and correspond to an HA molecule that is rod-like as an oligosaccharide and behaves as a stiffened random coil at large molecular mass, in close agreement with previous hydrodynamic observations. This new description of the solution conformation of HA is consistent with all available experimental data and accounts for its viscoelastic space-filling properties. This representation can be used as a basis for modeling the association between HA and proteins, which will elucidate important aspects of extracellular matrix assembly.

Biological Water: Femtosecond Dynamics of Macromolecular Hydration

The unique features of a macromolecule and water as a solvent make the issue of solvation unconventional, with questions about the static versus dynamic nature of hydration and the physics of orientational and translational diffusion at the boundary. For proteins, the hydration shell that covers the surface is critical to the stability of its structure and function. Dynamically speaking, the residence time of water at the surface is a signature of its mobility and binding. With femtosecond time resolution it is possible to unravel the shortest residence times which are key for the description of the hydration layer, static or dynamic. In this article we review these issues guided by experimental studies, from this laboratory, of polar hydration dynamics at the surfaces of two proteins (Subtilisin Carlsberg (SC) and Monellin). The natural probe tryptophan amino acid was used for the interrogation of the dynamics, and for direct comparison we also studied the behavior in bulk watera complete hydration in 1 ps. We develop a theoretical description of solvation and relate the theory to the experimental observations. In this theoretical approach, we consider the dynamical equilibrium in the hydration shell, defining the rate processes for breaking and making the transient hydrogen bonds, and the effective friction in the layer which is defined by the translational and orientational motions of water molecules. The relationship between the residence time of water molecules and the observed slow component in solvation dynamics is a direct one. For the two proteins studied, we observed a “bimodal decay” for the hydration correlation function, with two primary relaxation times: ultrafast, typically 1 ps or less, and longer, typically 15−40 ps, and both are related to the residence time at the protein surface, depending on the binding energies. We end by making extensions to studies of the denatured state of the protein, random coils, and the biomimetic micelles, and conclude with our thoughts on the relevance of the dynamics of native structures to their functions.

Synthesis, biological activity, and solution structures of a cyclic dodecapeptide from the EGF-2 domain of blood coagulation factor VII.

The cyclic dodecapeptide, disulfide-cyclo-[H-Cys-Val-Asn-Glu-Asn-Gly-Gly-Cys(Acm)-Glu-Gln-Tyr-Cys-OH], which corresponds to the 91-102 sequence of the second epidermal growth factor domain of human blood coagulation factor VII, was synthesized using solid-phase procedures. It was shown to be an inhibitor at the key step in the induction of coagulation by the extrinsic pathway, i.e. the factor VII/tissue factor-catalyzed activation of coagulation factor X. The solution structure of this peptide was investigated by NMR spectroscopy and was computer-modeled via molecular mechanics. Structures were calculated based on 112 distance and nine dihedral angle constraints. The resulting backbone structures were classified into two structural subsets: one which exhibited a twisted '8'-shaped folding and another describing an open, circular 'O' outline. The local backbone structures of segments Asn3-Glu4-Asn5, Gly7-Cys8 and Gln10-Tyr11 were well preserved among the two subsets. Apart from the unrestrained N- and C-termini, Gly6 and Glu9 sites exhibited marked local disorder between the two subsets, suggesting localized flexible hinges likely to govern tertiary structure interconversion between the two subsets. Two transient hydrogen bonds were identified from pH chemical shift titrations by matching the pKa values of NH and carboxylate groups, which supported the occurrence of the '8' structure, and agreed with temperature coefficients of peptidyl NH resonances. Structure-function relationships of the peptide were discussed in terms of the likely physiological function of the disulfide-bonded loop in factor VII which the peptide represents.

Phosphorylation-induced structural changes in the amyloid precursor protein cytoplasmic tail detected by NMR

The cytoplasmic tail of the amyloid precursor protein (APPc) interacts with several cellular factors implicated in intracellular signaling or proteolytic production of amyloid β peptide found in senile plaques of Alzheimer’s disease patients. APPc contains two threonine residues (654 and 668 relative to APP695, or 6 and 20 relative to APPc) and a serine residue (655 or 7, respectively) that are known to be phosphorylated in vivo and may play regulatory roles in these events. We show by solution NMR spectroscopy of a 49 residue cytoplasmic tail peptide (APP-C) that in all three cases, phosphorylation induces changes in backbone dihedral angles that can be attributed to formation of local hydrogen bonds between the phosphate group and nearby amide protons. Phosphorylation of S7 also induces chemical shift changes in the hydrophobic cluster (residues I8-V13), indicating additional medium-range effects. The most pronounced changes occur upon phosphorylation of T20, a neuron-specific phosphorylation site, where the N-terminal helix capping box previously characterized for this region is altered. Characterization of torsion angles and transient hydrogen bonds indicates that prolyl isomerization of the pThr-Pro peptide bond results from both destabilization of the N-terminal helix capping box and stabilization of the cis isomer by transient hydrogen bonds. The significant population of the cis isomer (9 %) present after phosphorylation of T20 suggests a potential role of selective recognition of cis versus trans isomers in response to phosphorylation of APP. Together, these structural changes indicate that phosphorylation may act as a conformational switch in the cytoplasmic tail of APP to alter specificity and affinity of binding to cytosolic partners, particularly in response to the abnormal phosphorylation events associated with Alzheimer’s disease.

Transient structure of the amyloid precursor protein cytoplasmic tail indicates preordering of structure for binding to cytosolic factors.

The cytoplasmic tail of the amyloid precursor protein (APP) appears to play two important roles in the cell through participation in intracellular signaling and proteolytic processing of APP. Hence, knowledge of the structure of the 47 residue cytoplasmic tail of APP is important for understanding the molecular interactions involved in normal cell function as well as in the pathogenesis of Alzheimer's disease. Multidimensional solution NMR spectroscopy has been applied to examine the structural features of a 49-residue peptide (APP-C) containing two N-terminal residues (GS) and the APP cytoplasmic tail, over the pH range of 4.2-7.1. Although the peptide does not adopt a stable folded structure, regions of unstable structure exist over the pH range examined and have been characterized by a combination of H(alpha) chemical shifts, NOE analysis, and (3)J(HNH)(alpha) coupling constants and by identification of transient hydrogen bonds between amide protons and titrating carboxylate groups. These studies extend the work of others [Kroenke et al. (1997) Biochemistry 36, 8145-8152] by identifying an additional nascent helix and a hydrophobic cluster within the N-terminal 20 amino acid residues and by further characterizing the TPEE turn as a helix capping box. The transient structure of APP-C provides insight into the importance of preordering of this cytoplasmic tail in governing specificity and affinity for cytosolic binding partners.

Structure of an analog of fusion peptide from hemagglutinin.

A 20-residue peptide E5 containing five glutamates, an analog of the fusion peptide of influenza virus hemagglutinin (HA) exhibiting fusion activity at acidic pH lower than 6.0-6.5 was studied by circular dichroism (CD), Fourier transform infrared, and 1H-NMR spectroscopy in water, water/trifluoroethanol (TFE) mixtures, dodecylphosphocholine (DPC) micelles, and phospholipid vesicles. E5 became structurally ordered at pH < or = 6 and the helical content in the peptide increased in the row: water < water/TFE < DPC approximately = phospholipid vesicle while the amount of beta-structure was approximately reverse. 1H-NMR data and line-broadening effect of 5-, 16-doxylstearates on proton resonances of DPC bound peptide showed E5 forms amphiphilic alpha-helix in residues 2-18, which is flexible in 11-18 part. The analysis of the proton chemical shifts of DPC bound and CD intensity at 220 nm of phospholipid bound E5 showed that the pH dependence of helical content is characterized by the same pKa approximately 5.6. Only Glu11 and Glu15 in DPC bound peptide showed such elevated pKas, presumably due to transient hydrogen bond(s) Glu11 (Glu15) deltaCOO- (H+)...HN Glu15 that dispose(s) the side chain of Glu11 (Glu15) residue(s) close to the micelle/water interface. These glutamates are present in the HA-fusion peptide and the experimental half-maximal pH of fusion for HA and E5 peptides is approximately 5.6. Therefore, a specific anchorage of these peptides onto membrane necessary for fusion is likely driven by the protonation of the carboxylate group of Glu11 (Glu15) residue(s) participating in transient hydrogen bond(s).

A quantum molecular dynamics study of proton diffusion in SrTiO3 and CaTiO3

Protonic motion in cubic perovskite SrTiO3 and CaTiO3 is investigated by numerical simulations at higher temperatures. The protons are primarily found to form transient hydrogen bond complexes. The repulsive titanium/proton interaction causes a bending of the hydrogen bonds and, thus, aggravates proton transfer. However, as the proton interaction also extends to the next-next nearest oxygen sites the formation of transient, linear inter-octahedra hydrogen bonds, i.e. between the tips of neighbouring octahedra, is also possible. Whereas the time constants for proton reorientation are found to be of similar magnitude in both materials, the time constant for proton transfer is found to be larger by an order of magnitude in SrTiO3. Furthermore, the numerical simulations yield an activation energy for proton diffusion of 0.50±0.22 eV for SrTiO3 and 0.42±0.30 eV for CaTiO3.

The relation between crystal structure and the formation and mobility of protonic charge carriers in perovskite-type oxides: A case study of Y-doped BaCeO3 and SrCeO3

Proton conductivity phenomena in 10% Y-doped barium and strontium cerate are investigated experimentally and by quantum molecular dynamics simulations. In particular the impact of deviations from the cubic perovskite structure on the formation and mobility of protonic charge carriers is investigated. For Y: SrCeO3, which shows a larger deviation from the ideal cubic perovskite structure, the concentration and mobility of protonic defects is significantly lower than for Y: BaCeO3. The first is due to the decay of the oxygen position into two sites, only one of which is involved in the formation of protonic defects. The symmetry reduction also leads to the formation of different one-dimensional proton diffusion paths, and unfavourable jumps between such paths are supposed to control the macroscopic proton diffusion coefficient in Y: SrCeO3. The analysis suggests the formation of strong but transient hydrogen bonds and inter-octa-hedra proton transfer between vertices for SrCeO3 in contrast to just intra-octahedra proton transfer for BaCeO3. Whereas for BaCeO3 the proton transfer step is identified to be rate-limiting at T= 1000 K, for SrCeO3 both proton transfer and reorientation are found to be of similar magnitude.

Molecular dynamics simulations of synthetic peptide folding.

Because the time scale of protein folding is much greater than that of the widely used simulations of native structures, a detailed report of molecular dynamics simulations of folding has not been available. In this study, we included the average solvent effect in the potential functions to simplify the calculation of the solvent effect and carried out long molecular dynamics simulations of the alanine-based synthetic peptides at 274 K. From either an extended or a randomly generated conformation, the simulations approached a helix-coil equilibrium in about 3 ns. The multiple minima problem did not prevent helix folding. The calculated helical ratio of Ac-AAQ-AAAAQAAAAQAAY-NH2 was 47%, in good agreement with the circular dichroism measurement (about 50%). A helical segment with frayed ends was the most stable conformation, but the hydrophobic interaction favored the compact, distorted helix-turn-helix conformations. The transition between the two types of conformations occurred in a much larger time scale than helix propagation. The transient hydrogen bonds between the glutamine side chain and the backbone carbonyl group could reduce the free energy barrier of helix folding and unfolding. The substitution of a single alanine residue in the middle of the peptide with valine or glycine decreased the average helical ratio significantly, in agreement with experimental observations.

Evidence for a Transient Interresidue Hydrogen Bond in Sucrose in Aqueous Solution Obtained by Rotating-Frame Exchange NMR Spectroscopy Under Supercooled Conditions

We have used the hydroxyl protons of sucrose dissolved in supercooled water as NMR probes for the detection of intramolecular hydrogen bonds. Neither the measured OH temperature shift coefficients, 3 J HCOH scalar couplings, isotope-induced 13C chemical shifts (Δδ COH/COD) nor OH exchange rates allowed us to single out any hydroxyl group in sucrose with characteristics indicative of involvement in strong hydrogen bonding. However, two-dimensional rotating-frame exchange (ROESY) spectroscopy revealed a direct exchange between the glucosyl OH2 and fructosyl OH1 protons. We conclude that an O2g::H::O1f interresidue hydrogen bond transiently exists in sucrose in aqueous solution. ROESY of sugars in supercooled water is proposed as a novel method to detect this type of weak hydrogen bonding.

Role of Glutamine-61 in the Hydrolysis of GTP by p21H-ras: An Experimental and Theoretical Study

The active GTP-bound form of p21ras is converted to the biologically inactive GDP-bound form by enzymatic hydrolysis and this function serves to regulate the wild-type ras protein. The side chain of the amino acid at position 61 may play a key role in this hydrolysis of GTP by p21. Experimental studies that define properties of the Q61E mutant of p21H-ras are presented along with supporting molecular dynamics simulations. We find that under saturating concentrations of GTP the Q61E mutant of p21H-ras has a 20-fold greater rate of intrinsic hydrolysis (kcat = 0.57 min-1) than the wild type. The affinity of the Q61E variant for GTP (Kd = 115 microM) is much lower than that of the wild type. GTPase activating protein does not activate the variant. From molecular dynamics simulations, we find that both the wild type and Q61E mutant have the residue 61 side chain in transient contact with a water molecule that is well-positioned for hydrolytic attack on the gamma phosphate. Thr-35 also is found to form a transient hydrogen bond with this critical water. These elements may define the catalytic complex for hydrolysis of the GTP [Pai et al. (1990) EMBO J. 9, 2351]. Similarly, the G12P mutant, which also has an intrinsic hydrolysis rate similar to the wild type, is found to form the same complex in simulation. In contrast, molecular dynamics analysis of the mutants G12R, G12V, and Q61L, which have much lower intrinsic rates than the wild-type p21, do not show this complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Hydrogen Bond Formation and Molecular Motion in Solutions of Aniline with Nitroxide Free Radicals

Abstract1H NMR spin‐lattice relaxation rates and paramagnetic contact shifts have been measured at various temperatures and frequencies in solutions of aniline, aniline‐d2, and aniline‐d5 containing nitroxide free radicals. The data can be explained in terms of transient hydrogen bond formation between the amino protons and the NO group of the radical. Detailed information about the intermolecular transfer of electron spin density and the steric arrangement of the complex have been obtained. Molecular motions and interactions have been probed by relaxation measurements over a large frequency range between 100 kHz and 300 MHz. Relaxation is induced by dipolar interactions of the aniline protons with the unpaired electron spin and rotational tumbling of the associated system. The results are compared with other hydrogen‐bonded complexes of nitroxide radicals and proton donor solvent molecules.

Effects of ionization and counterion binding on the surface areas of phosphatidic acids in monolayers.

At 24-26 degrees C, force-area isotherms show that unionized dipalmitoyl phosphatidic acid forms a solid-condensed film while unionized egg and dioleoyl phosphatidic acids form liquid-expanded films. Surface area is a characteristic feature of a specific phosphatidic acid and the purity of a phosphatidic acid preparation can be established by the surface area of the unionized phosphatidic acid (acid subphase) at 17 dynes/cm (castor oil piston). Ionized dipalmitoyl phosphatidic acid desorbs from a monolayer at a measurable rate while ionized egg and dioleoyl phosphatidic acids desorb too slowly for rate studies. The apparent surface pK(2) for dipalmitoyl phosphatidic acid, calculated from desorption rates, is 9.4. Surface areas of the phosphatidic acids expand with ionization. Solid dipalmitoyl phosphatidic acid films expand only in the pK(2) region, showing one inflection point which indicates that the K(1)/K(2) ratio is less than 100 and that, as a consequence of this ratio, the apparent surface pK(1) is greater than 7.4. Liquid egg and dioleoyl phosphatidic acid films have two inflection points, expanding in both the pK(1) and pK(2) regions. The apparent surface pK(1) and pK(2) values, calculated from inflection points in surface area data, are 3.5 and 8.0, respectively. Film expansion with phosphatidate anions is less than anticipated, showing the presence of weak transient hydrogen bonds. Expanded phosphatidate anion films are condensed by alkaline earth cations. The Ca(2+) and Ba(2+) salts of completely ionized phosphatidic acids collapse from monolayers, showing that the phosphatidate anion may function as an ionophore for the transport of alkaline earth ions.-Patil, G. S., N. J. Dorman, and D. G. Cornwell. Effects of ionization and counterion binding on the surface areas of phosphatidic acids in monolayers.

Dynamic Nuclear Polarization in Phosphorus Compounds with Perchlorotriphenylmethyl and Other Radicals

Dynamic nuclear polarization has been examined in liquid solutions containing the radical perchlorotriphenylmethyl and 14 different phosphorus compounds. In addition, the radicals bisdiphenylenephenylallyl, tri-t-butylphenoxyl, and diphenylpicrylhydrazyl have been examined with five phosphorus compounds. The chlorocarbon radical shows on the average the least scalar coupling of the four. This is in accord with the well-shielded spin distribution on this radical and the great chemical stability of aromatic chlorocarbons. PCTM is found to be almost completely inert, both electronically and chemically, toward phosphines and phosphites. Exceptional cases of high coupling were observed with PCTM and dimethylphosphite, triphenylphosphine oxide, and hexamethylphosphoramide. The first case appears to be due to a transient hydrogen bond Cl···H–P, the second due to an exposed polarizable orbital system, and the last due to an obscure chemical complexation tendency. Exceptional cases with the other radicals serve to illustrate further stereospecific molecular couplings between sites of active chemical or electronic attraction for each other.