Ordered Solvent Molecules Research Articles

Ordering effect of protein surfaces on water dynamics: NMR relaxation study

Proteins in solution affect the structural and dynamic properties of the bulk water at the protein-water interface, resulting in a contribution to the order of the hydration water. Theoretical and experimental NMR relaxation methods were developed to study the dynamic properties of water molecules in the protein hydration shell. Water non-selective and selective relaxation rates, were shown to be sensitive to contributions from ordered solvent molecules at protein surface. The average rotational correlation time of water molecules in the protein hydration shell was determined for three protein systems of different size: ribonuclease A, human serum albumin and fibrinogen. The knowledge of these properties is an important step toward the determination of the size of the water ordering contributions originate in proteins systems.

Oligoamide Foldamers as Helical Chloride Receptors-the Influence of Electron-Withdrawing Substituents on Anion-Binding Interactions.

The anion-binding properties of three closely related oligoamide foldamers were studied using NMR spectroscopy, isothermal titration calorimetry and mass spectrometry, as well as DFT calculations. The 1 H NMR spectra of the foldamers in [D6 ]acetone solution revealed partial preorganization by intramolecular hydrogen bonding, which creates a suitable cavity for anion binding. The limited size of the cavity, however, enabled efficient binding by the inner amide protons only for the chloride anion resulting in the formation of a thermodynamically stable 1:1 complex. All 1:1 chloride complexes displayed a significant favourable contribution of the entropy term. Most likely, this is due to the release of ordered solvent molecules solvating the free foldamer and the anion to the bulk solution upon complex formation. The introduction of electron-withdrawing substituents in foldamers 2 and 3 had only a slight effect on the thermodynamic constants for chloride binding compared to the parent receptor. Remarkably, the binding of chloride to foldamer 3 not only produced the expected 1:1 complex but also open aggregates with 1:2 (host:anion) stoichiometry.

Engineering altered protein-DNA recognition specificity.

Protein engineering is used to generate novel protein folds and assemblages, to impart new properties and functions onto existing proteins, and to enhance our understanding of principles that govern protein structure. While such approaches can be employed to reprogram protein–protein interactions, modifying protein–DNA interactions is more difficult. This may be related to the structural features of protein–DNA interfaces, which display more charged groups, directional hydrogen bonds, ordered solvent molecules and counterions than comparable protein interfaces. Nevertheless, progress has been made in the redesign of protein–DNA specificity, much of it driven by the development of engineered enzymes for genome modification. Here, we summarize the creation of novel DNA specificities for zinc finger proteins, meganucleases, TAL effectors, recombinases and restriction endonucleases. The ease of re-engineering each system is related both to the modularity of the protein and the extent to which the proteins have evolved to be capable of readily modifying their recognition specificities in response to natural selection. The development of engineered DNA binding proteins that display an ideal combination of activity, specificity, deliverability, and outcomes is not a fully solved problem, however each of the current platforms offers unique advantages, offset by behaviors and properties requiring further study and development.

Solvent-Mediated Proton Transfer in Catalysis by Carbonic Anhydrase

Considerable attention has been focused on proton transfer through intervening water molecules in complex macromolecules of biological interest, such as bacteriorhodopsin, cytochrome c oxidase, and many others. Proton transfer in catalysis by carbonic anhydrase provides a useful model for the study of the properties of such proton translocations. High-resolution X-ray crystallography in combination with measurements of catalysis have revealed new details of this process. A prominent proton shuttle residue His64 shows evidence of structural mobility, which appears to enhance proton transfer between the active site and bulk solvent. Moreover, the properties of the imidazole side chain of His64, including its conformations and pK(a), are finely tuned by surrounding residues of the active-site cavity. The structure of a network of ordered solvent molecules located between His64 and the active site are also sensitive to surrounding residues. These features combine to provide efficient proton-transfer rates as great as 10(6) s(-1) necessary to sustain rapid catalysis.

Determination of solvent content in cavities in IL-1beta using experimentally phased electron density.

The extent to which water is present within apolar cavities in proteins remains unclear. In the case of interleukin-1beta (IL-1beta), four independent structures solved by x-ray crystallography indicate that water is not present in the central apolar cavity. In contrast, results from NMR spectroscopy suggest that water has high occupancy within the cavity but is positionally disordered, making it undetectable by standard crystallographic methods. A theoretically based crystallographic-phase refinement technique also suggested that there was the equivalent of two fully occupied water molecules within the apolar cavity. To resolve these discrepancies we sought to obtain an experimentally phased electron density map that was free of possible bias caused by mathematical modeling of the protein or the solvent. By combining native diffraction data with multiple wavelength anomalous data from a platinum derivative, accurate phases were obtained. Using these experimental phases, we estimate that occupancy of the apolar cavity in IL-1beta by solvent is close or equal to zero. Polar cavities in the protein that contain ordered solvent molecules serve as internal controls.

A Comparative Study of Uracil-DNA Glycosylases from Human and Herpes Simplex Virus Type 1

Uracil-DNA glycosylase (UNG) is the key enzyme responsible for initiation of base excision repair. We have used both kinetic and binding assays for comparative analysis of UNG enzymes from humans and herpes simplex virus type 1 (HSV-1). Steady-state fluorescence assays showed that hUNG has a much higher specificity constant (k(cat)/K(m)) compared with the viral enzyme due to a lower K(m). The binding of UNG to DNA was also studied using a catalytically inactive mutant of UNG and non-cleavable substrate analogs (2'-deoxypseudouridine and 2'-alpha-fluoro-2'-deoxyuridine). Equilibrium DNA binding revealed that both human and HSV-1 UNG enzymes bind to abasic DNA and both substrate analogs more weakly than to uracil-containing DNA. Structure determination of HSV-1 D88N/H210N UNG in complex with uracil revealed detailed information on substrate binding. Together, these results suggest that a significant proportion of the binding energy is provided by specific interactions with the target uracil. The kinetic parameters for human UNG indicate that it is likely to have activity against both U.A and U.G mismatches in vivo. Weak binding to abasic DNA also suggests that UNG activity is unlikely to be coupled to the subsequent common steps of base excision repair.

Excision of a proposed electron transfer pathway in cytochrome c peroxidase and its replacement by a ligand-binding channel.

A previously proposed electron transfer (ET) pathway in the heme enzyme cytochrome c peroxidase has been excised from the structure, leaving an open ligand-binding channel in its place. Earlier studies on cavity mutants of this enzyme have revealed structural plasticity in this region of the molecule. Analysis of these structures has allowed the design of a variant in which the specific section of protein backbone representing a previously proposed ET pathway is accurately extracted from the protein. A crystal structure verified the creation of an open channel that overlays the removed segment, extending from the surface of the protein to the heme at the core of the protein. A number of heterocyclic cations were found to bind to the proximal-channel mutant with affinities that can be rationalized based on the structures. It is proposed that small ligands bind more weakly to the proximal-channel mutant than to the W191G cavity due to an increased off rate of the open channel, whereas larger ligands are able to bind to the channel mutant without inducing large conformational changes. The structure of benzimidazole bound to the proximal-channel mutant shows that the ligand accurately overlays the position of the tryptophan radical center that was removed from the wild-type enzyme and displaces four of the eight ordered solvent molecules seen in the empty cavity. Ligand binding also caused a small rearrangement of the redesigned protein loop, perhaps as a result of improved electrostatic interactions with the ligand. The engineered channel offers the potential for introducing synthetic replacements for the removed structure, such as sensitizer-linked substrates. These installed "molecular wires" could be used to rapidly initiate reactions, trap reactive intermediates, or answer unresolved questions about ET pathways.

Description of ordered solvent molecules in a platinated decanucleotide duplex refined at 1.6 Å resolution against experimental MAD phases. Addendum

An acknowledgment is published for the paper by Coste et al. [Acta Cryst. (2002) D58, 431–440]. The acknowledgement is as follows: This work was supported by grants from l'Association pour la Recherche sur le Cancer, and la Ligue Contre le Cancer du Loiret; FC was the recipient of a PhD fellowship from the Région Centre.

Description of ordered solvent molecules in a platinated decanucleotide duplex refined at 1.6 Å resolution against experimental MAD phases

Accurate experimental phases derived from a MAD experiment may be useful to enable the identification of solvent molecules during the course of an atomic parameter refinement. The structure of a double-stranded DNA decanucleotide bearing a cisplatin interstrand cross-link at 1.6A resolution, whose phases were first determined experimentally using the L(III) edge of the Pt atom, was refined by various methods. The previously published structure resulted from a least-squares refinement using the structure-factor magnitudes and stereochemical restraints (program SHELX). In this paper, these previous results are compared with a model obtained by the likelihood-maximization method (program REFMAC) which allows the combination of the observed magnitudes with experimental MAD phases. This solution corresponded to a lower R(free) (18.8 compared with 20.3%), a lower R factor and accounted for 135 water molecules and one spermine molecule collected by the program wARP during refinement. The previously published SHELX solution exhibited no spermine molecule and accounted for 92 water molecules, only 74 of which are also present in the model obtained with the MAD phases. In order to verify that these improvements were actually related to the use of the MAD phases, the same type of procedure without the MAD phases was applied starting from the initial model. The resulting solution had a higher R(free) (20.3%), which could be related to the loss of 22 water molecules and the addition of 20 new ones. MAD phases therefore seem especially helpful in preventing the model bias which may affect the solvent molecules. All models have in common a hydration cage of nine water molecules which surround the platinum residue. In addition to the spermine molecule, the model obtained with the MAD phases allows description of the water-molecule organization, with reproducible motifs related to the base pairs and to the phosphodiester backbone.

Self-assembly of 2-(guanidiniocarbonyl)-pyrrole-4-carboxylate in dimethyl sulfoxide: an entropy driven oligomerization

The self-assembly of 2-(guanidiniocarbonyl)-pyrrole-4-carboxylate (2), which is a heteroditopic zwitterion with self-complementary binding groups, was studied in DMSO. Through intermolecular ion pairing between the carboxylate of one and the guanidinium group of another molecule, 2 forms linear chain like oligomers in solution as can be seen from the corresponding shift changes in the NMR spectrum. Concentration dependent NMR studies at different temperatures allowed us to calculate the corresponding binding constants and thermodynamic parameters for this association process. These data show that the oligomerization is endothermic and therefore an entropy-driven process. The release of ordered solvent molecules into the bulk solution during complexation is hence responsible for the oligomerization.

Crystal Structure of the Iron-dependent Regulator from Mycobacterium tuberculosis at 2.0-Å Resolution Reveals the Src Homology Domain 3-like Fold and Metal Binding Function of the Third Domain

Iron-dependent regulators are primary transcriptional regulators of virulence factors and iron scavenging systems that are important for infection by several bacterial pathogens. Here we present the 2.0-A crystal structure of the wild type iron-dependent regulator from Mycobacterium tuberculosis in its fully active holorepressor conformation. Clear, unbiased electron density for the Src homology domain 3-like third domain, which is often invisible in structures of iron-dependent regulators, was revealed by density modification and averaging. This domain is one of the rare examples of Src homology domain 3-like folds in bacterial proteins, and, in addition, displays a metal binding function by contributing two ligands, one Glu and one Gln, to the pentacoordinated cobalt atom at metal site 1. Both metal sites are fully occupied, and tightly bound water molecules at metal site 1 ("Water 1") and metal site 2 ("Water 2") are identified unambiguously. The main chain carbonyl of Leu4 makes an indirect interaction with the cobalt atom at metal site 2 via Water 2, and the adjacent residue, Val5, forms a rare gamma turn. Residues 1-3 are well ordered and make numerous interactions. These ordered solvent molecules and the conformation and interactions of the N-terminal pentapeptide thus might be important in metal-dependent activation.

Solvent Organization in an Oligonucleotide Crystal: THE STRUCTURE OF d(GCGAATTCG)2 AT ATOMIC RESOLUTION

We describe the crystal structure of d(GCGAATTCG) determined by x-ray diffraction at atomic resolution level (0.89 A). The duplex structure is practically identical to that described at 2.05 A resolution (Van Meervelt, L., Vlieghe, D., Dautant, A., Gallois, B., Précigoux, G., and Kennard, O. (1995) Nature 374, 742-744), however about half of the phosphate groups show multiple conformations. The crystal has three regions with different solvent structure. One of them contains several ordered Mg(+2) ions and can be considered as an ionic crystal. A second region is formed by a network of ordered water molecules with a polygonal organization that binds three duplexes. The third region is formed by channels of solvent in which very few ordered solvent molecules are visible. The less ordered phosphates are found facing this channel. The latter region provides a view of DNA with highly movable charges, both negative phosphates and counterions, without a precise location.

Asp-170 Is Crucial for the Redox Properties of Vanillyl-alcohol Oxidase

Vanillyl-alcohol oxidase is a flavoprotein containing a covalent flavin that catalyzes the oxidation of 4-(methoxymethyl)phenol to 4-hydroxybenzaldehyde. The reaction proceeds through the formation of a p-quinone methide intermediate, after which, water addition takes place. Asp-170, located near the N5-atom of the flavin, has been proposed to act as an active site base. To test this hypothesis, we have addressed the properties of D170E, D170S, D170A, and D170N variants. Spectral and fluorescence analysis, together with the crystal structure of D170S, suggests that the Asp-170 replacements do not induce major structural changes. However, in D170A and D170N, 50 and 100%, respectively, of the flavin is non-covalently bound. Kinetic characterization of the vanillyl-alcohol oxidase variants revealed that Asp-170 is required for catalysis. D170E is 50-fold less active, and the other Asp-170 variants are about 10(3)-fold less active than wild type enzyme. Impaired catalysis of the Asp-170 variants is caused by slow flavin reduction. Furthermore, the mutant proteins have lost the capability of forming a stable complex between reduced enzyme and the p-quinone methide intermediate. The redox midpoint potentials in D170E (+6 mV) and D170S (-91 mV) are considerably decreased compared with wild type vanillyl-alcohol oxidase (+55 mV). This supports the idea that Asp-170 interacts with the protonated N5-atom of the reduced cofactor, thus increasing the FAD redox potential. Taken together, we conclude that Asp-170 is involved in the process of autocatalytic flavinylation and is crucial for efficient redox catalysis.

Comparison of binding energies of SrcSH2-phosphotyrosyl peptides with structure-based prediction using surface area based empirical parameterization.

The prediction of binding energies from the three-dimensional (3D) structure of a protein-ligand complex is an important goal of biophysics and structural biology. Here, we critically assess the use of empirical, solvent-accessible surface area-based calculations for the prediction of the binding of Src-SH2 domain with a series of tyrosyl phosphopeptides based on the high-affinity ligand from the hamster middle T antigen (hmT), where the residue in the pY+ 3 position has been changed. Two other peptides based on the C-terminal regulatory site of the Src protein and the platelet-derived growth factor receptor (PDGFR) are also investigated. Here, we take into account the effects of proton linkage on binding, and test five different surface area-based models that include different treatments for the contributions to conformational change and protein solvation. These differences relate to the treatment of conformational flexibility in the peptide ligand and the inclusion of proximal ordered solvent molecules in the surface area calculations. This allowed the calculation of a range of thermodynamic state functions (deltaCp, deltaS, deltaH, and deltaG) directly from structure. Comparison with the experimentally derived data shows little agreement for the interaction of SrcSH2 domain and the range of tyrosyl phosphopeptides. Furthermore, the adoption of the different models to treat conformational change and solvation has a dramatic effect on the calculated thermodynamic functions, making the predicted binding energies highly model dependent. While empirical, solvent-accessible surface area based calculations are becoming widely adopted to interpret thermodynamic data, this study highlights potential problems with application and interpretation of this type of approach. There is undoubtedly some agreement between predicted and experimentally determined thermodynamic parameters: however, the tolerance of this approach is not sufficient to make it ubiquitously applicable.

Refined crystal structure (2.3 A) of a double-headed winged bean alpha-chymotrypsin inhibitor and location of its second reactive site.

The crystal structure of a double-headed alpha-chymotrypsin inhibitor, WCI, from winged bean seeds has now been refined at 2.3 A resolution to an R-factor of 18.7% for 9,897 reflections. The crystals belong to the hexagonal space group P6(1)22 with cell parameters a = b = 61.8 A and c = 212.8 A. The final model has a good stereochemistry and a root mean square deviation of 0.011 A and 1.14 degrees from ideality for bond length and bond angles, respectively. A total of 109 ordered solvent molecules were localized in the structure. This improved structure at 2.3 A led to an understanding of the mechanism of inhibition of the protein against alpha-chymotrypsin. An analysis of this higher resolution structure also helped us to predict the location of the second reactive site of the protein, about which no previous biochemical information was available. The inhibitor structure is spherical and has twelve anti-parallel beta-strands with connecting loops arranged in a characteristic beta-trefoil fold common to other homologous serine protease inhibitors in the Kunitz (STI) family as well as to some non homologous functionally unrelated proteins. A wide variation in the surface loop regions is seen in the latter ones.

Crystal structure of MHC class II-associated p41 Ii fragment bound to cathepsin L reveals the structural basis for differentiation between cathepsins L and S.

The lysosomal cysteine proteases cathepsins S and L play crucial roles in the degradation of the invariant chain during maturation of MHC class II molecules and antigen processing. The p41 form of the invariant chain includes a fragment which specifically inhibits cathepsin L but not S. The crystal structure of the p41 fragment, a homologue of the thyroglobulin type-1 domains, has been determined at 2.0 A resolution in complex with cathepsin L. The structure of the p41 fragment demonstrates a novel fold, consisting of two subdomains, each stabilized by disulfide bridges. The first subdomain is an alpha-helix-beta-strand arrangement, whereas the second subdomain has a predominantly beta-strand arrangement. The wedge shape and three-loop arrangement of the p41 fragment bound to the active site cleft of cathepsin L are reminiscent of the inhibitory edge of cystatins, thus demonstrating the first example of convergent evolution observed in cysteine protease inhibitors. However, the different fold of the p41 fragment results in additional contacts with the top of the R-domain of the enzymes, which defines the specificity-determining S2 and S1' substrate-binding sites. This enables inhibitors based on the thyroglobulin type-1 domain fold, in contrast to the rather non-selective cystatins, to exhibit specificity for their target enzymes.

Distance-Dependent Enhanced ENDOR Phase (DEEP) Spectroscopy and Its Application to Determination of Solvation Structure around Paramagnetic Ions in Disordered Solids: The Three Ordered Hydration Shells of Aquated Transition Ions

We present a method that improves the spectral resolution and extends the distance range for detection of ordered solvent molecules surrounding a paramagnetic ion in frozen solutions using 1H electron−nuclear double-resonance (ENDOR) spectroscopy. This method is based on the R-3 distance dependence of the proton−electron spin hyperfine interaction and its effect on the electron−nuclear cross relaxation rate. This relaxation effect dramatically influences the ENDOR phase spectrum and appears through the radio-frequency (rf) phase angle dependence (using the conventional rf modulation detection scheme), and thus it is termed distance-dependent enhanced ENDOR phase (DEEP) spectroscopy. Applying DEEP spectroscopy to the conventional CW-ENDOR experiment, one is able to eliminate the large proton matrix peak from disordered solvent molecules. We observe a 4-fold increase in spectral resolution with DEEP spectroscopy for distant protons. When applied to solvation of aquo Mn(II) ions, we resolve 1H hyperfine coup...

Structure of Cubic Insulin Crystals in Glucose Solutions

X-ray structures of cubic insulin crystals in high concentrations of glucose at different pH levels and temperatures have been refined to high resolution. We have identified one glucose-binding site near the N-terminus of the A-chain whose occupancy is pH dependent. The effects of reduced water activity on the ordered protein and solvent structures have been examined. Our analysis showed no notable conformational changes in the ordered protein structures or ordered solvent molecules near the protein surface, but the presence of glucose does have a significant effect on the overall density distribution of the bulk solvent in the solvent-accessible volume. We compared the structure of cubic insulin at room temperature and liquid-nitrogen temperature, under identical solvent conditions, using glucose as a cryoprotectant. In this case, we found that the average temperature factor of the protein is reduced and more water molecules can be identified, but there are no significant changes in the protein conformation.

Refined Atomic Model of the Four-Layer Aggregate of the Tobacco Mosaic Virus Coat Protein at 2.4-Å Resolution

Previous x-ray studies (2.8-Å resolution) on crystals of tobacco mosaic virus coat protein grown from solutions containing high salt have characterized the structure of the protein aggregate as a dimer of a bilayered cylindrical disk formed by 34 chemically identical subunits. We have determined the crystal structure of the disk aggregate at 2.4-Å resolution using x-ray diffraction from crystals maintained at cryogenic temperatures. Two regions of interest have been extensively refined. First, residues of the low-radius loop region, which were not modeled previously, have been traced completely in our electron density maps. Similar to the structure observed in the virus, the right radial helix in each protomer ends around residue 87, after which the protein chain forms an extended chain that extends to the left radial helix. The left radial helix appears as a long α-helix with high temperature factors for the main-chain atoms in the inner portion. The side-chain atoms in this region (residues 90–110) are not visible in the electron density maps and are assumed to be disordered. Second, interactions between subunits in the symmetry-related central A pair have been determined. No direct protein-protein interactions are observed in the major overlap region between these subunits; all interactions are mediated by two layers of ordered solvent molecules. The current structure emphasizes the importance of water in biological macromolecular assemblies.

An eye lens protein-water structure: 1.2 A resolution structure of gammaB-crystallin at 150 K.

gammabeta-crystallin is a structural protein of the eye lens with a role in the maintenance of an even distribution of protein and water over distances around the wavelength of light, preserving lens transparency. The structure of the 174-residue bovine protein has already been determined at room temperature to 1.47 A resolution. By flash freezing the protein crystals, data have now been collected to a nominal resolution limit of 1.2 A as radiation damage was essentially eliminated. The protein-water model has been refined against this data using the program RESTRAIN converging to an R factor of 18.5% with all data. Atomic positions are clearly indicated in the electron-density maps. Discrete bimodal disorder has been visualized for a few side chains. Out of a total of 498 water molecules present in the crystal asymmetric unit, 394 have been modelled and refined at unit occupancy. The solvent structure is extremely well ordered with an average B value of 23.4 A(2). Partially occupied sites have been identified where disorder in the protein induces concomitant disorder in the local solvent structure. The solvent structure covers 97% of the solvent-exposed surface of the protein in the crystal. 126 water molecules are distributed in second and higher hydration shells. There are networks of hydrogen-bonded solvent extending up to 64 molecules in a network, comprising trimers and tetramers as well as five- and six-membered water-ring structures. The hydration of the protein surface is dominated by arginine and aspartate side chains. Extensive cages of highly ordered solvent molecules are also observed around exposed non-polar groups.