Eduardo Howard

Interaction of water with α,α-trehalose in solution: molecular dynamics simulation approach

Molecular simulations of an aqueous solution of α,α-trehalose (α-D-glucopyranosylα-D-glucopyranoside) have been carried out to further the understanding of the effect of α,α-trehalose as a protecting agent against water stress in biological systems. The hydrogen-bond network and water dynamics were found to be only slightly altered compared with pure water (SPC/E model). Some internal hydrogen bonds in trehalose stabilize the conformation that was found to have glycosysidic dihedral angles of 215° and 216°. It is found that trehalose can fit into a water structure involving at least ten water molecules per trehalose. Results support the view that the ability of trehalose to protect against water stress is due to the stabilization of biological structures and not to modification of the properties of water.

The effect of stereochemistry upon carbohydrate hydration. A molecular dynamics simulation of β-d-galactopyranose and (α,β)-d-talopyranose

Abstract This paper reports a molecular dynamics simulation study of β- d -galactopyranose and (α,β)- d -talopyranose in aqueous solution. Special emphasis was placed on the intramolecular next-nearest neighbour oxygen distances in the carbohydrate molecule and the hydrogen bonding of the hydroxy functionalities of the carbohydrates with water. The average number of hydrogen bonds of the hydroxy groups of the carbohydrates depends on the stereochemistry of the molecule. In contrast to the HO-2 and HO-4 of d - galactopyranose, those of d -talopyranose are shielded. This is a consequence of an intramolecular hydrogen bond between the HO-2 and HO-4 in d -talopyranose, which also explains why the apparent hydrophobicity of d -talose is found to be greater than that of d -galactose.

Neutron structure of type III Antifreeze Protein allows the reconstruction of AFP-ice interface

Antifreeze proteins (AFPs) inhibit ice growth at sub‐zero temperatures. The prototypical type‐III AFPs have been extensively studied, notably by X‐ray crystallography, solid‐state and solution NMR, and mutagenesis, leading to the identification of a compound ice‐binding surface (IBS) composed of two adjacent ice‐binding sections, each which binds to particular lattice planes of ice crystals, poisoning their growth. This surface, including many hydrophobic and some hydrophilic residues, has been extensively used to model the interaction of AFP with ice. Experimentally observed water molecules facing the IBS have been used in an attempt to validate these models. However, these trials have been hindered by the limited capability of X‐ray crystallography to reliably identify all water molecules of the hydration layer. Due to the strong diffraction signal from both the oxygen and deuterium atoms, neutron diffraction provides a more effective way to determine the water molecule positions (as D2O). Here we report the successful structure determination at 293u2009K of fully perdeuterated type‐III AFP by joint X‐ray and neutron diffraction providing a very detailed description of the protein and its solvent structure. X‐ray data were collected to a resolution of 1.05u2009Å, and neutron Laue data to a resolution of 1.85u2009Å with a “radically small” crystal volume of 0.13u2009mm3. The identification of a tetrahedral water cluster in nuclear scattering density maps has allowed the reconstruction of the IBS‐bound ice crystal primary prismatic face. Analysis of the interactions between the IBS and the bound ice crystal primary prismatic face indicates the role of the hydrophobic residues, which are found to bind inside the holes of the ice surface, thus explaining the specificity of AFPs for ice versus water. Copyright

Neutron macromolecular crystallography with LADI‐III

At the Institut Laue-Langevin, a new neutron Laue diffractometer LADI-III has been fully operational since March 2007. LADI-III is dedicated to neutron macromolecular crystallography at medium to high resolution (2.5-1.5 Å) and is used to study key H atoms and water structure in macromolecular structures. An improved detector design and readout system has been incorporated so that a miniaturized reading head located inside the drum scans the image plate. From comparisons of neutron detection efficiency (DQE) with the original LADI-I instrument, the internal transfer of the image plates and readout system provides an approximately threefold gain in neutron detection. The improved performance of LADI-III, coupled with the use of perdeuterated biological samples, now allows the study of biological systems with crystal volumes of 0.1-0.2 mm(3), as illustrated here by the recent studies of type III antifreeze protein (AFP; 7 kDa). As the major bottleneck for neutron macromolecular studies has been the large crystal volumes required, these recent developments have led to an expansion of the field, extending the size and the complexity of the systems that can be studied and reducing the data-collection times required.

Perdeuteration, purification, crystallization and preliminary neutron diffraction of an ocean pout type III antifreeze protein.

The highly homologous type III antifreeze protein (AFP) subfamily share the capability to inhibit ice growth at subzero temperatures. Extensive studies by X-ray crystallography have been conducted, mostly on AFPs from polar fishes. Although interactions between a defined flat ice-binding surface and a particular lattice plane of an ice crystal have now been identified, the fine structural features underlying the antifreeze mechanism still remain unclear owing to the intrinsic difficulty in identifying H atoms using X-ray diffraction data alone. Here, successful perdeuteration (i.e. complete deuteration) for neutron crystallographic studies of the North Atlantic ocean pout (Macrozoarces americanus) AFP in Escherichia coli high-density cell cultures is reported. The perdeuterated protein (AFP D) was expressed in inclusion bodies, refolded in deuterated buffer and purified by cation-exchange chromatography. Well shaped perdeuterated AFP D crystals have been grown in D(2)O by the sitting-drop method. Preliminary neutron Laue diffraction at 293 K using LADI-III at ILL showed that with a few exposures of 24 h a very low background and clear small spots up to a resolution of 1.85 A were obtained using a ;radically small perdeuterated AFP D crystal of dimensions 0.70 x 0.55 x 0.35 mm, corresponding to a volume of 0.13 mm(3).

Conformations of erythritol and L-threitol in aqueous solution in relation to sweetness properties: a molecular dynamics simulation

Erythritol and L-threitol in aqueous solution have been studied by molecular dynamics simulation. Computed NMR proton–proton coupling parameters agree fairly well with published experimental results. The analysis of the mean conformations of both polyalcohols shows that in both compounds it is possible to identify the sweetness triangle characteristic of substances capable of stimulating the sensation of a sweet taste. A close look into the mean structure suggests that erythritol should be a better sweetener than L-threitol. The hydration structure and dynamics were studied, analysing the mean orientation of water molecules through angular distribution functions and hydrogen-bond lifetimes. The water surrounding the solute exhibits, in both cases, higher mobility as measured by comparison of the average lifetimes of hydrogen bonds between water and the polyalcohols with those of bulk water–water hydrogen bonds.

Perdeuteration: improved visualization of solvent structure in neutron macromolecular crystallography

The 1.8u2005Å resolution neutron structure of deuterated type III antifreeze protein in which the methyl groups of leucine and valine residues are selectively protonated is presented. Comparison between this and the 1.85u2005Å resolution neutron structure of perdeuterated type III antifreeze protein indicates that perdeuteration improves the visibility of solvent molecules located in close vicinity to hydrophobic residues, as cancellation effects between H atoms of the methyl groups and nearby heavy-water molecules (D2O) are avoided.

Crystal packing modifies ligand binding affinity: The case of aldose reductase

The relationship between the structures of protein–ligand complexes existing in the crystal and in solution, essential in the case of fragment‐based screening by X‐ray crystallography (FBS‐X), has been often an object of controversy. To address this question, simultaneous co‐crystallization and soaking of two inhibitors with different ratios, Fidarestat (FID; Kd = 6.5 nM) and IDD594 (594; Kd = 61 nM), which bind to h‐aldose reductase (AR), have been performed. The subatomic resolution of the crystal structures allows the differentiation of both inhibitors, even when the structures are almost superposed. We have determined the occupation ratio in solution by mass spectrometry (MS) Occ(FID)/Occ(594) = 2.7 and by X‐ray crystallography Occ(FID)/Occ(594) = 0.6. The occupancies in the crystal and in solution differ 4.6 times, implying that ligand binding potency is influenced by crystal contacts. A structural analysis shows that the Loop A (residues 122–130), which is exposed to the solvent, is flexible in solution, and is involved in packing contacts within the crystal. Furthermore, inhibitor 594 contacts the base of Loop A, stabilizing it, while inhibitor FID does not. This is shown by the difference in B‐factors of the Loop A between the AR–594 and AR–FID complexes. A stable loop diminishes the entropic energy barrier to binding, favoring 594 versus FID. Therefore, the effect of the crystal environment should be taken into consideration in the X‐ray diffraction analysis of ligand binding to proteins. This conclusion highlights the need for additional methodologies in the case of FBS‐X to validate this powerful screening technique, which is widely used. Proteins 2012.

Incorporation of methyl-protonated valine and leucine residues into deuterated ocean pout type III antifreeze protein: expression, crystallization and preliminary neutron diffraction studies.

Antifreeze proteins (AFPs) are found in different species from polar, alpine and subarctic regions, where they serve to inhibit ice-crystal growth by adsorption to ice surfaces. Recombinant North Atlantic ocean pout (Macrozoarces americanus) AFP has been used as a model protein to develop protocols for amino-acid-specific hydrogen reverse-labelling of methyl groups in leucine and valine residues using Escherichia coli high-density cell cultures supplemented with the amino-acid precursor alpha-ketoisovalerate. Here, the successful methyl protonation (methyl reverse-labelling) of leucine and valine residues in AFP is reported. Methyl-protonated AFP was expressed in inclusion bodies, refolded in deuterated buffer and purified by cation-exchange chromatography. Crystals were grown in D(2)O buffer by the sitting-drop method. Preliminary neutron Laue diffraction at 293 K using LADI-III at ILL showed in a few 24 h exposures a very low background and clear small spots up to a resolution of 1.80 A from a crystal of dimensions 1.60 x 0.38 x 0.38 mm corresponding to a volume of 0.23 mm(3).

Post-translational modifications in DNA topoisomerase 2α highlight the role of a eukaryote-specific residue in the ATPase domain

Type 2 DNA topoisomerases (Top2) are critical components of key protein complexes involved in DNA replication, chromosome condensation and segregation, as well as gene transcription. The Top2 were found to be the main targets of anticancer agents, leading to intensive efforts to understand their functional and physiological role as well as their molecular structure. Post-translational modifications have been reported to influence Top2 enzyme activities in particular those of the mammalian Top2α isoform. In this study, we identified phosphorylation, and for the first time, acetylation sites in the human Top2α isoform produced in eukaryotic expression systems. Structural analysis revealed that acetylation sites are clustered on the catalytic domains of the homodimer while phosphorylation sites are located in the C-terminal domain responsible for nuclear localization. Biochemical analysis of the eukaryotic-specific K168 residue in the ATPase domain shows that acetylation affects a key position regulating ATP hydrolysis through the modulation of dimerization. Our findings suggest that acetylation of specific sites involved in the allosteric regulation of human Top2 may provide a mechanism for modulation of its catalytic activity.