Proton transfer drives protein radical formation in Helicobacter pylori catalase but not in Penicillium vitale catalase.

Heme catalases prevent cells from oxidative damage by decomposing hydrogen peroxide into water and molecular oxygen. Here we investigate the factors that give rise to an undesirable side reaction competing with normal catalase activity, the migration of a radical from the heme active site to the protein in the principal reaction intermediate compound I (Cpd I). Recently, it has been proposed that this electron transfer reaction takes place in Cpd I of Helicobacter pylori catalase (HPC), but not in Cpd I of Penicillium vitale catalase (PVC), where the oxidation equivalent remains located on the heme active site. Unraveling the factors determining the different radical locations could help engineer enzymes with enhanced catalase activity for detection or removal of hydrogen peroxide. Using quantum mechanics/molecular mechanics metadynamics simulations, we show that radical migration in HPC is facilitated by the large driving force (-0.65 eV) of the subsequent proton transfer from a histidine residue to the ferryl oxygen atom of reduced Cpd I. The corresponding free energy in PVC is significantly smaller (-0.19 eV) and, as we argue, not sufficiently high to support radical migration. Our results suggest that the energetics of oxoferryl protonation is a key factor regulating radical migration in catalases and possibly also in hydroperoxidases.

Genome-wide association identifies SKIV2L and MYRIP as protective factors for age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly in the developed world. We conducted a genome-wide association study in a series of families enriched for AMD and completed a meta-analysis of this new data with results from reanalysis of an existing study of a late-stage case-control cohort. We tested the top findings for replication in 1896 cases and 1866 controls and identified two novel genetic protective factors for AMD. In addition to the complement factor H (CFH) (P=2.3 × 10⁻64) and age-related maculopathy susceptibility 2 (ARMS2) (P=1.2 × 10⁻6°) loci, we observed a protective effect at rs429608, an intronic SNP in SKIV2L (P=5.3 × 10⁻¹5), a gene near the complement component 2 (C2)/complement factor B (BF) locus, that indicates the protective effect may be mediated by variants other than the C2/BF variants previously studied. Haplotype analysis at this locus identified three protective haplotypes defined by the rs429608 protective allele. We also identified a new potentially protective effect at rs2679798 in MYRIP (P=2.9 × 10⁻4), a gene involved in retinal pigment epithelium melanosome trafficking. Interestingly, MYRIP was initially identified in the family-based scan and was confirmed in the case-control set. From these efforts, we report the identification of two novel protective factors for AMD and confirm the previously known associations at CFH, ARMS2 and C3.

Polymorphisms in C2, CFB and C3 are associated with progression to advanced age related macular degeneration associated with visual loss.

BACKGROUND: Age related macular degeneration (AMD) is a leading cause of blindness. AMD is a complex disorder caused by genetic and environmental factors in which single nucleotide polymorphisms (SNPs) in the genes CFH and LOC387715/HTRA1/ARMS2 have prognostic importance for progression to advanced AMD (with visual loss). CFH may also have a pharmacogenetic role by affecting treatment response to widely used nutritional supplements. This paper examines other AMD susceptibility genes to determine if these genotypes influenced disease progression and treatment response. METHODS: Three cohorts, totalling 3137 individuals, were genotyped for SNPs in 13 genes previously published to be associated with advanced AMD (other than CFH and LOC387715/ARMS2/HTRA1). Those genes found associated were then evaluated for their involvement in disease progression. Interactions between the genes and with AREDS (Age-Related Eye Disease Study) nutritional supplements were investigated. RESULTS: Positive independent associations were noted in SNPs in the genes C2 (p = 0.0001, odds ratio (OR) 0.35, 95% confidence interval (CI) 0.2 to 0.6), CFB (p = 0.0001, OR 0.35, 95% CI 0.2 to 0.6), C3 (p = 0.0001, OR 3.91, 95% CI 1.94 to 7.88), APOE (epsilon4, p = 0.01, OR 0.50, 95% CI 0.29 to 0.86) and VEGFA (p = 0.01, OR 2.23, 95% CI 1.06 to 4.68). C2/CFB and C3 were independently related to progression from early/intermediate to advanced AMD with OR 0.32 (95% CI 0.14 to 0.73) and 3.32 (95% CI 1.46 to 7.59), respectively. Gene-gene and pharmacogenetic interactions were not observed. No preferential associations were observed with geographic atrophy or choroidal neovascularisation. CONCLUSION: This study provides insights into the genetic pathogenesis of AMD. Five genes have now been shown to be independently involved in progression from intermediate disease (before vision loss has occurred) to advanced disease in which blindness is frequent.

X-ray diffraction and computation yield the structure of alkanethiols on gold(111).

The structure of self-assembled monolayers (SAMs) of long-chain alkyl sulfides on gold(111) has been resolved by density functional theory-based molecular dynamics simulations and grazing incidence x-ray diffraction for hexanethiol and methylthiol. The analysis of molecular dynamics trajectories and the relative energies of possible SAM structures suggest a competition between SAM ordering, driven by the lateral van der Waals interaction between alkyl chains, and disordering of interfacial Au atoms, driven by the sulfur-gold interaction. We found that the sulfur atoms of the molecules bind at two distinct surface sites, and that the first gold surface layer contains gold atom vacancies (which are partially redistributed over different sites) as well as gold adatoms that are laterally bound to two sulfur atoms.

Genetic and phenotypic heterogeneity in pattern dystrophy.

BACKGROUND: The pattern dystrophies (PD) represent a clinically heterogeneous family of inherited macular diseases frequently caused by mutations in the peripherin/RDS gene. Most previous studies have detailed the clinical findings in single families, making it difficult to derive data from which progression and visual outcome can be generalised. METHODS: Families were ascertained and clinically evaluated including angiography and electrophysiology where appropriate. RESULTS: In each of the six families with autosomal dominant PD, a mutation in the peripherin/RDS gene was identified, including a novel Cys250Phe variant. These data suggest that the condition is characterised by the accumulation of yellow to grey subretinal flecks, followed by pigmentary change accompanied by patches of chorioretinal atrophy. Subsequently, 50% (16/32) of individuals with PD developed poor central vision because of chorioretinal geographic atrophy or subretinal neovascularisation. The risk of these complications appears to increase with age. CONCLUSION: PD should not necessarily be considered a benign condition. Instead, patients should be counselled that there is a significant chance of losing central vision in their later years. Some elderly patients with probands showing PD may be misdiagnosed with age related macular degeneration owing to the phenotypic similarities between these conditions in the advanced state.

Novel mutation of the initiation codon of PAX9 causes oligodontia.

Tooth development is under strict genetic control. Oligodontia is defined as the congenital absence of 6 or more permanent teeth, excluding the third molar. The occurrence of non-syndromic oligodontia is poorly understood, but in recent years several cases have been described where a single gene mutation is associated with oligodontia. Several studies have shown that MSX1 and PAX9 play a role in early tooth development. We screened one family with non-syndromic oligodontia for mutations in MSX1 and PAX9. The pedigree showed an autosomal-dominant pattern of inheritance. Direct sequencing and restriction enzyme analysis revealed a novel heterozygous A to G transition mutation in the AUG initiation codon of PAX9 in exon 1 in the affected members of the family. This is the first mutation found in the initiation codon of PAX9, and we suggest that it causes haploinsufficiency.

Molecular dynamics simulations of a hydrated protein vectorially oriented on polar and nonpolar soft surfaces.

We present a collection of molecular dynamics computer simulation studies on a model protein-membrane system, namely a cytochrome c monolayer attached to an organic self-assembled monolayer (SAM). Modifications of the system are explored, including the polarity of the SAM endgroups, the amount of water present for hydration, and the coordination number of the heme iron atom. Various structural parameters are measured, e.g., the protein radius of gyration and eccentricity, the deviation of the protein backbone from the x-ray crystal structure, the orientation of the protein relative to the SAM surface, and the profile structures of the SAM, protein, and water. The polar SAM appears to interact more strongly with the protein than does the nonpolar SAM. Increased hydration of the system tends to reduce the effects of other parameters. The choice of iron coordination model has a significant effect on the protein structure and the heme orientation. The overall protein structure is largely conserved, except at each end of the sequence and in one loop region. The SAM structure is only perturbed in the region of its direct contact with the protein. Our calculations are in reasonably good agreement with experimental measurements (polarized optical absorption/emission spectroscopy, x-ray interferometry, and neutron interferometry).

Short wavelength collective dynamics in phospholipid bilayers: a molecular dynamics study.

Short wavelength density fluctuations of hydrated multilamellar phospholipid bilayers have been studied by molecular dynamics simulations in both the gel and liquid crystalline phases, and the results compared to recent inelastic x-ray scattering data [Phys. Rev. Lett. 86, 740 (2001)]. We confirm the existence of a highly dispersive sound mode, whose frequency and damping depend on the lipid phase, that the scattering arises mainly from in-plane motion of hydrocarbon chains, and we also identify a nondispersive mode attributed to motions of the chain terminal carbons.

Membrane structural perturbations caused by anesthetics and nonimmobilizers: a molecular dynamics investigation.

The structural perturbations of the fully hydrated dimyristoyl-phosphatidylcholine bilayer induced by the presence of hexafluoroethane C(2F6), a "nonimmobilizer," have been examined by molecular dynamics simulations and compared with the effects produced by halothane CF3CHBrCl, an "anesthetic," on a similar bilayer (DPPC) (Koubi et al., Biophys. J. 2000. 78:800). We find that the overall structure of the lipid bilayer and the zwitterionic head-group dipole orientation undergo only a slight modification compared with the pure lipid bilayer, with virtually no change in the potential across the interface. This is in contrast to the anesthetic case in which the presence of the molecule led to a large perturbation of the electrostatic potential across to the membrane interface. Similarly, the analysis of the structural and dynamical properties of the lipid core are unchanged in the presence of the nonimmobilizer although there is a substantial increase in the microscopic viscosity for the system containing the anesthetic. These contrasting perturbations of the lipid membrane caused by those quite similarly sized molecules may explain the difference in their physiological effects as anesthetics and nonimmobilizers, respectively.

Dynamical properties of a hydrated lipid bilayer from a multinanosecond molecular dynamics simulation.

A fully hydrated dimiristoylphosphatidylcholine (DMPC) bilayer has been studied by a molecular dynamics simulation. The system, which consisted of 64 DMPC molecules and 1792 water molecules, was run in the NVE ensemble at a temperature of 333 K for a total of 10 ns. The resulting trajectory was used to analyze structural and dynamical quantities. The electron density, bilayer spacing, and order parameters (S(CD)), based on the AMBER forcefield and SPCE water model are in good agreement with previous calculations and experimental data. The simulation reveals evidence for two types of lateral diffusive behavior: cage hopping and that of a two-dimensional liquid. The lateral diffusion coefficient is 8 x 10(-8) cm(2)/s. We characterize the rotational motion, and find that the lipid tail rotation (D(rot_tail) = -0.04 rad(2)/ns) is slower then the head group rotation (D(rot_hg) = 2.2 rad(2)/ns), which is slower than the overall in plane (D(rot) = 3.2 rad(2)/ns) for the lipid molecule.

Influence of highly polyunsaturated lipid acyl chains of biomembranes on the NMR order parameters.

We investigate the effect of specific conformations of double-bond segments in highly polyunsaturated acyl chains on the deuterium (2)H NMR order parameters of a fully hydrated 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (SDPC, 18:0/22:6 PC) lipid bilayer. The system is analyzed by performing a molecular dynamics simulation study at ambient conditions in the fluid lamellar phase. By separately calculating the different partial contributions to the total order parameter profiles measurable experimentally, we are able to get insights into the molecular origin of earlier experimental and theoretical observations. The effect of the position of the different conformations of double-bond segments along the polyunsaturated acyl chain is also examined. As in experiments performed in a series of lipid bilayers with an increasing number of cis double bonds per lipid molecule [Holte, L. L., et al. Biophys. J. 1995, 68, 2396], we find that unsaturations influence mainly the order of the bottom half of the saturated chain. Specific conformations of the polyunsaturated chain close to the lipid headgroups have a distinct effect on the order of the bottom half of the saturated chain and on the top half of the polyunsaturated chain. Our results indicate that for SDPC the conformation of the region of the polyunsaturated chain located between the first three cis double bonds is responsible for the major effects on the orientational order of both the saturated and the polyunsaturated chains.

Molecular genetic heterogeneity in autosomal dominant drusen.

OBJECTIVE: Autosomal dominant drusen is of particular interest because of its phenotypic similarity to age related macular degeneration. Currently, mutation R345W of EFEMP1 and, in a single pedigree, linkage to chromosome 6q14 have been causally related to the disease. We proposed to investigate and quantify the roles of EFEMP1 and the 6q14 locus in dominant drusen patients from the UK and USA. DESIGN: Molecular genetic analysis. PARTICIPANTS: Ten unrelated families and 17 young drusen patients. MAIN OUTCOME MEASURES: Exons 1 and 2 of EFEMP1 were characterised by 5' rapid amplification of cDNA ends and direct sequencing. Exons 1-12 of EFEMP1 were then investigated for mutation by direct sequencing. A HpaII restriction digest test was constructed to detect the EFEMP1 R345W mutation. Marker loci spanning the two dominant drusen linked loci were used to generate haplotype data. RESULTS: Only seven of the 10 families (70%) and one of the 17 sporadic patients (6%) had the R345W mutation. The HpaII restriction digest test was found to be a reliable and quick method for detecting this. No other exonic or splice site mutation was identified. Of the three families without EFEMP1 mutation, two were linked to the 2p16 region. CONCLUSIONS: EFEMP1 R345W accounts for only a proportion of the dominant drusen phenotype. Importantly, other families linked to chromosome 2p16 raise the possibility of EFEMP1 promoter sequence mutation or a second dominant drusen gene at this locus. Preliminary haplotype data suggest that the disease gene at the 6q14 locus is responsible for only a minority of dominant drusen cases.

Structural properties of a highly polyunsaturated lipid bilayer from molecular dynamics simulations.

The structure of a fully hydrated mixed (saturated/polyunsaturated) chain lipid bilayer in the biologically relevant liquid crystalline phase has been examined by performing a molecular dynamics study. The model membrane, a 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (SDPC, 18:0/22:6 PC) lipid bilayer, was investigated at constant (room) temperature and (ambient) pressure, and the results obtained in the nanosecond time scale reproduced quite well the available experimental data. Polyunsaturated fatty acids are found in high concentrations in neuronal and retinal tissues and are essential for the development of human brain function. The docosahexaenoic fatty acid, in particular, is fundamental for the proper function of the visual receptor rhodopsin. The lipid bilayer order has been investigated through the orientational order parameters. The water-lipid interface has been explored thoroughly in terms of its dimensions and the organization of the different components. Several types of interactions occurring in the system have been analyzed, specifically, the water-hydrocarbon chain, lipid-lipid and lipid-water interactions. The distribution of dihedral angles along the chains and the molecular conformations of the polyunsaturated chain of the lipids have also been studied. Special attention has been focused on the microscopic (molecular) origin of the effects of polyunsaturations on the different physical properties of membranes.

Clinicopathologic correlation of fluorescein and indocyanine green angiography in exudative age-related macular degeneration.

PURPOSE: To correlate the clinical and histopathologic features of an eye with age-related macular degeneration studied with fluorescein (FA) and indocyanine green (ICG) angiography 4.5 months before the patient's death. METHODS: Histopathologic features from serial sections through the macula of a 76-year-old man with occult choroidal neovascularization (CNV) were reconstructed in a scaled two-dimensional map and compared with FA and ICG angiogram images obtained 4.5 months before his death. RESULTS: The region of prior laser photocoagulation was identified as a well-demarcated hypofluorescent region in the early frames of the FA and the early and late phases of the ICG angiogram. This corresponded histopathologically to a well-circumscribed area of absence of the choriocapillaris, loss of the outer retina and retinal pigment epithelium, and scarring of the choroid. Occult CNV characterized by elevated late hyperfluorescence on the FA and intense well-defined hyperfluorescence on the ICG angiogram corresponded to a thick fibrovascular membrane in the subretinal space and within Bruch's membrane. Thin extensions of both the subretinal and intra-Bruch's membrane fibrovascular membrane components corresponded to nonelevated stippled late hyperfluorescence on the FA and mild late hyperfluorescence on the ICG angiogram. CONCLUSION: Histopathologic mapping revealed a large fibrovascular complex located subretinally and within Bruch's membrane with thin and thick components that correlate well with findings of occult CNV on FA and ICG angiography.

Epidermolysis bullosa, pyloric atresia, and obstructive uropathy: a report of two case reports with molecular correlation and clinical management.

The epidermolysis bullosa-pyloric atresia-obstructive uropathy (EB-PA-OU) association is a rare, but well-described multisystem disease. While the prognosis at this time is still poor, an increasing number of patients are surviving to adolescence with aggressive care. It is important to understand this syndrome in order to anticipate medical complications and offer preventive strategies where possible. Prompt and expectant management of obstructive uropathy is crucial in these patients. Evidence of ureterovesicular obstruction may require bowel diversion, as excision of the obstructed ureterovesicular junction with reimplantation is often associated with a high risk of reobstruction. Many newborns succumb to sepsis or dehydration and electrolyte imbalance. Those infants who survive need close monitoring for the development of obstructive uropathy, failure to thrive, protein-losing enteropathy, respiratory compromise, and increased susceptibility to invasive infections. Once a clinical diagnosis is made, mutational analysis can confirm it and facilitate genetic counseling, as recurrence risks are 25% for this autosomal recessive condition. Mutational analysis enables direct genetic testing and accurate prenatal diagnosis. As more patients are studied, genotype/phenotype correlations may be possible.

A comparison of stereoscopic fluorescein angiography with indocyanine green videoangiography in age-related macular degeneration.

PURPOSE: To correlate features of indocyanine green (ICG) videoangiography with stereo film fluorescein angiography (FA) in the analysis of age-related macular degeneration (AMD). DESIGN: A retrospective study with concurrent comparison of two methods of assessment on one set of patients with AMD undergoing sequential FA-ICG videoangiography. METHODS: One hundred four patients with AMD who had undergone immediately sequential FA-ICG videoangiography were selected in a consecutive fashion from the photographic files of the Casey Eye Institute. Three interpreters independently graded in an unbiased fashion the FA features of AMD. These were compared with ICG videoangiography features of hypocyanescence (ICG videoangiography hypofluorescence), moderate or intense hypercyanescence (ICG videoangiography hyperfluorescence), or absence of fluorescence over background (ICG videoangiography isocyanescence). We also assessed ICG videoangiography features in the opposite eye of those with choroidal neovascularization (CNV) by FA in one eye by examining an additional 96 FA-ICG videoangiography scans. MAIN OUTCOME MEASURES: Outcomes were the ICG videoangiography characteristics of classic and occult CNV, serous pigment epithelial detachments (SPEDs) both with and without CNV, macular hemorrhage, and the comparative size of these features. RESULTS: There were 25 eyes that had significant macular hemorrhage by FA. Of these, the FA revealed CNV in its entirety under or near the hemorrhage in 11. The ICG examination revealed all 11 plus an additional 12 hypercyanescent features that were not visible by FA. Although 26 of 32 SPEDs had CNV by FA analysis, 31 of 32 had well-defined hypercyanescence under or at the SPED edge by ICG videoangiography. Eighty-seven percent of eyes with classic choroidal neovascular membranes (CCNV; 20 of 23) and 93% of eyes with fibrovascular pigment epithelial detachments (FVPED; 66 of 71) were hypercyanescent with distinct edges. Fifty percent of eyes with only late leakage of undetermined source (LLUS) were hypercyanescent, whereas 50% were isocyanescent on ICG videoangiography. There were four of 104 eyes in which ICG videoangiography revealed poorly defined hypercyanescent areas when, by FA, there were only drusen or elevated blocked fluorescence. Indocyanine green videoangiography revealed only three eyes with poorly defined hypercyanescence of 200 fellow eyes in patients with unilateral AMD. CONCLUSIONS: Indocyanine green videoangiography correlated fairly well with stereoscopic FA for the presence of CCNV or FVPED, but correlated poorly when LLUS was present. In certain cases where CNV was associated with SPED or macular hemorrhage, ICG videoangiography demonstrated features not apparent on FA.

Molecular dynamics simulation of four-alpha-helix bundles that bind the anesthetic halothane.

The mutation of a single leucine residue (L38) to methionine (M) is known experimentally to significantly increase the affinity of the synthetic four-alpha-helix bundle (Aalpha(2))(2) for the anesthetic halothane. We present a molecular dynamics study of the mutant (Aalpha(2)-L38M)(2) peptide, which consists of a dimer of 62-residue U-shaped di-alpha-helical monomers assembled in an anti topology. A comparison between the simulation results and those obtained for the native (Aalpha(2))(2) peptide indicates that the overall secondary structure of the bundle is not affected by the mutation, but that the side chains within the monomers are better packed in the mutant structure. Unlike the native peptide, binding of a single halothane molecule to the hydrophobic core of (Aalpha(2)-L38M)(2) deforms the helical nature of one monomer in a region close to the mutation site. Increased exposure of the cysteine side chain to the hydrophobic core in the mutant structure leads to the enhancement of the attractive interaction between halothane and this specific residue. Since the mutated residues are located outside the hydrophobic core the observed increased affinity for halothane appears to be an indirect effect of the mutation.

Two possible conducting states of the influenza A virus M2 ion channel.

Molecular dynamics simulations have been performed on protonated four-helix bundles based on the 25-residue Duff-Ashley transmembrane sequence of the M2 channel of the influenza A virus. Well-equilibrated tetrameric channels, with one, two and four of the H37 residues protonated, were investigated. The protonated peptide bundles were immersed in the octane portion of a phase-separated water/octane system, which provided a membrane-mimetic environment. The simulations suggest that there could be two conducting states of the M2 channel corresponding to tetramers containing one or two protonated histidines. The more open structure of the doubly protonated state suggests it would have the higher conductance.