Impact of Nucleotide Mutations at the HNF3- and HNF4-Binding Sites in Enhancer 1 on Viral Replication in Patients with Chronic Hepatitis B Virus Infection

Background/Aims

The hepatitis B virus (HBV) genome contains binding sites for hepatocyte nuclear factors (HNF) 3 and 4 in the core domain of enhancer 1 (Enh1), and mutations in this domain have a strong impact on virus replication. We aimed to identify frequent base-mutation sites in the core domain of Enh1 and to examine the impact of these mutations on viral replication.

Methods

We studied virological characteristics and genetic sequences in 387 patients with chronic hepatitis B. We evaluated functional differences associated with specific mutations within the core domain of Enh1.

Results

Mutations in the core domain were found with significant frequency in C1126 (122/387 [31.5%], the binding site for HNF3) and in C1134 (106/387 [27.4%], the binding site for HNF4). A single mutation at nt 1126 (C1126) was identified in 17/123 (13.8%), and 105/123 (85.4%) had double mutations (C1126/1134). The level of HBV DNA (log10 copies/mL) was lower in single mutants (C1126, 5.81±1.25) than in wild (6.80±1.65) and double mutants (C1126/1134, 6.81±1.54). Similarly, the relative luciferase activity of C1126 and C1126/C1134 was 0.18 and 1.12 times that of the wild-type virus, respectively.

Conclusions

Mutations in the HNF3 binding site inhibit viral replication, whereas mutations at the HNF4 binding site restore viral replication.     

Semirational design of active tumor suppressor p53 DNA binding domain with enhanced stability

We have designed a p53 DNA binding domain that has virtually the same binding affinity for the gadd45 promoter as does wild-type protein but is considerably more stable. The design strategy was based on molecular evolution of the protein domain. Naturally occurring amino acid substitutions were identified by comparing the sequences of p53 homologues from 23 species, introducing them into wild-type human p53, and measuring the changes in stability. The most stable substitutions were combined in a multiple mutant. The advantage of this strategy is that, by substituting with naturally occurring residues, the function is likely to be unimpaired. All point mutants bind the consensus DNA sequence. The changes in stability ranged from +1.27 (less stable Q165K) to −1.49 (more stable N239Y) kcal mol⁻¹, respectively. The changes in free energy of unfolding on mutation are additive. Of interest, the two most stable mutants (N239Y and N268D) have been known to act as suppressors and restored the activity of two of the most common tumorigenic mutants. Of the 20 single mutants, 10 are cancer-associated, though their frequency of occurrence is extremely low: A129D, Q165K, Q167E, and D148E are less stable and M133L, V203A and N239Y are more stable whereas the rest are neutral. The quadruple mutant (M133LV203AN239YN268D), which is stabilized by 2.65 kcal mol⁻¹ and T_m raised by 5.6°C is of potential interest for trials in vivo.     

Targeted rescue of a destabilized mutant of p53 by an in silico screened drug

The tumor suppressor p53 is mutationally inactivated in ≈50% of human cancers. Approximately one-third of the mutations lower the melting temperature of the protein, leading to its rapid denaturation. Small molecules that bind to those mutants and stabilize them could be effective anticancer drugs. The mutation Y220C, which occurs in ≈75,000 new cancer cases per annum, creates a surface cavity that destabilizes the protein by 4 kcal/mol, at a site that is not functional. We have designed a series of binding molecules from an in silico analysis of the crystal structure using virtual screening and rational drug design. One of them, a carbazole derivative (PhiKan083), binds to the cavity with a dissociation constant of ≈150 μM. It raises the melting temperature of the mutant and slows down its rate of denaturation. We have solved the crystal structure of the protein–PhiKan083 complex at 1.5-Å resolution. The structure implicates key interactions between the protein and ligand and conformational changes that occur on binding, which will provide a basis for lead optimization. The Y220C mutant is an excellent “druggable” target for developing and testing novel anticancer drugs based on protein stabilization. We point out some general principles in relationships between binding constants, raising of melting temperatures, and increase of protein half-lives by stabilizing ligands.     

In vivo protein stabilization based on fragment complementation and a split GFP system

Protein stabilization was achieved through in vivo screening based on the thermodynamic linkage between protein folding and fragment complementation. The split GFP system was found suitable to derive protein variants with enhanced stability due to the correlation between effects of mutations on the stability of the intact chain and the effects of the same mutations on the affinity between fragments of the chain. PGB1 mutants with higher affinity between fragments 1 to 40 and 41 to 56 were obtained by in vivo screening of a library of the 1 to 40 fragments against wild-type 41 to 56 fragments. Colonies were ranked based on the intensity of green fluorescence emerging from assembly and folding of the fused GFP fragments. The DNA from the brightest fluorescent colonies was sequenced, and intact mutant PGB1s corresponding to the top three sequences were expressed, purified, and analyzed for stability toward thermal denaturation. The protein sequence derived from the top fluorescent colony was found to yield a 12 °C increase in the thermal denaturation midpoint and a free energy of stabilization of -8.7 kJ/mol at 25 °C. The stability rank order of the three mutant proteins follows the fluorescence rank order in the split GFP system. The variants are stabilized through increased hydrophobic effect, which raises the free energy of the unfolded more than the folded state; as well as substitutions, which lower the free energy of the folded more than the unfolded state; optimized van der Waals interactions; helix stabilization; improved hydrogen bonding network; and reduced electrostatic repulsion in the folded state.     

Mutually compensatory mutations during evolution of the tetramerization domain of tumor suppressor p53 lead to impaired hetero-oligomerization

We have measured the stability and stoichiometry of variants of the human p53 tetramerization domain to assess the effects of mutation on homo- and hetero-oligomerization. The residues chosen for mutation were those in the hydrophobic core that we had previously found to be critical for its stability but are not conserved in human p73 or p51 or in p53-related proteins from invertebrates or vertebrates. The mutations introduced were either single natural mutations or combinations of mutations present in p53-like proteins from different species. Most of the mutations were substantially destabilizing when introduced singly. The introduction of multiple mutations led to two opposite effects: some combinations of mutations that have occurred during the evolution of the hydrophobic core of the domain in p53-like proteins had additive destabilizing effects, whereas other naturally occurring combinations of mutations had little or no net effect on the stability, there being mutually compensating effects of up to 9.5 kcal/mol of tetramer. The triple mutant L332V/F341L/L344I, whose hydrophobic core represents that of the chicken p53 domain, was nearly as stable as the human domain but had impaired hetero-oligomerization with it. Thus, engineering of a functional p53 variant with a reduced capacity to hetero-oligomerize with wild-type human p53 can be achieved without any impairment in the stability and subunit affinity of the engineered homo-oligomer.     

A peptide that binds and stabilizes p53 core domain: Chaperone strategy for rescue of oncogenic mutants

Conformationally compromised oncogenic mutants of the tumor suppressor protein p53 can, in principle, be rescued by small molecules that bind the native, but not the denatured state. We describe a strategy for the rational search for such molecules. A nine-residue peptide, CDB3, which was derived from a p53 binding protein, binds to p53 core domain and stabilizes it in vitro. NMR studies showed that CDB3 bound to p53 at the edge of the DNA binding site, partly overlapping it. The fluorescein-labeled peptide, FL-CDB3, binds wild-type p53 core domain with a dissociation constant of 0.5 microM, and raises the apparent melting temperatures of wild-type and a representative oncogenic mutant, R249S core domain. gadd45 DNA competes with CDB3 and displaces it from its binding site. But this competition does not preclude CDB3 from being a lead compound. CDB3 may act as a "chaperone" that maintains existing or newly synthesized destabilized p53 mutants in a native conformation and then allows transfer to specific DNA, which binds more tightly. Indeed, CDB3 restored specific DNA binding activity to a highly destabilized mutant I195T to close to that of wild-type level.     

Temperature dependence of protein motions in a thermophilic dihydrofolate reductase and its relationship to catalytic efficiency

We report hydrogen deuterium exchange by mass spectrometry (HDX-MS) as a function of temperature in a thermophilic dihydrofolate reductase from Bacillus stearothermophilus (Bs-DHFR). Protein stability, probed with circular dichroism, established an accessible temperature range of 10 °C to 55 °C for the interrogation of HDX-MS. Although both the rate and extent of HDX are sensitive to temperature, the majority of peptides showed rapid kinetics of exchange, allowing us to focus on plateau values for the maximal extent of exchange at each temperature. Arrhenius plots of the ratio of hydrogens exchanged at 5 h normalized to the number of exchangeable hydrogens vs. 1/T provides an estimate for the apparent enthalpic change of local unfolding, ΔH°_unf(avg). Most regions in the enzyme show ΔH°_unf(avg) ≤ 2.0 kcal/mol, close to the value of kT; by contrast, significantly elevated values for ΔH°_unf(avg) are observed in regions within the core of protein that contain the cofactor and substrate-binding sites. Our technique introduces a new strategy for probing the temperature dependence of local protein unfolding within native proteins. These findings are discussed in the context of the demonstrated role for nuclear tunneling in hydride transfer from NADPH to dihydrofolate, and relate the observed enthalpic changes to two classes of motion, preorganization and reorganization, that have been proposed to control the efficiency of hydrogenic wave function overlap. Our findings suggest that the enthalpic contribution to the heavy atom environmental reorganizations controlling the hydrogenic wave function overlap will be dominated by regions of the protein proximal to the bound cofactor and substrate.     

Molecular defects of the glycine 41 variants of alanine glyoxylate aminotransferase associated with primary hyperoxaluria type I

G41 is an interfacial residue located within the α-helix 34–42 of alanine:glyoxylate aminotransferase (AGT). Its mutations on the major (AGT-Ma) or the minor (AGT-Mi) allele give rise to the variants G41R-Ma, G41R-Mi, and G41V-Ma causing hyperoxaluria type 1. Impairment of dimerization in these variants has been suggested to be responsible for immunoreactivity deficiency, intraperoxisomal aggregation, and sensitivity to proteasomal degradation. However, no experimental evidence supports this view. Here we report that G41 mutations, besides increasing the dimer-monomer equilibrium dissociation constant, affect the protein conformation and stability, and perturb its active site. As compared to AGT-Ma or AGT-Mi, G41 variants display different near-UV CD and intrinsic emission fluorescence spectra, larger exposure of hydrophobic surfaces, sensitivity to Met53-Tyr54 peptide bond cleavage by proteinase K, decreased thermostability, reduced coenzyme binding affinity, and catalytic efficiency. Additionally, unlike AGT-Ma and AGT-Mi, G41 variants under physiological conditions form insoluble inactive high-order aggregates (∼5,000 nm) through intermolecular electrostatic interactions. A comparative molecular dynamics study of the putative structures of AGT-Mi and G41R-Mi predicts that G41 → R mutation causes a partial unwinding of the 34–42 α-helix and a displacement of the first 44 N-terminal residues including the active site loop 24–32. These simulations help us to envisage the possible structural basis of AGT dysfunction associated with G41 mutations. The detailed insight into how G41 mutations act on the structure-function of AGT may contribute to achieve the ultimate goal of correcting the effects of these mutations.     

Temperature dependence of the reduction potential of blue copper in fungal laccase

Thin-layer spectroelectrochemical methods have been employed to measure the reduction potentials of the blue copper in Polyporus versicolor laccase (EC 1.10.3.2) between 7°C and 41°C (0.2 M sodium phosphate, pH 5.4). Thermodynamic parameters are: ΔS° = -13.9 ± 2 cal/mol-K; ΔH° = -22.1 ± 0.5 kcal/mol; E° (25°C) = 780 ± 3 mV vs. the normal hydrogen electrode. Comparison of the ΔS° and ΔH° values with those for single-site proteins suggests that the high potential of the blue copper in fungal laccase is attributable mainly to stabilization of the copper (I) center by enhanced ligand binding interactions and that protein solvation effects play a lesser role.     

In vitro unfolding of yeast multicopper oxidase Fet3p variants reveals unique role of each metal site

Fet3p from Saccharomyces cerevisiae is a multicopper oxidase (MCO) that contains 3 cupredoxin-like β-barrel domains and 4 copper ions located in 3 distinct metal sites (T1 in domain 3, T2, and the binuclear T3 at the interface between domains 1 and 3). To better understand how protein structure and stability is defined by cofactor coordination in MCO proteins, we assessed thermal unfolding of apo and metallated forms of Fet3p by using spectroscopic and calorimetric methods in vitro (pH 7). We find that unfolding reactions of apo and different holo forms of Fet3p are irreversible reactions that depend on the scan rate. The domains in apo-Fet3p unfold sequentially [thermal midpoint (T_m) of 45 °C, 62 °C, and 72 °C; 1 K/min]. Addition of T3 imposes strain in the apo structure that results in coupled domain unfolding and low stability (T_m of 50 °C; 1 K/min). Further inclusion of T2 (i.e., only T1 absent) increases overall stability by ≈5 °C but unfolding remains coupled in 1 step. Introduction of T1, producing fully-loaded holo-Fet3p (or in the absence of T2), results in stabilization of domain 3, which uncouples unfolding of the domains; unfolding of domain 2 occurs first along with Cu-site perturbations (T_m 50–55 °C; 1 K/min), followed by unfolding of domains 1 and 3 (≈65–70 °C; 1 K/min). Our results suggest that there is a metal-induced tradeoff between overall protein stability and metal coordination in members of the MCO family.     

SAXS Studies of the Endoglucanase Cel12A from Gloeophyllum trabeum Show Its Monomeric Structure and Reveal the Influence of Temperature on the Structural Stability of the Enzyme

Endoglucanases are key enzymes applied to the conversion of biomass aiming for second generation biofuel production. In the present study we obtained the small angle X-ray scattering (SAXS) structure of the G. trabeum endo-1,4-β-glucanase Cel12A and investigated the influence of an important parameter, temperature, on both secondary and tertiary structure of the enzyme and its activity. The CD analysis for GtCel12A revealed that changes in the CD spectra starts at 55 °C and the T_m calculated from the experimental CD sigmoid curve using the Boltzmann function was 60.2 ± 0.6 °C. SAXS data showed that GtCel12A forms monomers in solution and has an elongated form with a maximum diameter of 60 ± 5 Å and a gyration radius of 19.4 ± 0.1 Å as calculated from the distance distribution function. Kratky analysis revealed that 60 °C is the critical temperature above which we observed clear indications of denaturation. Our results showed the influence of temperature on the stability and activity of enzymes and revealed novel structural features of GtCel12A.     

Co-evolving stability and conformational homogeneity of the human adenosine A2a receptor

Structural studies on mammalian integral membrane proteins have long been hampered by their instability in detergent. This is particularly true for the agonist conformation of G protein-coupled receptors (GPCRs), where it is thought that the movement of helices that occurs upon agonist binding results in a looser and less stable packing in the protein. Here, we show that mutagenesis coupled to a specific selection strategy can be used to stabilize the agonist and antagonist conformations of the adenosine A_2a receptor. Of the 27 mutations identified that improve the thermostability of the agonist conformation, only three are also present in the 17 mutations identified that improve the thermostability of the antagonist conformation, suggesting that the selection strategies used were specific for each conformation. Combination of the stabilizing mutations for the antagonist- or agonist-binding conformations resulted in mutants that are more stable at higher temperatures than the wild-type receptor by 17°C and 9°C, respectively. The mutant receptors both showed markedly improved stability in short-chain alkyl-glucoside detergents compared with the wild-type receptor, which will facilitate their structural analysis.     

Vibrational vs. translational energy in promoting a prototype metal-hydrocarbon insertion reaction

The reaction Y + CH₄ → HYCH₃ → YCH₂ + H₂ is initiated by C–H insertion involving a 20 ± 3 kcal/mol potential energy barrier. The reaction is studied in crossed molecular beams under two different conditions with nearly the same total energy. One experiment is carried out at a collision energy of 15.1 kcal/mol with one quantum of CH₄ antisymmetric (ν₃) stretching vibrational excitation (8.63 kcal/mol), the other at a collision energy of 23.8 kcal/mol. The reaction cross-section for C–H stretch excited methane (σ_s) is found to be at least a factor of 2.2 times larger than for ground-state methane (σ_g) at the same total energy.     

Unusual kinetics of thermal decay of dim-light photoreceptors in vertebrate vision

We present measurements of rate constants for thermal-induced reactions of the 11-cis retinyl chromophore in vertebrate visual pigment rhodopsin, a process that produces noise and limits the sensitivity of vision in dim light. At temperatures of 52.0–64.6 °C, the rate constants fit well to an Arrhenius straight line with, however, an unexpectedly large activation energy of 114 ± 8 kcal/mol, which is much larger than the 60-kcal/mol photoactivation energy at 500 nm. Moreover, we obtain an unprecedentedly large prefactor of 10^72±5 s⁻¹, which is roughly 60 orders of magnitude larger than typical frequencies of molecular motions! At lower temperatures, the measured Arrhenius parameters become more normal: E_a = 22 ± 2 kcal/mol and A_pref = 10^9±1 s⁻¹ in the range of 37.0–44.5 °C. We present a theoretical framework and supporting calculations that attribute this unusual temperature-dependent kinetics of rhodopsin to a lowering of the reaction barrier at higher temperatures due to entropy-driven partial breakup of the rigid hydrogen-bonding network that hinders the reaction at lower temperatures.Rhodopsin is a vertebrate dim-light photoreceptor. Molecular studies of rhodopsin in recent decades have largely focused on its photochemistry and photoactivation (1–3). However, complete understanding of rhodopsin’s function requires characterization of its thermal properties because thermal isomerization of the 11-cis retinyl chromophore can trigger the same physiological response as photo-isomerization, generating false visual signals as dark noise that jeopardizes photosensitivity (4–6). To enhance dim-light vision, rhodopsin has evolved to acquire remarkable thermal stability with a half-life of 420 y as determined by electrophysiological experiments using the outer segments of rod cells at 36 °C (4). However, the molecular mechanism for the thermal stability has remained unclear. Here, we have addressed this by exploring the temperature dependence of thermal decay rate constants k(T) associated with isomerization of the retinyl chromophore and hydrolysis of the chromophore protonated Schiff-base (PSB) linkage, for temperatures ranging from the physiological temperature of 37.0 °C to 64.6 °C. In the upper range of temperatures from 52.0 °C to 64.6 °C, we find that the rate constants determined by UV-visible spectroscopy follow a linear Arrhenius model, k(T) = A_pref exp(−E_a/k_BT), where k_B is the Boltzmann constant (Fig. 1A). The slope, however, is very steep, giving an elevated activation energy, E_a = 114 ± 8 kcal/mol. Surprisingly, this value is much higher than the photoactivation energy at visible wavelengths (60 kcal/mol at 500 nm). E_a is also much higher than the reaction enthalpy change (32–35 kcal/mol) (7, 8) (Fig. 1B). In the lower temperature range of our rate constant measurements, 37.0–44.5 °C, the slope of the Arrhenius plot decreases abruptly, as shown in Fig. 1A. Fitting a straight line through the low temperature points produces an activation energy E_a = 22 ± 2 kcal/mol, although with only three data points, the precise value must be viewed with caution. In the upper temperature range of our measurements, the Arrhenius prefactor was found to be enormous, A_pref = 10^72±5 s⁻¹, which is many orders of magnitude larger than the 10¹²–10¹⁵ s⁻¹ timescale of molecular motions. In fact, the largest Arrhenius prefactor we have been able to find in the literature for a thermal unimolecular reaction is ∼10³⁸ s⁻¹ (9). By contrast, in the lower temperature range of our measurements, the prefactor was found to have a more typical value, estimated from the three data points to be about 10^9±1 s⁻¹. Similar activation energies can be inferred from Arrhenius plots previously obtained by Hubbard in 1958 (∼100 kcal/mol) (10) and by Janz and Farrens in 2004 (∼103 kcal/mol) (11). However, they did not explicitly report prefactors, and the origins of the large E_a and the sharp bending of the Arrhenius plot remained unexplored. Here, we present a model to describe the molecular origins of the observed rate parameters, including the extraordinarily large prefactor and dramatic inflection in the Arrhenius plot, and discuss the implications of this behavior to rhodopsin’s dim-light photoreceptor function. We tackle this puzzling phenomenon by combining the kinetic and thermodynamic analysis with theoretical and molecular modeling. The comparative analysis of the unusual kinetics observed at 52.0–64.6 °C to the more normal Arrhenius behavior observed at lower temperatures (T < 46 °C) provides insights into the potential role of hydrogen bonds (H-bonds) in the reaction mechanism.Open in a separate windowFig. 1.(A) Natural logarithm of our measured rate constants plotted vs. inverse temperature. Error bars represent 1 SD. Straight lines are fitted separately to the lower-temperature (blue dots) and upper-temperature (red dots) regions. (B) Schematic of energy surfaces for ground and excited electronic states of rhodopsin and decomposition of Arrhenius activation energy.     

Effect of Temperature on the Growth of Silver Nanoparticles Using Plasmon-Mediated Method under the Irradiation of Green LEDs

Plasmon-mediated shape conversion of spherical silver nanoparticles (NPs) to nanostructures with other shapes under the irradiation of green LEDs (520 ± 20 nm, 35 mw/cm²) at various temperatures (60, 40, 20, 10, 5, and 0 °C) was performed in this study. It was found that the bath temperature used in the reaction can influence the reaction rates, i.e., the times needed for the shape transformation process were 5, 11.5, 25, 45, 72, and 100 h at 60, 40, 20, 10, 5, and 0 °C, respectively. In addition, the bath temperature can also alter the morphologies of the final products. The major products are silver nanoplates at 60, 40 and 20 °C. However, they became decahedral silver NPs at 5 and 0 °C. The percentages of decahedral silver NPs synthesized at 60, 40, 20, 10, 5, and 0 °C are 0%, 1%, 5%, 45%, 73%, and 89%, respectively. Measuring the surface-enhanced Raman spectroscopy (SERS) spectra of the probe molecule R6G in the presence of KBr showed that both silver nanoplate colloids synthesized at 60 °C and decahedral silver NP colloids synthesized at 0 °C in the absence of PVP had good SERS activities.     

Low Activity of β-Galactosidase in Frameshift Mutants of Escherichia coli

16 lac frameshift mutants induced by an acridine derivative, ICR-191D, in E. coli are leaky for β-galactosidase activity. Activities of all mutants differ from each other and from the wild type in their stability to thermal denaturation. The leakiness is under ribosomal control, since it is strongly reduced by strA restrictive mutations and is restored by ram mutations that reverse restriction. Addition of streptomycin during growth has an effect similar to the presence of the ram mutation. These ribosomal alterations do not modify the thermal stability of the enzyme.     

Polydispersity vs. Monodispersity. How the Properties of Ni-Ag Core-Shell Nanoparticles Affect the Conductivity of Ink Coatings

The effect of polydispersity of nickel-silver core-shell nanoparticles (Ni-Ag NPs) on the conductivity of ink coatings was studied. Ni-Ag NPs of various average diameters (100, 220, and 420 nm) were synthesized and utilized for the preparation of conductive inks composed of monodisperse NPs and their polydisperse mixtures. The shell thickness of synthesized Ni-Ag NPs was found to be in the range of 10–20 nm and to provide stability of a core metal to oxidation for at least 6 months. The conductivity of metallic films formed by inks with monodisperse Ni-Ag NPs was compared with those formed by polydisperse inks. In all cases, the optimal conditions for the formation of conductive patterns (weight ratio of monodisperse NPs for polydisperse composition, the concentration of the wetting agent, sintering temperature, and duration) were determined. It was found that metallic films formed by polydisperse ink containing 100, 220, and 420 nm Ni-Ag NPs with a mass ratio of 1:1.5:0.5, respectively, are characterized by the lowest resistivity, 10.9 µΩ·cm, after their thermal post-coating sintering at 300 °C for 30 min that is only 1.6 higher than that of bulk nickel.     

Annealing Effect on the Characteristics of Co40Fe40W10B10 Thin Films on Si(100) Substrate

This research explores the behavior of Co₄₀Fe₄₀W₁₀B₁₀ when it is sputtered onto Si(100) substrates with a thickness (t_f) ranging from 10 nm to 100 nm, and then altered by an annealing process at temperatures of 200 °C, 250 °C, 300 °C, and 350 °C, respectively. The crystal structure and grain size of Co₄₀Fe₄₀W₁₀B₁₀ films with different thicknesses and annealing temperatures are observed and estimated by an X-ray diffractometer pattern (XRD) and full-width at half maximum (FWHM). The XRD of annealing Co₄₀Fe₄₀W₁₀B₁₀ films at 200 °C exhibited an amorphous status due to insufficient heating drive force. Moreover, the thicknesses and annealing temperatures of body-centered cubic (BCC) CoFe (110) peaks were detected when annealing at 250 °C with thicknesses ranging from 80 nm to 100 nm, annealing at 300 °C with thicknesses ranging from 50 nm to 100 nm, and annealing at 350 °C with thicknesses ranging from 10 nm to 100 nm. The FWHM of CoFe (110) decreased and the grain size increased when the thickness and annealing temperature increased. The CoFe (110) peak revealed magnetocrystalline anisotropy, which was related to strong low-frequency alternative-current magnetic susceptibility (χ_ac) and induced an increasing trend in saturation magnetization (Ms) as the thickness and annealing temperature increased. The contact angles of all Co₄₀Fe₄₀W₁₀B₁₀ films were less than 90°, indicating the hydrophilic nature of Co₄₀Fe₄₀W₁₀B₁₀ films. Furthermore, the surface energy of Co₄₀Fe₄₀W₁₀B₁₀ presented an increased trend as the thickness and annealing temperature increased. According to the results, the optimal conditions are a thickness of 100 nm and an annealing temperature of 350 °C, owing to high χ_ac, large Ms, and strong adhesion; this indicates that annealing Co₄₀Fe₄₀W₁₀B₁₀ at 350 °C and with a thickness of 100 nm exhibits good thermal stability and can become a free or pinned layer in a magnetic tunneling junction (MTJ) application.     

Structure, thermostability, and conformational flexibility of hen egg-white lysozyme dissolved in glycerol

Hen egg-white lysozyme dissolved in glycerol containing 1% water was studied by using CD and amide proton exchange monitored by two-dimensional ¹H NMR. The far- and near-UV CD spectra of the protein showed that the secondary and tertiary structures of lysozyme in glycerol were similar to those in water. Thermal melting of lysozyme in glycerol followed by CD spectral changes indicated unfolding of the tertiary structure with a T_m of 76.0 ± 0.2°C and no appreciable loss of the secondary structure up to 85°C. This is in contrast to the coincident denaturation of both tertiary and secondary structures with T_m values of 74.8 ± 0.4°C and 74.3 ± 0.7°C, respectively, under analogous conditions in water. Quenched amide proton exchange experiments revealed a greater structural protection of amide protons in glycerol than in water for a majority of the slowly exchanging protons. The results point to a highly ordered, native-like structure of lysozyme in glycerol, with the stability exceeding that in water.     

Structure and function in rhodopsin: correct folding and misfolding in point mutants at and in proximity to the site of the retinitis pigmentosa mutation Leu-125-->Arg in the transmembrane helix C.

L125R is a mutation in the transmembrane helix C of rhodopsin that is associated with autosomal dominant retinitis pigmentosa. To probe the orientation of the helix and its packing in the transmembrane domain, we have prepared and studied the mutations E122R, I123R, A124R, S127R, L125F, and L125A at, and in proximity to, the above mutation site. Like L125R, the opsin expressed in COS-1 cells from E122R did not bind 11-cis-retinal, whereas those from I123R and S127R formed the rhodopsin chromophore partially. A124R opsin formed the rhodopsin chromophore (lambda max 495 nm) in the dark, but the metarhodopsin II formed on illumination decayed about 6.5 times faster than that of the wild type and was defective in transducin activation. The mutant opsins from L125F and L125A bound 11-cis-retinal only partially, and in both cases, the mixtures of the proteins produced were separated into retinal-binding and non-retinal-binding (misfolded) fractions. The purified mutant rhodopsin from L125F showed lambda max at 500 nm, whereas that from L125A showed lambda max at 503 nm. The mutant rhodopsin L125F showed abnormal bleaching behavior and both mutants on illumination showed destabilized metarhodopsin II species and reduced transducin activation. Because previous results have indicated that misfolding in rhodopsin is due to the formation of a disulfide bond other than the normal disulfide bond between Cys-110 and Cys-187 in the intradiscal domain, we conclude from the misfolding in mutants L125F and L125A that the folding in vivo in the transmembrane domain is coupled to that in the intradiscal domain.