Pressure-tuning FT-IR spectroscopic study on the helix–coil transition of Ala-rich oligopeptide in aqueous solution

We investigated the effect of pressure on the helix–coil transition of an Ala-rich peptide (AK16: YGAAKAAAAKAAAAKA-NH₂) in aqueous solution by FT-IR spectroscopy. The spectra of the amide I' region of AK16 in aqueous solution was decomposed into some component bands using a curve fitting method. The peak at around 1635 cm ^?1 corresponding to the solvent exposed α-helix conformer increases with increasing pressures, while the peak at around 1655 cm ^?1 corresponding to the random coil conformer decreases. From the pressure dependence of the band intensities, we determined the volume change from the α-helix to random coil conformers of AK16 to be + 10.5 ± 0.3 cm³/mol. The positive volume change is different from the negative volume change generally observed in the pressure denaturation of proteins.     

Pressure-dependent changes in the structure of the melittin α-helix determined by NMR

A novel method is described, which uses changes in NMR chemical shifts to characterise the structural change in a protein with pressure. Melittin in methanol is a small -helical protein, and its chemical shifts change linearly and reversibly with pressure between 1 and 2000 bar. An improved relationship between structure and HN shift has been calculated, and used to drive a molecular dynamics-based calculation of the change in structure. With pressure, the helix is compressed, with the H—O distance of the NH—O=C hydrogen bonds decreased by 0.021 ± 0.039 Å, leading to an overall compression along the entire helix of about 0.4 Å, corresponding to a static compressibility of 6 ×10^–6 bar^–1. The backbone dihedral angles and are altered by no more than ± 3° for most residues with a negative correlation coefficient of –0.85 between _i and _i–1, indicating that the local conformation alters to maintain hydrogen bonds in good geometries. The method is shown to be capable of calculating structural change with high precision, and the results agree with structural changes determined using other methodologies.     

Redesign of the monomer–monomer interface of Cre recombinase yields an obligate heterotetrameric complex

Cre recombinase catalyzes the cleavage and religation of DNA at loxP sites. The enzyme is a homotetramer in its functional state, and the symmetry of the protein complex enforces a pseudo-palindromic symmetry upon the loxP sequence. The Cre-lox system is a powerful tool for many researchers. However, broader application of the system is limited by the fixed sequence preferences of Cre, which are determined by both the direct DNA contacts and the homotetrameric arrangement of the Cre monomers. As a first step toward achieving recombination at arbitrary asymmetric target sites, we have broken the symmetry of the Cre tetramer assembly. Using a combination of computational and rational protein design, we have engineered an alternative interface between Cre monomers that is functional yet incompatible with the wild-type interface. Wild-type and engineered interface halves can be mixed to create two distinct Cre mutants, neither of which are functional in isolation, but which can form an active heterotetramer when combined. When these distinct mutants possess different DNA specificities, control over complex assembly directly discourages recombination at unwanted half-site combinations, enhancing the specificity of asymmetric site recombination. The engineered Cre mutants exhibit this assembly pattern in a variety of contexts, including mammalian cells.     

Backbone dynamics of an alamethicin in methanol and aqueous detergent solution determined by heteronuclear 1H−15N NMR spectroscopy

Summary The ¹⁵N relaxation rates of the -aminoisobutyric acid (Aib)-rich peptide alamethicin dissolved in methanol at 27°C and 5°C, and dissolved in aqueous sodium dodecylsulfate (SDS) at 27°C, were measured using inverse-detected one-and two-dimensional ¹H–¹⁵N NMR spectroscopy. Measurements of ¹⁵N longitudinal (R_N(N_z)) and transverse (R_N(N_x,y)) relaxation rates and the {¹H} ¹⁵N nuclear Overhauser enhancement (NOE) at 11.7 Tesla were used to calculate (quasi-) spectral density values at 0, 50, and 450 MHz for the peptide in methanol and in SDS. Spectral density mapping at 0, 50, 450, 500, and 550 MHz was done using additional measurements of the ¹H–¹⁵N lingitudinal two-spin order, R_NH(2H _infZ ^supN N_Z), two-spin antiphase coherence, R_NH(2H _infN ^supZ N_x,y), and the proton longitudinal relaxation rate, R_H(H _infN ^supZ ), for the peptide dissolved in methanol only. The spectral density of motions was also modeled using the three-parameter Lipari-Szabo function. The overall rotational correlation times were determined to be 1.1, 2.5, and 5.7 ns for alamethicin in methanol at 27°C and 5°C, and in SDS at 27°C, respectively. From the rotational correlation time determined in SDS the number of detergent molecules associated with the peptide was estimated to be about 40. The average order parameter was about 0.7 and the internal correlation times were about 70 ps for the majority of backbone amide ¹⁵N sites of alamethicin in methanol and in SDS. The relaxation data, spectral densities, and order parameters suggest that the peptide N-H vectors of alamethicin are not as highly constrained as the core regions of folded globular proteins. However, the peptide backbone is clearly not as mobile as the most unconstrained regions of folded proteins, such as those found in the frayed C-and N-termini of some proteins, or in randomcoil peptides. The data also suggest significant mobility at both ends of the peptide dissolved in methanol. In SDS the mobility in the middle and at the ends of the peptide is reduced. The implications of the results with respect to the sterically hindered Aib residues and the biological activities of the peptide are discussed.To whom correspondence should be addressed.     

Cell-based and in-silico studies on the high intrinsic activity of two boron-containing salbutamol derivatives at the human β₂-adrenoceptor

Salbutamol is a well-known β(2) adrenoceptor (β(2)AR) partial agonist. We synthesized two boron-containing salbutamol derivatives (BCSDs) with greater potency and efficacy, compared to salbutamol, for inducing β(2)AR-mediated smooth-muscle relaxation in guinea-pig tracheal rings. However, the mechanism involved in this pharmacological effect remains unclear. In order to gain insight, we carried out binding and functional assays for BCSDs in HEK-293T cells transfected with the human β(2)AR (hβ(2)AR). The transfected hβ(2)AR showed similar affinity for BCSDs and salbutamol, but adenosine 3',5'-cyclic phosphate (cAMP) accumulation induced by both BCSDs was similar to that elicited by isoproterenol and greater than that induced by salbutamol. The boron-containing precursors (boric and phenylboronic acids, 100 μM) had no significant effect on salbutamol binding or salbutamol-induced cAMP accumulation. These experimental results are in agreement with theoretical docking simulations on lipid bilayer membrane-embedded hβ(2)AR structures. These receptors showed slightly higher affinity for BCSDs than for salbutamol. An essential change between putative active and inactive conformational states depended on the interaction of the tested ligands with the fifth, sixth and seventh transmembrane domains. Overall, these data suggest that BCSDs induce and stabilize conformational states of the hβ(2)AR that are highly capable of stimulating cAMP production.     

The effects of γ-rays on poly-L-glutamic acid in aqueous solution

Summary Salt-free and 0.2 M NaCl oxygenated aqueous solutions of poly-L-glutamic acid were irradiated with⁶⁰Co--radiation at variouspH's to examine whether or not the changes caused by the exposure to ionizing radiation depend onpH, that is, the conformations of polypeptide.TheG-values (the number of main-chain scissions per 100 eV of energy absorbed) in both salt-free and 0.2 M NaCl solutions of poly-L-glutamic acid were found to change sharply withpH. and to have a maximum value at thepH of a mid-point of helix-coil transition. The change ofG-values withpH was discussed in terms of the conformational change of poly-L-glutamic acid.     

The influence of β-cyclodextrin on acid-base and tautomeric equilibrium of fluorescein dyes in aqueous solution

The protolytic equilibrium of fluorescein in aqueous solutions was studied in the presence of cycloheptaamylose (β-cyclodextrin, or β-CD). The constants of stepwise ionization of the dye (), K_a0, K_a1, and K_a2 were determined using vis-spectroscopy at ionic strength 0.05 M (NaCl + buffer) and 25 °C. In the presence of 0.0086 M β-CD, the indices of ionization constants are as follows: pK_a0 = 1.21 ± 0.12, pK_a1 = 5.08 ± 0.03, pK_a2 = 6.35 ± 0.02. The changes in these pK_as, as compared with the values determined without cyclodextrin, are unequal. Namely, the pK_a0 value decreases by 1.0, while the pK_a1 value increases by 0.7. Thus, the introduction of β-CD allows to govern the ratios K_a0/K_a1 and K_a1/K_a2, which are equal to, respectively, 141 and 151 in water, and 7.4 × 10³ and 18.6 with cyclodextrin added. Rationalization of the observed phenomenon is possible taking into account the detailed scheme of protolytic equilibrium. Conclusions concerning tautomerism of dye molecules were deduced from absorption spectra; the fractions of tautomers, tautomerization constants, and microscopic ionization constants were evaluated. These data allow concluding that the main reason for the aforementioned pK_a alterations is the binding of H₂R by the cyclodextrin cavity accompanied by turning these neutral species into the colorless lactone. The host-guest interaction of neutral species of fluorescein isothiocyanate, 2,7-dichlorofluorescein, and 3′,4′,5′,6′-tetrachlorofluorescein also results in the cyclodextrin-assisted shift of tautomeric equilibrium. Such nature of interactions is proved by the addition of competing agents, camphor-4-carboxylic acid and sodium n-nonylsulfonate, which results in the removing of neutral dye species from the cycloheptaamylose cavity.     

Influence of multiple action–outcome associations on the transition dynamics toward an optimal choice in rats

When faced with familiar versus novel options, animals may exploit the acquired action–outcome associations or attempt to form new associations. Little is known about which factors determine the strategy of choice behavior in partially comprehended environments. Here we examine the influence of multiple action–outcome associations on choice behavior in the context of rewarding outcomes (food) and aversive outcomes (electric foot-shock). We used a nose-poke paradigm with rats, incorporating a dilemma between a familiar option and a novel, higher-value option. In Experiment 1, two groups of rats were trained with different outcome schedules: either a single action–outcome association (“Reward-Only”) or dual action–outcome associations (“Reward-Shock”; with the added opportunity to avoid an electric foot-shock). In Experiment 2, we employed the same paradigm with two groups of rats performing the task under dual action–outcome associations, with different levels of threat (a low- or high-amplitude electric foot-shock). The choice behavior was clearly influenced by the action–outcome associations, with more efficient transition dynamics to the optimal choice with dual rather than single action–outcome associations. The level of threat did not affect the transition dynamics. Taken together, the data suggested that the strategy of choice behavior was modulated by the information complexity of the environment.     

A computational analysis of ordering in ABCHN at the nematic–isotropic transition

A computational analysis of ordering in the nematogenic compound 4-alkenyl bicyclohexylnitrile has been carried out based on quantum mechanics and intermolecular forces. The evaluation of atomic charge and dipole moment at each atomic center has been carried out using the complete neglect differential overlap (CNDO/2) method. Modified Rayleigh–Schrodinger perturbation theory along with a multicentered- multipole expansion method has been employed to evaluate long-range intermolecular interactions, while a ‘6-exp‘ potential function has been assumed for short-range interactions. The total interaction energy values obtained through these computations were used to calculate the probability of each configuration at room temperature (300 K), the nematic–isotropic transition temperature (364.7 K) and above transition temperature (450 K) using the Maxwell–Boltzmann formula. The various possible configurations during the different modes (i.e., stacking, in-plane and terminal) of interactions have been studied in terms of variation of probability due to small departures from the most probable configurations. An attempt has been made to analyze the characteristic features of liquid crystallinity in terms of their relative order with molecular parameters introduced in this paper.     

Disproportionately high N-mineralisation rates from green manures at low temperatures – implications for modeling and management in cool temperate agro-ecosystems

We examined the decomposition of Medicago lupulina, Melilotus alba and Poa pratensis at 3, 9, and 25 °C during 4 weeks. There was a strong temperature effect on the rate of CO₂ evolution, and thus the extent of energy exhaustion from the added substrates. However, there was no concomitant retardation of N mineralisation at low temperatures. In the analysis of variance of mineralized N the residue type gave a 10 times larger contribution to the regression than the temperature (T), whereas for CO₂ evolution residue type and temperature were equally important contributors. This indicates that although the temperature has a statistically significant effect on N-mineralisation it is substantially less than compared with the effect on carbon mineralisation in the materials examined. The retardation of carbon mineralisation was least strong in Melilotus alba that had a relatively low cellulose content, and a higher content of low molecular compounds. Though more research will be necessary to consolidate and explain this phenomena, it is likely that an important factor is a decrease in the bioavailability of C-rich polymers at low temperatures, and thus a preferential utilization of N-rich low molecular substances. Nitrification was not effectively deterred at 3 °C. Thus, in terms of management, it is pertinent to reconsider the timing of green manure and catch crop incorporation in cool temperate climate regions, since the rapid release of nitrogen, coupled with the relatively undeterred nitrification may result in a high N leaching risk by early incorporation, but a low risk for N immobilization at late incorporation, if N rich residues are used.     

The effect of ”living high–training low” on physical performance in rats

In this research, we hypothesized that, in rats, adaptation to high altitude (2500 m) plus training at low altitude (610 m), ”living high–training low”, improves physical performance at low altitude more than living and training at low altitude (610 m). Rats were divided into four groups: (1) living at low altitude (LL, n=12), (2) living and training at low altitude (LLTL, n=13), (3) living at high altitude (LH, n=12), (4) living at high altitude and training at low altitude (LHTL, n=13). The program for living at high altitude involved raising rats under hypobaric hypoxia (equivalent to 2500 m), and the training program consisted of running on a tread-mill at low altitude. All groups were raised at each altitude and trained to run at 35 m/min for 40 min/day, 6 days/week for 6 weeks. During this program, we measured heart rates both at rest and during exercise, and performed running-time trials. The mean heart rate during exercise was lower in groups with training than in groups without training, and the groups receiving training could run longer than the untrained groups. The LHTL group especially showed the lowest mean heart rate during exercise and the longest running time among all groups. After 6 weeks of the training program, all rats had a catheter implanted into the carotid artery, and the mean systemic arterial pressure was continuously measured during treadmill running. The rate of increase of this pressure as the running intensity increased was lower in groups with training than in groups without training, especially in the LHTL group. Finally, we anesthetized all the rats and extracted both the right and left ventricles, and the triceps surae and liver. Training increased the weight of the left ventricle, triceps surae, and liver. The increase in weight of the left ventricle and triceps surae was higher in the LHTL group than in the LLTL group in particular. It appeared that living high– training low may be an effective strategy to improve performance ability at low altitude. Received: 16 July 1999 / Revised: 24 January 2000 / Accepted: 25 January 2000     

NMR spin–echo studies of hydration properties of the molecular chaperone α-crystallin in the bovine lens

The water-binding properties of bovine lens α-crystallin, collagen from calf skin and bovine serum albumin (BSA), were investigated with various techniques. The water absorptive capacity was obtained in high vacuum desorption experiments volumetrically, and also gravimetrically in controlled atmosphere experiments. NMR spin–echo technique was used to study the hydration of protein samples and to determine the spin–spin relaxation times (T₂) from the protons of water, absorbed on the proteins. Isolated bovine lenses were sectioned into 11–12 morphological layers (from anterior cortex through nucleus to posterior cortex). Crystallin profiles were obtained for each lens layer using thin-layer isoelectric focusing in polyacrylamide gel (IEF). The water content in relation to dry weight of proteins was measured in individual morphological lens layers. During the water vapor uptake P/P₀=0.75, α-crystallin did not absorb water, suggesting that hydrophobic regions of the protein are exposed to the aqueous solvent. At P/P₀=1.0, the absorption of water by α-crystallin was 17% with a single component decay character of spin–echo (T₂=3 ms). Addition of water to α-crystallin to about 50% of its w/w in the protein sample showed T₂=8 ms with only one single component decay of the spin–echo signal. The single component decay character of the spin–echo indicates at the tightly bound water by α-crystallin. Under a relative humidity P/P₀=1.0, collagen and BSA absorbed correspondingly 19.3% and 28% of water and showed a two-component decay curve with T₂ of about 5 and 40 ms. The findings demonstrate the presence of two water fractions in collagen and BSA which are separated in space. The IEF data suggest a tight binding of water with α-crystallin with similar distribution patterns in the lens layers. The IEF data demonstrate a possible chaperone-like function for α-crystallin in the nucleus and inner cortex of the lens, but not in the outer cortex. To conclude, it was found that α-crystallin can immobilize and bind water to a greater extent than other proteins such as collagen and BSA. These results shed new light on structural properties of α-crystallin and have important implications for understanding the mechanism of the chaperone-like action of this protein in the lens and non-ocular tissues.     

Review of NMR and μSR studies in the molecular nanomagnet Mn12-ac

We present a comprehensive review of the NMR and μSR studies performed in the molecular nanomagnet Mn12 a system characterized and widely studied by Prof. Gatteschi’s group in Florence. The proton (¹H, ²D) NMR, the ⁵⁵Mn NMR and the μSR investigations have yielded important information regarding both static and dynamic magnetic properties of the molecule. The magnetic and quadrupole hyperfine interactions have been extracted from NMR data. The spin dynamics at high and intermediate temperature associated with the zero dimensionality and with the spin–phonon coupling has been studied together with the spin dynamics in the quantum tunneling low temperature regime. The local spin configuration in the giant S = 10 total spin ground state has been determined via ⁵⁵Mn NMR in zero magnetic field and with fields parallel and perpendicular to the anisotropy axis. Finally a novel method is described to monitor the relaxation of the magnetization from the time evolution of the NMR spectrum.     

Absorption and linear dichroism spectroscopy at room and low temperatures of single crystals of the B800–850 light-harvesting complex from Rhodopseudomonas acidophila strain 10050

The absorbance, polarized absorbance and linear dichroism spectra of single crystals of the B800–850 light-harvesting complex from Rhodopseudomonas acidophila strain 10050 taken at room (298 K) and low (85 K) temperatures are presented. The spectra are compared and contrasted with random phase solution spectra from the same complex. The single crystal spectra display a spectral narrowing at low temperatures in the BChl Q_x (550–650 nm) and carotenoid (450–550 nm) regions similar to that observed from the random phase solution. The single crystal absorption spectra in the BChl Q_y (750–900 nm) region are broader than the solution spectra and remain broad as the temperature is lowered. It is suggested that this broadening is the result of specific exciton interactions between the BChl chromophore Q_y transition dipoles and is a molecular feature which occurs only in the crystalline complex.     

Epithelial–mesenchymal transition in the formation of hypertrophic scars and keloids

Abnormal wound healing is likely to induce the formation of hypertrophic scars and keloids, which leads to dysfunction, deformity, and mental problem in the patients. Despite the advances in prevention and management of hypertrophic scar and keloids, the mechanism underlying scar and keloid formation has not been fully elucidated. Recent insights into the role of the epithelial–mesenchymal transition (EMT) in development, wound healing, stem cell regulation, fibrosis, and tumorigenesis have increased our understanding of the pathophysiology of hypertrophic scarring and keloids and suggested new therapeutic targets. This review summarizes recent progress in the elucidation of the role of EMT in physiologic wound healing and pathologic scar formation. This knowledge will facilitate an understanding of EMT roles in scar formation and shed new light on the modulation and potential treatment of hypertrophic scars and keloids.     

Ordering of p–n-alkoxybenzoic acids at phase transition temperatures: a comparative computational analysis

A comparative analysis of molecular ordering of nematogenic p-n-alkoxybenzoic acids has been carried out with respect to translatory and orientational motions for the acids with seven (7OBAC), eight (8OBAC), nine (9OBAC) and 10 (10OBAC) carbon atoms in the alkyl chain. The CNDO/2 method has been used to compute the net atomic charge and dipole moment components at each atomic center. Modified Rayleigh-Schrodinger perturbation theory with multicentered-multipole expansion method has been used to evaluate long-range intermolecular interactions while a '6-exp' potential function has been assumed for short-range interactions. The total interaction-energy values obtained by these computations were used to calculate the probability of each configuration at the phase-transition temperature using the Maxwell-Boltzmann formula. The flexibility of various configurations has been studied in terms of variation of probability due to small departures from the most probable configuration. A comparative picture of molecular parameters like total energy, binding energy and total dipole moment has been given. An attempt has been made to explain the nematogenicity of these acids in terms of their relative order with the molecular parameter introduced in this paper.