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.     

Lipopolysaccharide bound structures of the active fragments of fowlicidin-1, a cathelicidin family of antimicrobial and antiendotoxic peptide from chicken, determined by transferred nuclear Overhauser effect spectroscopy

Cathelicidins comprise a major family of host-defense antimicrobial peptides in vertebrates. The C-terminal part of the cathelicidins is bestowed with antimicrobial and lipopolysaccharide (LPS) neutralizing activities. In this work, we repot high resolution solution structures of two nontoxic active fragments, residues 1-16 or RG16 and residues 8-26 or LK19, of fowlicidin-1, a cathelicidin family of peptide from chicken, as a complex with LPS using two-dimensional transferred nuclear Overhauser effect (Tr-NOE) spectroscopy. Both peptides are highly flexible and do not assume any preferred conformations in their free states. Upon complexation with endotoxin or LPS, peptides undergo structural transitions towards folded conformations. Structure calculations reveal that the LK19 peptide adopts a well defined helical structure with a bend at the middle. By contrast, the first seven amino acids of RG16 are found to be flexible followed by a helical conformation for the residues L8-A15. In addition, a truncated version of LK19 encompassing residues A15-K26 or AK12 displays an amphipathic helical structure in LPS. Saturation transfer difference (STD) NMR studies demonstrate that all peptides, RG16, LK19, and AK12, are in close proximity with LPS, whereby the aromatic residues showed the strongest STD effects. Fluorescence studies with fluorescein isothiocyanate (FITC) labeled LPS in the presence of full-length fowlicidin-1, LK19, RG16, and AK12 indicated that LPS-neutralization property of these peptides may result from plausible dissociation of LPS aggregates. The helical structures of peptide fragments derived from fowlicidin-1 in LPS could be utilized to develop nontoxic antiendotoxic compounds.     

High-pressure NMR spectroscopy for characterizing folding intermediates and denatured states of proteins

Extensive structural studies using high-pressure NMR spectroscopy have recently been carried out on proteins, which potentially contribute to our understanding of the mechanisms of protein folding. Pressure shifts the conformational equilibrium from higher to lower volume conformers. If the pressure is varied, starting from the folded native structure, in many cases we observe intermediate conformers before the onset of total unfolding. This enables the investigation of details of the structure and thermodynamic characteristics of various intermediate conformers of proteins under equilibrium conditions. We can also examine pressure effects on the structure and stability of some typical denatured states such as helical denatured, molten globule, and unfolded states. The high-pressure NMR method can also be used to investigate association/dissociation equilibria of oligomeric or aggregated proteins. Beside direct observation of kinetic intermediates upon pressure jump, NMR structural investigations of equilibrium conformers under pressure provide information about the structures of kinetic intermediates during folding/unfolding reactions.     

Deceleration of Arginine Kinase Refolding by Induced Helical Structures

Arginine kinase (AK) is a key metabolic enzyme for keeping energy balance in invertebrates. Therefore, regulation of the enzymatic activity and the folding studies of AK from the various invertebrates have been the focus of investigation. We studied the effects of helical structures by using hexafluoroisopropanol (HFIP) on AK folding. Folding kinetic studies showed that the folding rates of the urea-denatured AKs were significantly decelerated after being induced in various concentrations of HFIP. AK lost its activity completely at concentrations greater than 60%. The results indicated that the HFIP-induced helical structures in the denatured state play a negative role in protein folding, and the helical structures induced in 5% (v/v) HFIP act as the most effective barrier against AK taking its native structure. The computational docking simulations (binding energies for −2.19 kcal/mol for AutoDock4.2 and −20.47 kcal/mol for Dock6.3) suggested that HFIP interacts with the several important residues that are predicted by both programs. The excessively pre-organized helical structures not only hampered the folding process, but also ultimately brought about changes in the three-dimensional conformation and biological function of AK.     

Fast collapse but slow formation of secondary structure elements in the refolding transition of E. coli adenylate kinase

The various models proposed for protein folding transition differ in their order of appearance of the basic steps during this process. In this study, steady state and time-resolved dynamic non-radiative excitation energy transfer (FRET and trFRET) combined with site specific labeling experiments were applied in order to characterize the initial transient ensemble of Escherichia coli adenylate kinase (AK) molecules upon shifting conditions from those favoring denaturation to refolding and from folding to denaturing. Three sets of labeled AK mutants were prepared, which were designed to probe the equilibrium and transient distributions of intramolecular segmental end-to-end distances. A 176 residue section (residues 28-203), which spans most of the 214 residue molecule, and two short secondary structure chain segments including an alpha-helix (residues 169-188) and a predominantly beta-strand region (residues 188-203), were labeled. Upon fast change of conditions from denaturing to folding, the end-to-end distance of the 176 residue chain section showed an immediate collapse to a mean value of 26 A. Under the same conditions, the two short secondary structure elements did not respond to this shift within the first ten milliseconds, and retained the characteristics of a fully unfolded state. Within the first 10 ms after changes of the solvent from folding to denaturing, only minor changes were observed at the local environments of residues 203 and 169. The response of these same local environments to the shift of conditions from denaturing to folding occurred within the dead time of the mixing device. Thus, the response of the CORE domain of AK to fast transfer from folding to unfolding conditions is slow at all three conformational levels that were probed, and for at least a few milliseconds the ensemble of folded molecules is maintained under unfolding conditions. A different order of the changes was observed upon initiation of refolding. The AK molecules undergo fast collapse to an ensemble of compact structures where the local environment of surface probes seems to be native-like but the two labeled secondary structure elements remain unfolded.     

Home Range,Body Condition,and Survival of Rehabilitated Raccoons (Procyon lotor) During Their First Winter

The effects of raccoon (Procyon lotor) rehabilitation on postrelease survivorship are unknown. Raccoon rehabilitation success was measured as differences in prewinter body condition, home range size, distance to manmade structures, and during-winter survival between raccoons in the wild and those who have been rehabilitated. Prewinter body condition did not differ between wild and rehabilitated raccoons, but there was a trend for rehabilitated raccoons to have better body conditions. There was no difference between wild and rehabilitated raccoon adaptive kernel (AK) home range for 95% and 90% AK home ranges, or for core (50% AK) use areas. There was no sex difference in distance traveled from the release site within rehabilitated raccoons. However, rehabilitated raccoons were found significantly closer (49.4 ± 4.7 m) to manmade structures than wild raccoons (92.2 ± 14.4 m), and female raccoons were found significantly closer (64.8 ± 4.5 m) to manmade structures than male raccoons (72.3 ± 17.6 m). The results of this study indicate that raccoons can be successfully rehabilitated, but they may occupy habitat closer to manmade structures than wild raccoons.     

Pressure-jump studies of the folding/unfolding of trp repressor.

The dimeric protein, trp apo-repressor of Escherichia coli has been subjected to high hydrostatic pressure under a variety of conditions, and the effects have been monitored by fluorescence spectroscopic and infra-red absorption techniques. Under conditions of micromolar protein concentration and low, non-denaturing concentrations of guanidinium hydrochloride (GuHCl), tryptophan and 8-anilino-1-naphthalene sulfonate (ANS) fluorescence detected high pressure profiles demonstrate that pressures below 3 kbar result in dissociation of the dimer to a monomeric species that presents no hydrophobic binding sites for ANS. The FTIR-detected high pressure profile obtained under significantly different solution conditions (30 mM trp repressor in absence of denaturant) exhibits a much smaller pressure dependence than the fluorescence detected profiles. The pressure-denatured form obtained under the FTIR conditions retains about 50 % alpha-helical structure. From this we conclude that the secondary structure present in the high pressure state achieved under the conditions of the fluorescence experiments is at least as disrupted as that achieved under FTIR conditions. Fluorescence-detected pressure-jump relaxation studies in the presence of non-denaturing concentrations of GuHCl reveal a positive activation volume for the association/folding reaction and a negative activation volume for dissociation/unfolding reaction, implicating dehydration as the rate-limiting step for association/folding and hydration as the rate-limiting step for unfolding. The GuHCl concentration dependence of the kinetic parameters place the transition state at least half-way along the reaction coordinate between the unfolded and folded states. The temperature dependence of the pressure-jump fluorescence-detected dissociation/unfolding reaction in the presence of non-denaturing GuHCl suggests that the curvature in the temperature dependence of the stability arises from non-Arrhenius behavior of the folding rate constant, consistent with a large decrease in heat capacity upon formation of the transition state from the unfolded state. The decrease in the equilibrium volume change for folding with increasing temperature (due to differences in thermal expansivity of the folded and unfolded states) arises from a decrease in the absolute value for the activation volume for unfolding, thus indicating that the thermal expansivity of the transition state is similar to that of the unfolded state.     

Unique Features of the Folding Landscape of a Repeat Protein Revealed by Pressure Perturbation

The volumetric properties of proteins yield information about the changes in packing and hydration between various states along the folding reaction coordinate and are also intimately linked to the energetics and dynamics of these conformations. These volumetric characteristics can be accessed via pressure perturbation methods. In this work, we report high-pressure unfolding studies of the ankyrin domain of the Notch receptor (Nank1-7) using fluorescence, small-angle x-ray scattering, and Fourier transform infrared spectroscopy. Both equilibrium and pressure-jump kinetic fluorescence experiments were consistent with a simple two-state folding/unfolding transition under pressure, with a rather small volume change for unfolding compared to proteins of similar molecular weight. High-pressure fluorescence, Fourier transform infrared spectroscopy, and small-angle x-ray scattering measurements revealed that increasing urea over a very small range leads to a more expanded pressure unfolded state with a significant decrease in helical content. These observations underscore the conformational diversity of the unfolded-state basin. The temperature dependence of pressure-jump fluorescence relaxation measurements demonstrated that at low temperatures, the folding transition state ensemble (TSE) lies close in volume to the folded state, consistent with significant dehydration at the barrier. In contrast, the thermal expansivity of the TSE was found to be equivalent to that of the unfolded state, indicating that the interactions that constrain the folded-state thermal expansivity have not been established at the folding barrier. This behavior reveals a high degree of plasticity of the TSE of Nank1-7.     

NMR studies of the backbone flexibility and structure of human growth hormone: a comparison of high and low pH conformations

(15)N NMR relaxation parameters and amide (1)H/(2)H-exchange rates have been used to characterize the structural flexibility of human growth hormone (rhGH) at neutral and acidic pH. Our results show that the rigidity of the molecule is strongly affected by the solution conditions. At pH 7.0 the backbone dynamics parameters of rhGH are uniform along the polypeptide chain and their values are similar to those of other folded proteins. In contrast, at pH 2.7 the overall backbone flexibility increases substantially compared to neutral pH and the average order parameter approaches the lower limit expected for a folded protein. However, a significant variation of the backbone dynamics through the molecule indicates that under acidic conditions the mobility of the residues becomes more dependent on their location within the secondary structure units. In particular, the order parameters of certain loop regions decrease dramatically and become comparable to those found in unfolded proteins. Furthermore, the HN-exchange rates at low pH reveal that the residues most protected from exchange are clustered at one end of the helical bundle, forming a stable nucleus. We suggest that this nucleus maintains the overall fold of the protein under destabilizing conditions. We therefore conclude that the acid state of rhGH consists of a structurally conserved, but dynamically more flexible helical core surrounded by an aura of highly mobile, unstructured loops. However, in spite of its prominent flexibility the acid state of rhGH cannot be considered a "molten globule" state because of its high stability. It appears from our work that under certain conditions, a protein can tolerate a considerable increase in flexibility of its backbone, along with an increased penetration of water into its core, while still maintaining a stable folded conformation.     

Order through disorder: hyper-mobile C-terminal residues stabilize the folded state of a helical peptide. a molecular dynamics study

Conventional wisdom has it that the presence of disordered regions in the three-dimensional structures of polypeptides not only does not contribute significantly to the thermodynamic stability of their folded state, but, on the contrary, that the presence of disorder leads to a decrease of the corresponding proteins' stability. We have performed extensive 3.4 μs long folding simulations (in explicit solvent and with full electrostatics) of an undecamer peptide of experimentally known helical structure, both with and without its disordered (four residue long) C-terminal tail. Our simulations clearly indicate that the presence of the apparently disordered (in structural terms) C-terminal tail, increases the thermodynamic stability of the peptide's folded (helical) state. These results show that at least for the case of relatively short peptides, the interplay between thermodynamic stability and the apparent structural stability can be rather subtle, with even disordered regions contributing significantly to the stability of the folded state. Our results have clear implications for the understanding of peptide energetics and the design of foldable peptides.     

Alkaline unfolding and salt-induced folding of arginine kinase from shrimp Feneropenaeus chinensis under high pH conditions

The structural and functional properties of arginine kinase (AK) in alkaline conditions in the absence or presence of salt have been investigated. The conformational changes of AK during alkaline unfolding and salt-induced folding at alkaline pH were monitored using intrinsic fluorescence emission, binding of the fluorescence probe 1-anilino-8-naphthalenesulfonate and circular dichroism. The results for the alkaline unfolded enzyme showed that much lower pH (11.0) was required to cause the complete loss of AK activity than was required to cause an obvious conformational change of the enzyme. Compared with the completely unfolded state in 5 M urea, the high pH denatured enzyme had some residual secondary and tertiary structure even at pH 13.0. Increasing the ionic strength by adding salt at pH 12.75 resulted in the formation of a relatively compact tertiary structure and a little new secondary structure with hydrophobic surface enhancement. These results indicate that the partially folded state formed under alkaline conditions may have similarities to the molten globule state which is compact, but it has a poorly defined tertiary structure and a native-like secondary structure.     

Numerical simulation of wave propagation in a realistic model of the human external ear

In this study, a numerical investigation is performed to evaluate the effects of high-pressure sinusoidal and blast wave's propagation around and inside of a human external ear. A series of computed tomography images are used to reconstruct a realistic three-dimensional (3D) model of a human ear canal and the auricle. The airflow field is then computed by solving the governing differential equations in the time domain using a computational fluid dynamics software. An unsteady algorithm is used to obtain the high-pressure wave propagation throughout the ear canal which is validated against the available analytical and numerical data in literature. The effects of frequency, wave shape, and the auricle on pressure distribution are then evaluated and discussed. The results clearly indicate that the frequency plays a key role on pressure distribution within the ear canal. At 4 kHz frequency, the pressure magnitude is much more amplified within the ear canal than the frequencies of 2 and 6 kHz, for the incident wave angle of 90° investigated in this study, attributable to the ‘4-kHz notch’ in patients with noise-induced hearing loss. According to the results, the pressure distribution patterns at the ear canal are very similar for both sinusoidal pressure waveform with the frequency of 2 kHz and blast wave. The ratio of the peak pressure value at the eardrum to that at the canal entrance increases from about 8% to 30% as the peak pressure value of the blast wave increases from 5 to 100 kPa for the incident wave angle of 90° investigated in this study. Furthermore, incorporation of the auricle to the ear canal model is associated with centerline pressure magnitudes of about 50% and 7% more than those of the ear canal model without the auricle throughout the ear canal for sinusoidal and blast waves, respectively, without any significant effect on pressure distribution pattern along the ear canal for the incident wave angle of 90° investigated in this study.     

A model for the 77 K excited state dynamics in Chlamydomonas reinhardtii in state 1 and state 2

The regulatory mechanism of state transitions was studied in Chlamydomonas reinhardtii (C.r.) wild type (WT) as well as mutant strains deficient in the photosystem I (PSI) or the photosystem II (PSII) core. Time-resolved fluorescence measurements were obtained on instantly frozen cells incubated beforehand in the dark in aerobic or anaerobic conditions which leads to state 1 (S1) or state 2 (S2). WT data contains information on the light-harvesting complex (LHC) connected to PSI and PSII. The mutants' data contain information on either LHCII-LHCI-PSI or LHCII-PSII, plus information on LHC antennas devoid of a PS core. In a simultaneous analysis of the data from all strains under S1 or S2 conditions a unified model for the excited state dynamics at 77 K was created. This yielded the completely resolved LHCII-LHCI-PSI and LHCII-PSII dynamics and quantified the state transitions. In WT cells the fraction of light absorbed by LHCII connected to PSII decreases from 45% in S1 to 29% in S2, while it increases from 0% to 16% for LHCII connected to PSI. Thus (16/45 =) 36% of all LHCII is involved in the state transition. In the mutant strains deficient in the PSI core, the red most species peaking at 716 nm disappears completely, indicating that this far red Chl pigment is located in the PSI core. In the mutant strain deficient in the PSII core, red shifted species with maxima at 684 and 686 nm appear in the LHCII antenna. LHCII-684 is quenched and decays with a rate of (310 ps)^? 1.     

Reduced Efficiency of Magnetotaxis in Magnetotactic Coccoid Bacteria in Higher than Geomagnetic Fields

Magnetotactic bacteria are microorganisms that orient and migrate along magnetic field lines. The classical model of polar magnetotaxis predicts that the field-parallel migration velocity of magnetotactic bacteria increases monotonically with the strength of an applied magnetic field. We here test this model experimentally on magnetotactic coccoid bacteria that swim along helical trajectories. It turns out that the contribution of the field-parallel migration velocity decreases with increasing field strength from 0.1 to 1.5 mT. This unexpected observation can be explained and reproduced in a mathematical model under the assumption that the magnetosome chain is inclined with respect to the flagellar propulsion axis. The magnetic disadvantage, however, becomes apparent only in stronger than geomagnetic fields, which suggests that magnetotaxis is optimized under geomagnetic field conditions. It is therefore not beneficial for these bacteria to increase their intracellular magnetic dipole moment beyond the value needed to overcome Brownian motion in geomagnetic field conditions.     

Periodic density functional theory study of the high-pressure behavior of energetic crystalline 1,4-dinitrofurazano[3, 4-b]piperazine

A detailed study of the structural, electronic, and optical absorption properties of crystalline 1,4-dinitrofurazano[3,4-b]piperazine (DNFP) under hydrostatic pressures of 0–100 GPa was performed using periodic density functional theory. As the pressure increases, the lattice constants and cell volumes calculated by LDA gradually approach those obtained by GGA-PW91. It was found that the structure of DNFP is much stiffer in the b direction than along the a and c axes, indicating that the compressibility of the crystal is anisotropic. As the pressure increases, the band gap gradually decreases, and this decrease is more pronounced in the low-pressure range than in the high-pressure region. An analysis of the density of states showed that the electronic delocalization in DNFP gradually increases under the influence of pressure. DNFP exhibits relatively high optical activity at high pressure. As the pressure increases, the bands in the fundamental absorption region of the absorption spectrum of DNFP become more numerous and intense.     

Susceptibility of sheep,human, and pig erythrocytes to haemolysis by the antimicrobial peptide Modelin 5

Modelin-5-CONH₂, a synthetic antimicrobial peptide, was used to gain an insight into species-selective haemolytic activity. The peptide displayed limited haemolytic activity against sheep (12 %), human (2 %), and pig (2 %) erythrocytes. Our results show that Modelin-5-CONH₂ had a disordered structure in the presence of vesicles formed from sheep, human, and pig erythrocyte lipid extract (<26 % helical) yet folded to form helices in the presence of a phosphatidylcholine (PC) membrane interface (e.g. >42 % in the presence of 1,2-dimyristoyl-sn-glycero-3-phosphocholine). Monolayer studies showed a strong correlation between anionic lipid content and monolayer insertion and lysis inducing surface pressure changes of 9.17 mN m^?1 for 1,2-dimyristoyl-sn-glycero-3-phospho-l-serine compared with PC monolayers, which induced pressure changes of ca. 3 mN m^?1. The presence of cholesterol in the membrane is shown to increase the packing density as the PC:sphingomyelin (SM) ratio increases so preventing the peptide from forming a stable association with the membrane. The data suggests that the key driver for membrane interaction for Modelin-5-CONH₂ is the anionic lipid attraction. However, the key factors in the species-specific haemolysis level for this peptide are the differing packing densities which are influenced by the SM:PC:cholesterol ratio.     

Bilirubin Oxidase from Trachyderma tsunodae K-2593, a Multi-copper Enzyme

The gel-setting and gel-melting temperatures of aqueous gelatin solutions (0.5 and 1.0 wt%) were measured by a steady state hot-wire method during treatment under high hydrostatic pressure up to 200 MPa. The high-pressure treatment caused both the gel-setting and gel-melting temperatures to increase with increasing pressure. The hot-wire method was proved to be effective for assessing the coagulation or gelation of food materials under pressurized conditions.     

Kinetics of Low-Temperature Oxidation of Enzymatic Lignin from Pine Wood (Pinus silvestris) in an Aqueous Alkaline Medium

The kinetics of oxidation of the enzymatic lignin from pine wood (brown rotted wood) by oxygen in an aqueous alkaline medium at 90–160°C was investigated. It was established that the vanillin yield increased gradually with the temperature increase in this range from 3.4 to 5.6 wt % with respect to lignin. The observed activation energy of the process of oxygen consumption varies in the range 6–19 kJ/mol depending on the process conditions. The process order with respect to oxygen pressure calculated from the rates of O₂ consumption during the first two hours of oxidation at 90–120°C is 1.02 ± 0.05. The low value of activation energy of the process as well as the first order in oxygen indicates a diffusion-controlled process under these conditions. The kinetic data demonstrate that the role of degenerate chain branching processes increases with the increase in the degree of oxidation as a result of decomposition of the hydroperoxides formed. The hydroperoxide decomposition during oxidation in quantity of more than 16 mol % with respect to the initial lignin was registered using a volumetric method based on the oxygen release. The main causes and features determining the monotonically increasing function of the dependence of the selectivity of lignin oxidation to aromatic aldehydes on temperature were discussed.