527 research outputs found
EPR of Cu\u3csup\u3e2+\u3c/sup\u3e Prion Protein Constructs at 2 GHz Using the \u3cem\u3eg\u3c/em\u3e\u3csub\u3eâ„\u3c/sub\u3e Region to Characterize Nitrogen Ligation
A double octarepeat prion protein construct, which has two histidines, mixed with copper sulfate in a 3:2 molar ratio provides at most three imidazole ligands to each copper ion to form a square-planar Cu2+ complex. This work is concerned with identification of the fourth ligand. A new (to our knowledge) electron paramagnetic resonance method based on analysis of the intense features of the electron paramagnetic resonance spectrum in the gâ„ region at 2 GHz is introduced to distinguish between three and four nitrogen ligands. The methodology was established by studies of a model system consisting of histidine imidazole ligation to Cu2+. In this spectral region at 2 GHz (S-band), g-strain and broadening from the possible rhombic character of the Zeeman interaction are small. The most intense line is identified with the MI = +1/2 extra absorption peak. Spectral simulation demonstrated that this peak is insensitive to cupric Ax and Ay hyperfine interaction. The spectral region to the high-field side of this peak is uncluttered and suitable for analysis of nitrogen superhyperfine couplings to determine the number of nitrogens. The spectral region to the low-field side of the intense extra absorption peak in the gâ„ part of the spectrum is sensitive to the rhombic distortion parameters Ax and Ay. Application of the method to the prion protein system indicates that two species are present and that the dominant species contains four nitrogen ligands. A new loop-gap microwave resonator is described that contains âŒ1 mL of frozen sample
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Charge Characteristics of Agouti-Related Protein Implicate Potent Involvement of Heparan Sulfate Proteoglycans in Metabolic Function.
The endogenous melanocortin peptide agouti-related protein (AgRP) plays a well-known role in foraging, but its contribution to metabolic regulation is less understood. Mature AgRP(83-132) has distinct residues for melanocortin receptor binding and heparan sulfate interactions. Here, we show that AgRP increases ad libitum feeding and operant responding for food in mice, decreases oxygen consumption, and lowers body temperature and activity, indicating lower energy expenditure. AgRP increased the respiratory exchange ratio, indicating a reduction of fat oxidation and a shift toward carbohydrates as the primary fuel source. The duration and intensity of AgRP's effects depended on the density of its positively charged amino acids, suggesting that its orexigenic and metabolic effects depend on its affinity for heparan sulfate. These findings may have major clinical implications by unveiling the critical involvement of interactions between AgRP and heparan sulfate to the central regulation of energy expenditure, fat utilization, and possibly their contribution to metabolic disease
The Octarepeat Domain of the Prion Protein Binds Cu(II) with Three Distinct Coordination Modes at pH 7.4
The prion protein (PrP) binds Cu2+ in its N-terminal octarepeat domain. This unusual domain is comprised of four or more tandem repeats of the fundamental sequence PHGGGWGQ. Previous work from our laboratories demonstrates that at full copper occupancy, each HGGGW segment binds a single Cu2+. However, several recent studies suggest that low copper occupancy favors different coordination modes, possibly involving imidazoles from histidines in adjacent octapeptide segments. This is investigated here using a combination of X-band EPR, S-band EPR, and ESEEM, along with a library of modified peptides designed to favor different coordination interactions. At pH 7.4, three distinct coordination modes are identified. Each mode is fully characterized to reveal a series of copper-dependent octarepeat domain structures. Multiple His coordination is clearly identified at low copper stoichiometry. In addition, EPR detected copperâcopper interactions at full occupancy suggest that the octarepeat domain partially collapses, perhaps stabilizing this specific binding mode and facilitating cooperative copper uptake. This work provides the first complete characterization of all dominant copper coordination modes at pH 7.4
The role of conformational diffusion in ion channel gating
We consider an exactly tractable model of the Kramers type for the
voltage-dependent gating dynamics of single ion channels. It is assumed that
the gating dynamics is caused by the thermally activated transitions in a
bistable potential. Moreover, the closed state of the channel is highly
degenerate and embraces the whole manifold of closed substates. Opening of the
ion channel is energetically prohibited from most of the closed substates and
requires a special conformation where the voltage sensor can move along an
activation pathway and trigger the transition into the open conformation. When
the corresponding activation barrier towards the channel's opening is removed
by the applied voltage, the statistics of non-conducting time intervals become
strongly influenced by the conformational diffusion. For the corresponding
supra-threshold voltages, our model explains the origin of the power law
distribution of the closed time intervals. The exponential-linear dependence of
the opening rate on voltage, often used as an experimental fit, is also
reproduced by our model
When translocation dynamics becomes anomalous
Recent single molecule experiments probing the passage process of a short
single-stranded DNA (ssDNA) through a membrane channel (translocation) allow to
measure the passage time distribution. Building on a recent modelling approach
(D. K. Lubensky and D. R. Nelson, Biophys. J. 77, 1824 (1999)), which has been
demonstrated to be valid for chains of up to nucleotides and
therefore well applies to the system we have in mind, we discuss the
consequences if the associated dynamics is not of Markov origin, but if strong
memory effects prevail during the translocation. Motivation is drawn from
recent results indicating that the distribution of translocation times is
broader than predicted by simple Markovian models based on Brownian motion.Comment: 5 pages, 2 figures, RevTeX4, submitted to Biophys.
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Photocatalytic Generation of Singlet Oxygen by Graphitic Carbon Nitride for Antibacterial Applications
Carbon-based functional nanocomposites have emerged as potent antimicrobial agents and can be exploited as a viable option to overcome antibiotic resistance of bacterial strains. In the present study, graphitic carbon nitride nanosheets are prepared by controlled calcination of urea. Spectroscopic measurements show that the nanosheets consist of abundant carbonyl groups and exhibit apparent photocatalytic activity under UV photoirradiation towards the selective production of singlet oxygen. Therefore, the nanosheets can effectively damage the bacterial cell membranes and inhibit the growth of bacterial cells, such as Gram-negative Escherichia coli, as confirmed in photodynamic, fluorescence microscopy, and scanning electron microscopy measurements. The results from this research highlight the unique potential of carbon nitride derivatives as potent antimicrobial agents
Distance-Dependent Fluorescence Quenching and Binding of CdSe Quantum Dots by Functionalized Nitroxide Radicals
Quantum dot (QD) fluorescence is effectively quenched at low concentration by nitroxides bearing amine or carboxylic acid ligands. The association constants and fluorescence quenching of CdSe QDs with these derivatized nitroxides have been examined using electron paramagnetic resonance (EPR) and fluorescence spectroscopy. The EPR spectra in the non-protic solvent toluene are extremely sensitive to intermolecular and intramolecular hydrogen bonding of the functionalized nitroxides. Fluorescence measurements show that quenching of QD luminescence is nonlinear, with a strong dependence on the distance between the radical and the QD. The quenched fluorescence is restored when the surface-bound nitroxides are converted to hydroxylamines by mild reducing agents, or trapped by carbon radicals to form alkoxyamines. EPR studies indicate that photoreduction of the nitroxide occurs in toluene solution upon photoexcitation at 365 nm. However, photolysis in benzene solution gives no photoreduction, suggesting that photoreduction in toluene is independent of the quenching mechanism. The fluorescence quenching of QDs by nitroxide binding is a reversible process
A minimalist chemical model of matrix metalloproteinases- Can small peptides mimic the more rigid metal binding sites of proteins?
In order to develop a minimalist chemical model of matrix metalloproteinases (MMPs), we
synthesized a pentadecapeptide (Ac-KAHEFGHSLGLDHSK-NH2) corresponding to the
catalytic zinc(II) binding site of human MMP-13. The multi-domain structural organization of
MMPs fundamentally determines their metal binding affinity, catalytic activity and
selectivity. Our potentiometric, UV-VIS, CD, EPR, NMR, ESI-MS and kinetic study are
aimed to explore the usefulness of flexible peptides to mimic the more rigid metal binding
sites of proteins, to examine the intrinsic metal binding properties of this naked sequence, as
well as to contribute the development of a minimalist, peptide-based chemical model of
MMPs, including the catalytic properties. Since multiimidazole environment is also
characteristic for copper(II), and recently copper(II) containing variants of MMPs have been
identified, we also studied the copper(II) complexes of the above peptide.
Around pH 6-7 the peptide, similarly to MMPs, offers {3Nim} coordinated binding site
for both zinc(II) and copper(II). In the case of copper(II), the formation of amide coordinated
species at higher pH ceased the analogy with the copper(II) containing MMP variant. On the
other hand, the zinc(II)-peptide system mimics some basic features of the MMP active sites:
the main species around pH 7 (ZnH2L) possesses {3Nim,H2O} coordination environment, the
deprotonation of the zinc-bound water takes place near to the physiological pH, it forms
relatively stable ternary complexes with hydroxamic acids, and the species ZnH2L(OH) and
ZnH2L(OH)2 have notable hydrolytic activity between pH 7-9
Comparing ion conductance recordings of synthetic lipid bilayers with cell membranes containing TRP channels
In this article we compare electrical conductance events from single channel
recordings of three TRP channel proteins (TRPA1, TRPM2 and TRPM8) expressed in
human embryonic kidney cells with channel events recorded on synthetic lipid
membranes close to melting transitions. Ion channels from the TRP family are
involved in a variety of sensory processes including thermo- and
mechano-reception. Synthetic lipid membranes close to phase transitions display
channel-like events that respond to stimuli related to changes in intensive
thermodynamic variables such as pressure and temperature. TRP channel activity
is characterized by typical patterns of current events dependent on the type of
protein expressed. Synthetic lipid bilayers show a wide spectrum of electrical
phenomena that are considered typical for the activity of protein ion channels.
We find unitary currents, burst behavior, flickering, multistep-conductances,
and spikes behavior in both preparations. Moreover, we report conductances and
lifetimes for lipid channels as described for protein channels. Non-linear and
asymmetric current-voltage relationships are seen in both systems. Without
further knowledge of the recording conditions, no easy decision can be made
whether short current traces originate from a channel protein or from a pure
lipid membraneComment: 13 pages, 9 Figure
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