Chemistry

Knowledge of the influence of chain length and amino acid sequence on the structural and dynamic properties of small peptides in solution provides essential information on protein folding pathways. The combination of time-resolved optical spectroscopy and molecular dynamics (MD) simulation methods has become a powerful tool to investigate the kinetics of end-to-end collisions (looping rates) in short peptides, which are relevant in early protein folding events. We applied the combination of both techniques to study temperature-dependent
(280-340 K) looping rates of the Dbo-AlaGlyGln-Trp-NH2 peptide, where Dbo represents a 2,3-diazabicyclo-[2.2.2]oct-2-ene-labeled asparagine, which served as a fluorescent probe in the time-resolved spectroscopic experiments. The experimental looping rates increased from 4.8  107 s-1 at 283 K to 2.0  108 s-1 at 338 K in H2O. The corresponding Arrhenius plot provided as activation parameters Ea ) 21.5 ( 1.0 kJ mol-1 and ln(A/s-1) ) 26.8 ( 0.2 in H2O. The results in D2O were consistent with a slight solvent viscosity effect, i.e., the looping rates were 10-20% slower. MD simulations were performed with the GROMOS96 force field in a water solvent model, which required first a parametrization of the synthetic amino acid Dbo. After corrections for solvent viscosity effects, the calculated looping rates varied from 1.5  108 s-1 at 280 K to 8.2  108 s-1
at 340 K in H2O, which was about four times larger than the experimental data. The calculated activation parameters were Ea ) 24.7 ( 1.5 kJ mol-1 and ln(A/s-1) ) 29.4 ( 0.1 in H2O.

Fluorescence resonance energy transfer (FRET) from the amino acid tryptophan (Trp) as donor and a 2,3-diazabicyclo[2.2.2]oct-2-ene-labeled asparagine (Dbo) as acceptor in peptides of the general structure Trp-(Pro)n-Dbo-NH2 (n = 1−6) was investigated by steady-state and time-resolved fluorescence, CD, and NMR spectroscopy as well as by molecular dynamics (MD) simulations (GROMOS96 force field). The Trp/Dbo FRET pair is characterized by a very short Förster radius (R0 ca. 9 Å), which allowed distance determinations in such short peptides. Water and propylene glycol were investigated as solvents. The peptides were designed to show an early nucleation of the poly(Pro)II (PPII) secondary helix structure for n ≥ 2, which was confirmed by their CD spectra. The shortest peptide (n = 1) adopts preferentially the trans conformation about the Trp-Pro bond, as confirmed by NMR spectra. The FRET efficiencies ranged 2−72% and were found to depend sensitively on the peptide length, i.e., the number of intervening proline residues. The analysis of the FRET data at different levels of theory (assuming either a fixed distance or distance distributions according to a wormlike chain or Gaussian model) afforded donor−acceptor distances between ca. 8 Å (n = 1) and ca. 16 Å (n = 6) in water, which were found to be similar or slightly higher in propylene glycol. The distances afforded by the Trp/Dbo FRET pair were found to be reasonable in comparison to literature data, expectations from the PPII helix structure, and the results from MD simulations. The persistence lengths for the longer peptides were found to lie at 30−70 Å in water and 220 ± 40 Å in propylene glycol, suggesting a more rigid PPII helical structure in propylene glycol. A detailed comparison with literature data on FRET in polyprolines demonstrates that the donor−acceptor distances extracted by FRET are correlated with the Förster radii of the employed FRET pairs. This demonstrates the limitations of using FRET as a spectroscopic ruler for short polyprolines, which is presumably due to the breakdown of the point dipole approximation in Förster theory, when the size of the chromophores becomes comparable or larger than the distances under investigation.

The collision-induced fluorescence quenching of a 2,3-diazabicyclo[2.2.2]oct-2-ene-labeled asparagine (Dbo) by hydrogen atom abstraction from the tyrosine residue in peptide substrates was introduced as a single-labeling strategy to assay the activity of tyrosine kinases and phosphatases. The assays were tested for 12 different combinations of Dbo-labeled substrates and with the enzymes p60c-Src Src kinase, EGFR kinase, YOP protein tyrosine phosphatase, as well as acid and alkaline phosphatases, thereby demonstrating a broad application potential. The steady-state fluorescence changed by a factor of up to 7 in the course of the enzymatic reaction, which allowed for a sufficient sensitivity of continuous monitoring in steady-state experiments. The fluorescence lifetimes (and intensities) were found to be rather constant for the phosphotyrosine peptides (ca. 300 ns in aerated water), while those of the unphosphorylated peptides were as short as 40 ns (at pH 7) and 7 ns (at pH 13) as a result of intramolecular quenching. Owing to the exceptionally long fluorescence lifetime of Dbo, the assays were alternatively performed by using nanosecond time-resolved fluorescence (Nano-TRF) detection, which leads to an improved discrimination of background fluorescence and an increased sensitivity. The potential for inhibitor screening was demonstrated through the inhibition of acid and alkaline phosphatases by molybdate.

Looping rates in short polypeptides can be determined by intramolecular fluorescence quenching of a 2,3-diazabicyclo[2.2.2]oct-2-ene-labeled asparagine (Dbo) by tryptophan. By this methodology, the looping rates in glycine-serine peptides with the structure Trp-(Gly-Ser)n-Dbo-NH2 of different lengths (n = 0–10) were determined in dependence on temperature in D2O and the activation parameters were derived. In general, the looping rate increases with decreasing peptide length, but the shortest peptide (n=0) shows exceptional behavior because its looping rate is slower than that for the next longer ones (n=1,2). The activation energies increase from 17.5 kJ mol−1 for the longest peptide (n=10) to 20.5 kJ mol−1 for the shortest one (n=0), while the pre-exponential factors (log⁡(A/s−1)) range from 10.20 to 11.38. The data are interpreted in terms of an interplay between internal friction (stiffness of the biopolymer backbone and steric hindrance effects) and solvent friction (viscosity-limited diffusion). For the longest peptides, the activation energies resemble more and more the value expected for solvent viscous flow. Internal friction is most important for the shortest peptides, causing a negative curvature and a smaller than ideal slope (ca. –1.1) of the double-logarithmic plots of the looping rates versus the number of peptide chain segments (N). Interestingly, the corresponding plot for the pre-exponential factors (logA versus logN) shows the ideal slope (–1.5). While the looping rates can be used to assess the flexibility of peptides in a global way, it is suggested that the activation energies provide a measure of the “thermodynamic” flexibility of a peptide, while the pre-exponential factors reflect the “dynamic” flexibility.

A complete understanding of a protein-folding landscape requires detailed characterization of intermediates that are populated during a folding reaction. We are exploring the folding intermediates sampled by a 136-amino acid β-barrel
protein, cellular retinoic acid-binding protein I (CRABP I). This model protein is made up of two five-stranded orthogonal β-sheets wrapped around a central ligand-binding cavity. Previous work has shown that the folding of CRABP I involves well-defined stages: An early intermediate forms in ca. 300 μs by
hydrophobic collapse, next (~100 ms) an intermediate is populated that has native topology including the ligand-binding cavity, and lastly, in ca. 1 s, interstrand hydrogen bonds form and native packing of side chains develops. The nature of
the intermediates is relatively poorly understood, including structural details, compactness, and the size of the intermediate ensembles. To address these questions, we have designed CRABP I mutants with solvent-accessible Cys residues (M1C, S55C, N64C, K106C, and D103C) suitable for attachment of
thiol-reactive fluorophores, as well as transglutaminase (TGase) tags at their Ctermini for enzymatically mediated labeling with a second fluorophore. The Cys residues have been labeled with HyLite488 or BODIPY-FL (donor), and the TGase tags have been labeled with tetramethylrhodamine (acceptor). As an
additional strategy to deduce the nature of the folding intermediates, we are varying solvent conditions using salts that differentially affect species stabilized by hydrophobic, electrostatic, or hydrogen bonding interactions. Taken together,
ensemble and single-molecule FRET studies of doubly-labeled variants are providing a increasingly detailed picture of the CRABP I folding landscape

Fluorescence resonance energy transfer (FRET) between tryptophan (Trp) as donor and 2,3-diazabicyclo[2.2.2]oct-2-ene (Dbo) as acceptor was studied by steady-state and time-resolved fluorescence spectroscopy. The unique feature of this FRET pair is its exceptionally short Förster radius (10 Å), which allows one to recover distance distributions in very short structureless peptides. The technique was applied to Trp-(GlySer)n-Dbo-NH2 peptides with n = 0−10, for which the average probe/quencher distance ranged between 8.7 and 13.7 Å experimentally (in propylene glycol, analysis according to wormlike chain model) and 8.6−10.2 Å theoretically (for n = 0−6, GROMOS96 molecular dynamics simulations). The larger FRET efficiency in steady-state compared to time-resolved fluorescence experiments was attributed to a static quenching component, suggesting that a small but significant part (ca. 10%) of the conformations are already in van der Waals contact when excitation occurs.

Fluorescence Correlation Spectroscopy (FCS) and Förster Resonance Energy Transfer (FRET) are both scientific concepts that are frequently discussed in the context of single molecule fluorescence techniques. In contrast to FCS, FRET is strictly not a true technique but a photophysical phenomenon, which can be employed in combination with any method that probes fluorescence intensity or lifetime. Thus, the combination of FCS with FRET is possible, and, although these concepts are quite often treated as alternative approaches - particularly for the analysis of biological systems – quite attractive. However, under certain circumstances, e.g., for applications of fluorescence cross-correlation spectroscopy, FRET effects can cause significant complications for quantitative data analysis, and careful calibration has to be carried out to avoid FRET-induced artifacts. This can be most elegantly done if alternating excitation schemes such as PIE (pulsed interleaved excitation) are employed. In this short review, we discuss the potential and the caveats of FCS combined with FRET; and give a short record on successful and promising applications.

Förster resonance energy transfer (FRET) in association with the recent advancements in optical techniques provides a way to understand the detailed mechanisms in different biological systems at the molecular level. Improvements in wide-field, confocal and two-photon microscopy facilitate the measurements of two-dimensional spatial distribution in steady-state as well as dynamic bimolecular interactions. In the recent decade, FRET became an exceptional fluorescence-based technique due to its potential advantages for studying the biological processes in living cells and more for spatial resolution at nanometer scale. In particular, FRET investigations have shown that biomolecules adopt different conformational structures to perform their functions. In this review, the basic principles and applications of FRET in chemistry, biology, and physics are discussed. Along with, the recent improvements in fluorophore design and labeling and FRET measurement methods are briefly mentioned.

Recent advancements in analytical methods relating organic chemistry, molecular biology, spectroscopy, engineering, and chemical biology have provided powerful tools to explore different selective fluorescent labeling techniques for analyzing molecular events in cells. New fluorescent labeling techniques have been developed and improvised depending on requirements like non-specificity, selectivity, small size, and of course, high yield. Along with synthesized small fluorophores that are used mostly for chemical labeling, fluorescent proteins such as green fluorescent protein
(GFP) and its variants are used to label living cells genetically, both in vivo and in vitro. Fluorescent labeling has been extended over a large area of interdisciplinary research to visualize the functions and conditions of molecules using lower concentrations of the fluorescent material. Considering the wide applications of fluorescent techniques in various fields, the basic principles of different fluorescent labeling techniques relevant to chemistry and biology are discussed in this review.

In the present study, we report the F€orster resonance energy transfer (FRET) from fluorescent
copper nanoclusters (Cu NCs) as donor to cobalt complex (nitrate (Co (NO3)2)) as acceptor.
Fluorescent Cu NCs have been synthesized on bovine serum albumin template by wet chemistry
method; these NCs show fluorescence maxima at 435 nm. The fluorescence intensity of Cu NCs is
quenched in proximity presence of acceptors, and subsequently, energy is transferred. In such type
of system, these Cu NCs are found to be efficient donor with F€orster distance (R0) 8.9A ° and FRET
efficiency (E) up to 42%. The F€orster distance obtained is found to be the lowest among other
reported values for donor/acceptor pair till today.

Quasi all water soluble composites use graphene oxide (GO) or reduced graphene oxide (rGO) as graphene based additives despite the long and harsh conditions required for their preparation. Herein, polyvinyl alcohol (PVA) films containing few layer graphene (FLG) are prepared by the co-mixing of aqueous colloids and casting, where the FLG colloid is first obtained via an efficient, rapid, simple, and bio-compatible exfoliation method providing access to relatively large FLG flakes. The enhanced mechanical, electrical conductivity, and O2 barrier properties of the films are investigated and discussed together with the structure of the films. In four different series of the composites, the best Young’s modulus is measured for the films containing around 1% of FLG. The most significant enhancement is obtained for the series with the largest FLG sheets contrary to the elongation at break which is well improved for the series with the lowest FLG sheets. Relatively high one-side electrical...

Highly efficient graphene oxide–SnO2–TiO2 ternary nanocomposite was designed via one step solvothermal method considering the step-wise band edge energy levels of each component and the corresponding photocatalytic performance was investigated.

Few layer graphene/carbon (FLG/C) composites are prepared directly via the rapid and simple exfoliation of expanded graphite in the presence of carbon based natural precursors (i.e. protein, polysaccharide) in water, followed by carbonization process. Several parameters such as nature of C-precursor, FLG/C ratio and carbonization conditions (gas, temperature) are modified in order to optimize the morphology, composition and porosity of FLG/C and thereby investigate their impact on gravimetric and volumetric capacitance, their stability and contribution of pseudocapacitance (Ps) vs. double-layer capacitance (DL). Few composites exhibit extremely high capacitance considering their low BET-surface area ranging in 130-260 m 2 /g. The highest gravimetric and volumetric capacitance of 322 F/g and 467 F/cm 3 respectively (0.5 A/g); and energy/power performance is reached for FLG/C:1/2, synthesized from graphite-bovine serum albumin(BSA). Despite relatively high theoretical pseudocapacitance contribution of 69% (1.1V), this sample shows also high capacity retention at high current density and elevated energy -to-power densities. The overall great capacity performance is attributed to the high electrochemical surface area from combined structural features: ultramicroporosity, FLG alignment with high accessibility of FLG edges and elevated packing density. The transport limitation enhances however at higher scan rate (> 100 mV/s).

Few layer graphene (FLG) supported structurally flat polygonal and hexagonal iron platelet NPs are prepared by two different high temperature annealing approaches, i.e., under "conventional" and µ-wave assisted heating. Both methods confirm that FLG acts as a template for 2D NPs tailoring and flat Fe-based NPs with high surface-to-volume ratio are still well dispersed and stable at the temperatures as high as several hundreds of C° despite weakened interfacial interactions between FLG and NPs. The high surface-to-volume ratio benefit of 2D NPs geometry is highlighted by mathematical estimation. The additional morphology of grown carbon nanofibers (CNFs) confirms the flat active faceted structures of NPs catalysts.

Few layer graphene/carbon (FLG/C) composites are prepared directly via the rapid and simple exfoliation of expanded graphite in the presence of carbon based natural precursors (i.e. protein, polysaccharide) in water, followed by carbonization process. Several parameters such as nature of C-precursor, FLG/C ratio and carbonization conditions (gas, temperature) are modified in order to optimize the morphology, composition and porosity of FLG/C and thereby investigate their impact on gravimetric and volumetric capacitance, their stability and contribution of pseudocapacitance (Ps) vs. double-layer capacitance (DL). Few composites exhibit extremely high capacitance considering their low BET-surface area ranging in 130-260 m2/g. The highest gravimetric and volumetric capacitance of 322 F/g and 467 F/cm3 respectively (0.5 A/g); and energy/power performance is reached for FLG/C:1/2, synthesized from graphite-bovine serum albumin(BSA). Despite relatively high theoretical pseudocapacitance c...

In austempering, the microstructural end product of the spheroidal graphite
(SG) iron matrix is essentially bainite, a structure formed below the pearlite temperature
range but above the martensite range. Ductile cast iron undergoes a remarkable
transformation when subjected to the austempering process. Due to isothermal
transformation, it produces a microstructure that is  stronger and tougher than the
structures resulting from conventional heat treatment process. In the present investigation,
the SG iron was austempered with three different austempering temperatures
(2500
C,3000
C and 3500
C) with varying austempering time. The sample was taken for
XRD analysis to study the morphology of the matrix. It was found that both the austenite
(111) and ferrite (110) lines are identified nearly in all cases. The maximum intensity of
the austenite (111) line is increasing with increasing temperature but ferrite (110) line is
increasing with increasing austempering time and decreasing with austempering
temoerature. Hence austempering calls for very precise control of process times and
temperatures.

Austempererd ductile iron is the most recent development in the area of ductile iron. This is formed
by an isothermal heat treatment of the ductile iron. The newly developed austempered ductile iron is
now replacing steel in many fields so it has becoming very important to various aspects of this
material. In the present work the effect of copper along with the process variables (austempering
temperature and austempering time) on the properties (Tensile strength and Elongation) and
microstructure of ductile iron was studied. With increasing austempering time tensile strength and
elongation were increasing but with increasing austempering temperature tensile strength was
decreasing and elongation was increasing. Austempered ductile iron with copper was showing some
higher strength and lower elongation than the austempered ductile iron without copper. In
microstructure ferrite was increasing with increasing austempering time and austenite was increasing
with increasing austempering temperature in both the grades.