Temperature-dependent Circular Dichroism Research Articles

Chiral Tectonics: VCD and ECD Application for Epimerization of a Star-Burst Tetranuclear Complex with a Labile Central Core

The present article reports the application of vibrational circular dichroism (VCD) and temperature-dependent electronic circular dichroism (ECD) methods to reveal the dynamical aspects of a star-burst tetranuclear metal complex with a labile central core in a solution. One-handed chiral inert tecton, ∆- or Λ-[Ru(III)(acac)2(taetH)] (acacH = acetylacetone, taetH2 = tetraacetylethane), was prepared by reacting [Ru(acac)3] with taetH2 in solid at 120 °C. The ∆Λ-pair of pure enantiomers was obtained chromatographically. On adding Al(ClO4)3 to its enantiopure solution, three units of one-handed tecton were assembled spontaneously around an aluminum(III) ion to form a star-burst tetranuclear complex, [{∆- or Λ-Ru(acac)2(taet)}3Al(III)]. The VCD spectrum recorded on the CDCl3 solution of the complex showed that the central chirality around an Al(III) ion took dominantly the absolute configuration antipodal to those of peripheral Ru(III) units at the temperature lower than −10 °C. The complex underwent interconversion between the ∆- and Λ-configurations around a central Al(III) core (or epimerization) in solution. The activation energy barrier was determined from the time courses of ECD spectra in CHCl3 and CH3OH.

Sequence Reversal Prevents Chain Collapse and Yields Heat-Sensitive Intrinsic Disorder

Sequence patterns of charge, hydrophobicity, hydrogen bonding, and other amino acid physicochemical properties contribute to mechanisms of protein folding, but how sequence composition and patterns influence the conformational dynamics of the denatured state ensemble is not fully understood. To investigate structure-sequence relationships in the denatured state, we reversed the sequence of staphylococcal nuclease and characterized its structure, thermodynamic character, and hydrodynamic radius using circular dichroism spectroscopy, dynamic light scattering, analytical ultracentrifugation, and size-exclusion chromatography as a function of temperature. The macromolecular size of “Retro-nuclease” is highly expanded in solution with characteristics similar to biological intrinsically disordered proteins. In contradistinction to a disordered state, Retro-nuclease exhibits a broad sigmoid transition of its hydrodynamic dimensions as temperature is increased, indicating a thermodynamically controlled compaction. Counterintuitively, the magnitude of these temperature-induced hydrodynamic changes exceed that observed from thermal denaturation of folded unaltered staphylococcal nuclease. Undetectable by calorimetry and intrinsic tryptophan fluorescence, the lack of heat capacity or fluorescence changes throughout the thermal transition indicate canonical hydrophobic collapse did not drive the Retro-nuclease structural transitions. Temperature-dependent circular dichroism spectroscopy performed on Retro-nuclease and computer simulations correlate to temperature sensitivity in the intrinsic sampling of backbone conformations for polyproline II and α-helix. The experimental results indicate a role for sequence direction in mediating the collapse of the polypeptide chain, whereas the simulation trends illustrate the generality of the observed heat effects on disordered protein structure.

Thermal Stability of Type II Polyketide Acyl Carrier Proteins

Engineering biosynthetic pathways in microorganisms is a compelling route to gain access to novel molecules with drug‐like properties. Polyketide synthases (PKSs) utilize acyl carrier proteins (ACPs) to convert simple acetate building blocks into structurally complex molecules, many of which are antibiotics and anticancer agents. In a process known as combinatorial synthesis, biochemists have attempted to mix‐and‐match enzymes from different PKSs to create new hybrid synthases capable of synthesizing novel polyketide natural products. However, the ACP has proven to be a roadblock in these studies, as little is known about how the ACP functions and interacts with other enzymes due to its small and dynamic nature. To further our knowledge about the structure and function of ACPs, we cloned, expressed, and characterized a subset of 14 type II PKS ACPs. Temperature‐dependent circular dichroism (CD) experiments were conducted to determine the thermostability of the ACPs in the presence or absence of divalent cations. Our results indicate that despite strong sequence similarity, ACPs display a range in thermal stability. Further, the presence of metal ions confers additional stability to the ACP, presumably by decreasing electrostatic repulsive forces. Analytical ultracentrifugation‐sedimentation velocity experiments revealed that all ACPs sediment as monomers, and that despite sharing a common ancestor with fatty acid synthases, only one of the PKS ACPs studied interacted with the E. coli fatty acid ketosynthase, FabF.Support or Funding InformationWe would like to acknowledge an NSF CAREER Award #R15GM120704 and an NIH Award #CHE‐1652424 to Louise K. Charkoudian, as well as the Beckman Scholars Program, as sources of funding for this project.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Spectra and Simulation of Model Beta-Sheets and Hairpins. Impact of Turn Sequences and Aromatic Contacts on Equilibria and Dynamics

β-sheets can be modeled as antiparallel strands coupled by turns. Isolated model systems are less stable, due to amide solvation), but can be induced to fold into sheet-like structures by restricting the conformational space of turn residues, such as with DPro-Gly sequences, or by inclusion of cross-strand hydrophobic interactions, especially aromatic contacts, which tend to desolvate parts of the structure. Expanding to multistrand structures can provide different characteristics for study of physical properties. Larger structures have strand differentiation, since the center strand of a three-strand sheet has a different H-bonds than the outer ones. In larger structures, integral hairpin-like sequences of either the first two or the second two strands might have different stabilities and folding mechanisms, if it is overall a multistate process. To explore these factors, we have prepared a series of three-stranded sheet structures that are stabilized by either DPro-Gly or by Aib-Gly turns and contain various Trp-Tyr side-chain contacts. To assess the impact of the third strand we also separately synthesized the component hairpins and studied their relative properties with temperature dependent circular dichroism, fluorescence, infrared (IR) and vibrational circular dichroism for evaluation of global thermodynamic equilibria as well as IR–detected temperature jump relaxation dynamics of selected components (strands, turns). Where possible, NMR structures were obtained for comparison and MD simulations of unfolding were used to aid interpretations. While all the sequences showed evidence of β-structure formation, the extent and stability varied markedly. In terms of equilibrium properties, the turn residues (especially DPro-Gly) seemed to have the strongest influence, while the aromatic contacts had minor effects. However in terms of dynamics, the aromatic effects differentiated the structures more, suggesting a variation in the folding mechanisms.

Designability of Aromatic Interaction Networks at E. coli Bacterioferritin B-Type Channels.

The bacterioferritin from E. coli (BFR), a maxi-ferritin made of 24 subunits, has been utilized as a model to study the fundamentals of protein folding and self-assembly. Through structural and computational analyses, two amino acid residues at the B-site interface of BFR were chosen to investigate the role they play in the self-assembly of nano-cage formation, and the possibility of building aromatic interaction networks at B-type protein–protein interfaces. Three mutants were designed, expressed, purified, and characterized using transmission electron microscopy, size exclusion chromatography, native gel electrophoresis, and temperature-dependent circular dichroism spectroscopy. All of the mutants fold into α-helical structures and possess lowered thermostability. The double mutant D132W/N34W was 12 °C less stable than the wild type, and was also the only mutant for which cage-like nanostructures could not be detected in the dried, surface-immobilized conditions of transmission electron microscopy. Two mutants—N34W and D132W/N34W—only formed dimers in solution, while mutant D132W favored the 24-mer even more robustly than the wild type, suggesting that we were successful in designing proteins with enhanced assembly properties. This investigation into the structure of this important class of proteins could help to understand the self-assembly of proteins in general.

Temperature- and Composition-Dependent DNA Condensation by Thermosensitive Block Copolymers.

Successful intracellular delivery of genes requires an efficient carrier, as genes by themselves cannot diffuse across cell membranes. Because of the toxicity and immunogenicity of viral vectors, nonviral vectors are gaining tremendous interest in research. In this work, we have investigated the temperature-dependent DNA condensation efficiency of various compositions of a thermosensitive block copolymer viz., poly(N-isopropylacrylamide)-b-poly(2-(diethylamino)ethyl methacrylate) (PNIPA-b-PDMAEMA). Three different copolymer compositions of varying molecular weights were successfully synthesized via the RAFT polymerization technique. Steady-state fluorescence and circular dichroism (CD) spectroscopies, dynamic light scattering (DLS) and zeta potential measurements, agarose gel electrophoresis, and atomic force microscopy techniques were utilized to study the interaction of the copolymers with DNA at temperatures above and below the critical aggregation temperature (CAT). All these experiments revealed that, above the CAT, there was formation of highly stable and tight polymer–DNA complexes (polyplexes). The size of polyplexes was dependent on the temperature up to a certain charge ratio, as determined by the DLS results. The results obtained from temperature-dependent fluorescence spectroscopy, CD, and gel electrophoresis indicated that the DNA molecules were shielded more from aqueous exposure above the CAT because of the formation of relatively more compact complexes. The polyplexes also exhibited changes in the particle morphology below and above the CAT, with particles generated above CAT being more spherical in morphology. These results suggested at the possibility of modulating the complex formation by temperature modification. The present biophysical studies would provide new physical insight into the design of novel gene carriers.

The Helix to Super-Helix Transition in the Self-Assembly of π-Systems: Superseding of Molecular Chirality at Hierarchical Level.

Higher-order super-helical structures derived from biological molecules are known to evolve through opposite coiling of the initial helical fibers, as seen in collagen protein. A similar phenomenon is observed in a π-system self-assembly of chiral oligo(phenyleneethylene) derivatives (S)-1 and (R)-1 that explains the unequal formation of both left- and right-handed helices from molecule having a specific chiral center. Concentration- and temperature-dependent circular dichroism (CD) and UV/Vis spectroscopic studies revealed that the initial formation of helical aggregates is in accordance with the molecular chirality. At the next level of hierarchical self-assembly, coiling of the fibers occurs with opposite handedness, thereby superseding the command of the molecular chirality. This was confirmed by solvent-dependent decoiling of super-helical structures and concentration-dependent morphological analysis.

Ratio of ellipticities between 192 and 208 nm (R1 ): An effective electronic circular dichroism parameter for characterization of the helical components of proteins and peptides.

Circular dichroism (CD) spectroscopy represents an important tool for characterization of the peptide and protein secondary structures that mainly arise from the conformational disposition of the peptide backbone in solution. In 1991 Manning and Woody proposed that, in addition to the signal intensity, the ratio between [θ]nπ* and [θ]ππ*ǁ ((R2 ) ≅ [θ]222 /[θ]208 ), along with [θ]ππ*⊥ and [θ]ππ*ǁ ((R1 ) ≅ [θ]192 /[θ]208 ), may be utilized towards identifying the peptide/protein conformation (especially 310 - and α-helices). However, till date the use of the ratiometric ellipticity component for helical structure analysis of peptides and proteins has not been reported. We studied a series of temperature dependent CD spectra of a thermally stable, model helical peptide and its related analogs in water as a function of added 2,2,2-trifluoroethanol (TFE) in order to explore their landscape of helicity. For the first time, we have experimentally shown here that the R1 parameter can characterize better the individual helices, while the other parameter R2 and the signal intensity do not always converge. We emphasize the use of the R1 ratio of ellipticities for helical characterization because of the common origin of these two bands (exciton splitting of the amide π→ π* transition in a helical polypeptide). This approach may become worthwhile and timely with the increasing accessibility of CD synchrotron sources.

Platinum(II) and palladium(II) complexes of tridentate hydrazone-based ligands as selective guanine quadruplex binders

Several tridentate hydrazone-based ligands, synthesized by a condensation reaction of either 2-(1-methylhydrazinyl)pyridine or 2-(1-methylhydrazinyl)quinoline with an aldehyde (picolinaldehyde, 1H-pyrrole-2-carbaldehyde, 2-mercaptobenzaldehyde, 2-aminobenzaldehyde) have been reacted with palladium(II) and platinum(II) salts and precursor complexes to yield nine new metal complexes. These planar complexes were investigated with respect to their capability to interact with guanine quadruplex DNA. Two sequences (H-telo, derived from the human telomeric sequence, and c-myc, representing an excerpt of the promoter region of the c-Myc oncogene) were investigated. Circular dichroism (CD) spectroscopy, temperature-dependent CD spectroscopy, UV–Vis spectroscopy and a fluorescent intercalator displacement (FID) assay were applied in this respect. The spectroscopic data show that the complexes indeed interact with guanine quadruplex DNA. According to the FID assay, some of the complexes belong to the highest-affinity metal-containing quadruplex binders reported so far. Their affinity towards quadruplex DNA is up to 80-fold higher than to a reference double helix. These findings make the metal complexes good candidates as anticancer drugs, as guanine quadruplexes have been proposed as potential targets in anticancer drug design.

N-terminal diproline and charge group effects on the stabilization of helical conformation in alanine-based short peptides: CD studies with water and methanol as solvent.

Protein folding problem remains a formidable challenge as main chain, side chain and solvent interactions remain entangled and have been difficult to resolve. Alanine-based short peptides are promising models to dissect protein folding initiation and propagation structurally as well as energetically. The effect of N-terminal diproline and charged side chains is assessed on the stabilization of helical conformation in alanine-based short peptides using circular dichroism (CD) with water and methanol as solvent. A1 (Ac-Pro-Pro-Ala-Lys-Ala-Lys-Ala-Lys-Ala-NH2 ) is designed to assess the effect of N-terminal homochiral diproline and lysine side chains to induce helical conformation. A2 (Ac-Pro-Pro-Glu-Glu-Ala-Ala-Lys-Lys-Ala-NH2 ) and A3 (Ac-dPro-Pro-Glu-Glu-Ala-Ala-Lys-Lys-Ala-NH2 ) with N-terminal homochiral and heterochiral diproline, respectively, are designed to assess the effect of Glu...Lys (i, i+4) salt bridge interactions on the stabilization of helical conformation. The CD spectra of A1, A2 and A3 in water manifest different amplitudes of the observed polyproline II (PPII) signals, which indicate different conformational distributions of the polypeptide structure. The strong effect of solvent substitution from water to methanol is observed for the peptides, and CD spectra in methanol evidence A2 and A3 as helical folds. Temperature-dependent CD spectra of A1 and A2 in water depict an isodichroic point reflecting coexistence of two conformations, PPII and β-strand conformation, which is consistent with the previous studies. The results illuminate the effect of N-terminal diproline and charged side chains in dictating the preferences for extended-β, semi-extended PPII and helical conformation in alanine-based short peptides. The results of the present study will enhance our understanding on stabilization of helical conformation in short peptides and hence aid in the design of novel peptides with helical structures. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Structural and biophysical properties of h-FANCI ARM repeat protein

Fanconi anemia complementation groups – I (FANCI) protein facilitates DNA ICL (Inter-Cross-link) repair and plays a crucial role in genomic integrity. FANCI is a 1328 amino acids protein which contains armadillo (ARM) repeats and EDGE motif at the C-terminus. ARM repeats are functionally diverse and evolutionarily conserved domain that plays a pivotal role in protein–protein and protein–DNA interactions. Considering the importance of ARM repeats, we have explored comprehensive in silico and in vitro approach to examine folding pattern. Size exclusion chromatography, dynamic light scattering (DLS) and glutaraldehyde crosslinking studies suggest that FANCI ARM repeat exist as monomer as well as in oligomeric forms. Circular dichroism (CD) and fluorescence spectroscopy results demonstrate that protein has predominantly α- helices and well-folded tertiary structure. DNA binding was analysed using electrophoretic mobility shift assay by autoradiography. Temperature-dependent CD, Fluorescence spectroscopy and DLS studies concluded that protein unfolds and start forming oligomer from 30°C. The existence of stable portion within FANCI ARM repeat was examined using limited proteolysis and mass spectrometry. The normal mode analysis, molecular dynamics and principal component analysis demonstrated that helix-turn-helix (HTH) motif present in ARM repeat is highly dynamic and has anti-correlated motion. Furthermore, FANCI ARM repeat has HTH structural motif which binds to double-stranded DNA.

Bottom-Up Hierarchical Self-Assembly of Chiral Porphyrins through Coordination and Hydrogen Bonds.

A series of chiral synthetic compounds is reported that shows intricate but specific hierarchical assembly because of varying positions of coordination and hydrogen bonds. The evolution of the aggregates (followed by absorption spectroscopy and temperature-dependent circular dichroism studies in solution) reveal the influence of the proportion of stereogenic centers in the side groups connected to the chromophore ring in their optical activity and the important role of pyridyl groups in the self-assembly of these chiral macrocycles. The optical activity spans 2 orders of magnitude depending on composition and constitution. Two of the aggregates show very high optical activity even though the isolated chromophores barely give a circular dichroism signal. Molecular modeling of the aggregates, starting from the pyridine-zinc(II) porphyrin interaction and working up, and calculation of the circular dichroism signal confirm the origin of this optical activity as the chiral supramolecular organization of the molecules. The aggregates show a broad absorption range, between approximately 390 and 475 nm for the transitions associated with the Soret region alone, that spans wavelengths far more than the isolated chromophore. The supramolecular assemblies of the metalloporphyrins in solution were deposited onto highly oriented pyrolitic graphite in order to study their hierarchy in assembly by atomic force microscopy. Zero and one-dimensional aggregates were observed, and a clear dependence on deposition temperature was shown, indicating that the hierarchical assembly took place largely in solution. Moreover, scanning electron microscopy images of porphyrins and metalloporphyrins precipitated under out-of-equilibrium conditions showed the dependence of the number and position of chiral amide groups in the formation of a fibrillar nanomaterial. The combination of coordination and hydrogen bonding in the complicated assembly of these molecules-where there is a clear hierarchy for zinc(II)-pyridyl interaction followed by hydrogen-bonding between amide groups, and then van der Waals interactions-paves the way for the preparation of molecular materials with multiple chromophore environments.

Interplay between Lipid Interaction and Homo-coiling of Membrane-Tethered Coiled-Coil Peptides.

The designed coiled-coil-forming peptides E [(EIAALEK)3] and K [(KIAALKE)3] are known to trigger efficient membrane fusion when they are tethered to lipid vesicles in the form of lipopeptides. Knowledge of their secondary structure is a key element in understanding their role in membrane fusion. Special conditions can be found at the interface of the membrane, where the peptides are confined in close proximity to other peptide molecules as well as to the lipid interface. Consequently, different structural states were proposed for the peptides when tethered to this interface. Due to the multitude of possible states, determining the structure solely on the basis of circular dichroism (CD) spectra at a single temperature can be misleading. In addition, it has not yet been possible to unambiguously distinguish between the membrane-bound and the coiled-coil states of these peptides by means of infrared (IR) spectroscopy due to their very similar amide I' bands. Here, the molecular basis of this similarity is investigated by means of site-specific (13)C-labeled FTIR spectroscopy. Structural similarities between the membrane-interacting helix of K and the homo-coiled-coil-forming helix of E are shown to cause the similar spectroscopic properties. Furthermore, the peptide structure is investigated using temperature-dependent CD and IR spectroscopy, and it is shown that the different states can be distinguished on the basis of their thermal behavior. It is shown that the two peptides behave fundamentaly differently when tethered to the lipid membrane, which implies that their role during membrane fusion is different and the mechanism of this process is asymmetric.

Thermal, Chemical and pH Induced Unfolding of Turmeric Root Lectin: Modes of Denaturation

Curcuma longa rhizome lectin, of non-seed origin having antifungal, antibacterial and α-glucosidase inhibitory activities, forms a homodimer with high thermal stability as well as acid tolerance. Size exclusion chromatography and dynamic light scattering show it to be a dimer at pH 7, but it converts to a monomer near pH 2. Circular dichroism spectra and fluorescence emission maxima are virtually indistinguishable from pH 7 to 2, indicating secondary and tertiary structures remain the same in dimer and monomer within experimental error. The tryptophan environment as probed by acrylamide quenching data yielded very similar data at pH 2 and pH 7, implying very similar folding for monomer and dimer. Differential scanning calorimetry shows a transition at 350.3 K for dimer and at 327.0 K for monomer. Thermal unfolding and chemical unfolding induced by guanidinium chloride for dimer are both reversible and can be described by two-state models. The temperatures and the denaturant concentrations at which one-half of the protein molecules are unfolded, are protein concentration-dependent for dimer but protein concentration-independent for monomer. The free energy of unfolding at 298 K was found to be 5.23 Kcal mol−1 and 14.90 Kcal mol−1 for the monomer and dimer respectively. The value of change in excess heat capacity upon protein denaturation (ΔCp) is 3.42 Kcal mol−1 K−1 for dimer. The small ΔCp for unfolding of CLA reflects a buried hydrophobic core in the folded dimeric protein. These unfolding experiments, temperature dependent circular dichroism and dynamic light scattering for the dimer at pH 7 indicate its higher stability than for the monomer at pH 2. This difference in stability of dimeric and monomeric forms highlights the contribution of inter-subunit interactions in the former.

Biochemical Analysis of Recombinant AlkJ from Pseudomonas putida Reveals a Membrane-Associated, Flavin Adenine Dinucleotide-Dependent Dehydrogenase Suitable for the Biosynthetic Production of Aliphatic Aldehydes

The noncanonical alcohol dehydrogenase AlkJ is encoded on the alkane-metabolizing alk operon of the mesophilic bacterium Pseudomonas putida GPo1. To gain insight into the enzymology of AlkJ, we have produced the recombinant protein in Escherichia coli and purified it to homogeneity using His6 tag affinity and size exclusion chromatography (SEC). Despite synthesis in the cytoplasm, AlkJ was associated with the bacterial cell membrane, and solubilization with n-dodecyl-β-D-maltoside was necessary to liberate the enzyme. SEC and spectrophotometric analysis revealed a dimeric quaternary structure with stoichiometrically bound reduced flavin adenine dinucleotide (FADH2). The holoenzyme showed thermal denaturation at moderate temperatures around 35°C, according to both activity assay and temperature-dependent circular dichroism spectroscopy. The tightly bound coenzyme was released only upon denaturation with SDS or treatment with urea-KBr and, after air oxidation, exhibited the characteristic absorption spectrum of FAD. The enzymatic activity of purified AlkJ for 1-butanol, 1-hexanol, and 1-octanol as well as the n-alkanol derivative ω-hydroxy lauric acid methyl ester (HLAMe) was quantified in the presence of the artificial electron acceptors phenazine methosulfate (PMS) and 2,6-dichlorophenolindophenol (DCPIP), indicating broad substrate specificity with the lowest activity on the shortest alcohol, 1-butanol. Furthermore, AlkJ was able to accept as cosubstrates/oxidants the ubiquinone derivatives Q0 and Q1, also in conjunction with cytochrome c, which suggests coupling to the bacterial respiratory chain of this membrane-associated enzyme in its physiological environment. Using gas chromatographic analysis, we demonstrated specific biocatalytic conversion by AlkJ of the substrate HLAMe to the industrially relevant aldehyde, thus enabling the biotechnological production of 12-amino lauric acid methyl ester via subsequent enzymatic transamination.

Ultrafast Spectroscopic Studies on the Interaction of a Potential Food Carcinogen with Biologically Relevant Macromolecules

Benzo[a]pyrene (BP), a potential carcinogen in a class of pyrene derivatives shows interesting photophysics including very sharp vibronic structures in the emission spectrum. A detail spectroscopic study on the close interaction of the carcinogenic molecule with other biologically relevant macromolecules through ultrafast energy/charge transfer reactions is the motive of the present review. Firstly, we present our picosecond resolved studies on the Forster resonance energy transfer (FRET) from various vibronic bands in BP, showing strong dependency on the spectral overlap of an energy acceptor in a confined environment. Our study on the dipolar interactions between BP and different acceptors ethidium (Et), acridine orange (AO) and crystal violet (CV) at the surface of a model anionic micelle reveals the Forster distance (R0) and the rate of energy transfer to be dependent on the individual spectral overlap of the vibronic bands of BP with the absorption spectra of different energy acceptors. The differential behaviour of the vibronic bands is compared with that of different dyes (quantum dots; QDs) in a ‘dye-blend’ (mixture) under FRET to an energy acceptor. Such comparison of the FRET of QDs with that of BP, not only confirms independent nature of dipolar interaction of the vibronic bands with other organic molecules, the use of deconvolution technique in the interpretation of the donor-acceptor (D-A) distance has also been justified. We have also shown that consideration of differential FRET from vibronic bands of BP and from the QDs in the ‘dye-blend’ is equally acceptable in the theoretical frameworks including ‘Infelta-Tachiya’ model and D-A distribution analysis in the nano-environments. While such energy transfer reactions act as “spectroscopic ruler” to measure the distance between two sites on a macromolecule, reactions involving electron transfer (ET)/charge transfer (CT) and reactive oxygen species (ROS) play a pivotal role in carcinogenesis and cancer biochemistry. The review further emphasizes our studies on UVA radiation induced ET reaction as one of the key aspects of BP in the presence of a wide variety of molecules covering organic para-benzoquinone (BQ), biological macromolecules like calf-thymus DNA (CT-DNA), human serum albumin (HSA) protein and inorganic zinc oxide (ZnO) nanorods (NRs). Steady-state and picosecond-resolved fluorescence spectroscopy have been used to monitor such ET reactions. Physical consequences of BP association with CT-DNA have been investigated through temperature-dependent circular dichroism (CD) spectroscopy. The temperature-dependent steady-state, picosecond-resolved fluorescence lifetime and anisotropy studies reveal the effect of temperature on the perturbation of such ET reactions from BP to biological macromolecules, highlighting their temperature-dependent association. Furthermore, the electron donating property of BP has been corroborated by measuring wavelength-dependent photocurrent in a BP-anchored ZnO NR-based photodevice, offering new physical insights for the carcinogenic study of BP

Mechanistic study of CuZn-SOD from Ipomoea carnea mutated at dimer interface: Enhancement of peroxidase activity upon monomerization

The enzymatically active monomeric form of CuZn-superoxide dismutase has always been of interest to decipher the structure-function relationship in this class of enzymes. In the present study, spectroscopic and enzymatic characteristics of the dimeric and monomeric forms of recombinant Ipomoea carnea CuZn-superoxide dismutase were made to decipher their stability and altered catalytic properties. The monomeric form of protein was produced through site directed mutagenesis by replacing a conserved hydrophobic leucine with a polar lysine residue at the dimer-interface. Spectral characteristics of both the forms (monomer and dimer) showed the presence of novel electronic transitions. Superoxide scavenging activity of the mutated form was reduced to nearly half of the activity found in the native enzyme. Concomitantly, compared to native form the mutated enzyme showed an increase in peroxidase activity. High temperature dependent circular dichroism spectral analysis, differential scanning calorimetric profile, and the measurement of temperature dependent superoxide scavenging activity indicated an increased susceptibility of the mutated form to higher temperature as compared to the native form. The inhibitor studies like hydrogen peroxide, diethyldithiocarbamate and phenylglyoxal also indicate higher susceptibility, which might be due to, altered arrangement of active site residues as a consequence of the mutation. Molecular modeling and MD simulation studies further indicated that this specific mutation induces loss of hydrophobic interaction at dimer interface, resulting in the observed instability of the dimeric form. Increased peroxidative activity of the enzyme, upon monomerization may have physiological implication essentially in presence of high concentration of H2O2, as in case of plant cells specifically under stress conditions.

Molecular recognition of a model globular protein apomyoglobin by synthetic receptor cyclodextrin: effect of fluorescence modification of the protein and cavity size of the receptor in the interaction

Labelling of proteins with some extrinsic probe is unavoidable in molecular biology research. Particularly, spectroscopic studies in the optical region require fluorescence modification of native proteins by attaching polycyclic aromatic fluoroprobe with the proteins under investigation. Our present study aims to address the consequence of the attachment of a fluoroprobe at the protein surface in the molecular recognition of the protein by selectively small model receptor. A spectroscopic study involving apomyoglobin (Apo-Mb) and cyclodextrin (CyD) of various cavity sizes as model globular protein and synthetic receptors, respectively, using steady-state and picosecond-resolved techniques, is detailed here. A study involving Förster resonance energy transfer, between intrinsic amino acid tryptophan (donor) and N, N-dimethyl naphthalene moiety of the extrinsic dansyl probes at the surface of Apo-Mb, precisely monitor changes in donor acceptor distance as a consequence of interaction of the protein with CyD having different cavity sizes (β and γ variety). Molecular modelling studies on the interaction of tryptophan and dansyl probe with β-CyD is reported here and found to be consistent with the experimental observations. In order to investigate structural aspects of the interacting protein, we have used circular dichroism spectroscopy. Temperature-dependent circular dichroism studies explore the change in the secondary structure of Apo-Mb in association with CyD, before and after fluorescence modification of the protein. Overall, the study well exemplifies approaches to protein recognition by CyD as a synthetic receptor and offers a cautionary note on the use of hydrophobic fluorescent labels for proteins in biochemical studies involving recognition of molecules.

Characterization and molecular modelling of an engineered organic solvent tolerant, thermostable lipase with enhanced enzyme activity

Abstract To evolve a variant with improved enzymatic properties i.e. thermostability, catalytic efficiency, by error prone mutagenesis, lipR1 was used as template. Using two tier screening protocol, a highly thermostable lipase variant (LipR3) with a single amino acid change (R214C) was identified. The LipR3 lipase demonstrated 9 folds enhanced catalytic efficiency in comparison to LipR1 enzyme. The variant demonstrated 90 folds higher thermostability at 60 °C, when compared with the parent enzyme. In addition it displayed appreciable tolerance to organic solvents. The variant gene was cloned in pQE30-UA and the protein was expressed in E. coli . The protein product of approximately 44.0 kDa was purified by affinity chromatography and characterized in detail. Temperature dependent circular dichroism and steady state fluorescence studies of LipR3 and LipR1 lipase supported the observation that LipR3 is more thermostable than LipR1. Structural analysis of the homology model of LipR3 showed that the Arg 214 was substituted with Cys at a large surface loop which connects lid helix (α6) to another helix (α7). The hydrophobic residues of the loop (Val 197, Tyr 204 and Phe 206) formed a compact hydrophobic region with residues from lid helix. It is suggested that replacement of Arg with Cys leads to packing of Cys favourably with hydrophobic core and provide structural stability to LipR3.

UVA Radiation Induced Ultrafast Electron Transfer from a Food Carcinogen Benzo[a]pyrene to Organic Molecules, Biological Macromolecules, and Inorganic Nano Structures

Reactions involving electron transfer (ET) and reactive oxygen species (ROS) play a pivotal role in carcinogenesis and cancer biochemistry. Our present study emphasizes UVA radiation induced ET reaction as one of the key aspects of a potential carcinogen, benzo[a]pyrene (BP), in the presence of a wide variety of molecules covering organic p-benzoquinone (BQ), biological macromolecules like calf-thymus DNA (CT-DNA), human serum albumin (HSA) protein, and inorganic zinc oxide (ZnO) nanorods (NRs). Steady-state and picosecond-resolved fluorescence spectroscopy have been used to monitor such ET reactions. Physical consequences of BP association with CT-DNA have been investigated through temperature-dependent circular dichroism (CD) spectroscopy. The temperature-dependent steady-state, picosecond-resolved fluorescence lifetime and anisotropy studies reveal the effect of temperature on the perturbation of such ET reactions from BP to biological macromolecules, highlighting their temperature-dependent association. Furthermore, the electron-donating property of BP has been corroborated by measuring wavelength-dependent photocurrent in a BP-anchored ZnO NR-based photodevice, offering new physical insights for the carcinogenic study of BP.