Measurement of energy landscape roughness of folded and unfolded proteins.

The dynamics of protein conformational changes, from protein folding to smaller changes, such as those involved in ligand binding, are governed by the properties of the conformational energy landscape. Different techniques have been used to follow the motion of a protein over this landscape and thus quantify its properties. However, these techniques often are limited to short timescales and low-energy conformations. Here, we describe a general approach that overcomes these limitations. Starting from a nonnative conformation held by an aromatic disulfide bond, we use time-resolved spectroscopy to observe nonequilibrium backbone dynamics over nine orders of magnitude in time, from picoseconds to milliseconds, after photolysis of the disulfide bond. We find that the reencounter probability of residues that initially are in close contact decreases with time following an unusual power law that persists over the full time range and is independent of the primary sequence. Model simulations show that this power law arises from subdiffusional motion, indicating a wide distribution of trapping times in local minima of the energy landscape, and enable us to quantify the roughness of the energy landscape (4-5 k(B)T). Surprisingly, even under denaturing conditions, the energy landscape remains highly rugged with deep traps (>20 k(B)T) that result from multiple nonnative interactions and are sufficient for trapping on the millisecond timescale. Finally, we suggest that the subdiffusional motion of the protein backbone found here may promote rapid folding of proteins with low contact order by enhancing contact formation between nearby residues.

Insight into the early stages of thermal unfolding of peanut agglutinin by molecular dynamics simulations.

Peanut agglutinin is a homotetrameric nonglycosylated protein. The protein has a unique open quaternary structure. Molecular dynamics simulations have been employed to follow the atomistic details of its unfolding at different temperatures. The early events of the deoligomerization of the protein have been elucidated in the present study. Simulation trajectories of the monomer as well as those of the tetramer have been compared and the tetramer is found to be substantially more stable than its monomeric counterpart. The tetramer shows retention of most of its secondary structure but considerable loss of the tertiary structure at high temperature. This observation implies the generation of a molten globule-like intermediate in the later stages of deoligomerization. The quaternary structure of the protein has weakened to a large extent, but none of the subunits are separated. In addition, the importance of the metal-binding to the stability of the protein structure has also been investigated. Binding of the metal ions not only enhances the local stability of the metal-ion binding loop, but also imparts a global stability to the overall structure. The dynamics of different interfaces vary significantly as probed through interface clusters. The differences are substantially enhanced at higher temperatures. The dynamics and the stability of the interfaces have been captured mainly by cluster analysis, which has provided detailed information on the thermal deoligomerization of the protein.

Dendritic Polynitrato Energetic Motifs: Development and Exploration of Physicochemical Behavior through Theoretical and Experimental Approach.

Considering the fundamental and most desirable characteristics of energetic materials, a series of 1,2,3-triazole-based heterocyclic energetic motifs nicely tuned with nitrato (-ONO2) functionality were synthesized by a microwave-assisted environmental friendly synthetic approach with good yields. Thermal stability and the nature of evolved gases on decomposition of structurally characterized energetic motifs were analyzed by thermogravimetric analysis (TGA), differential scanning calorimetry (DSC) analysis and Fourier transform infrared coupled with TGA-DSC. The explosiveness of these motifs was explored by calculation of enthalpy of formation and density employing density functional theory, and the detonation performances (detonation pressure and velocity) were explored using EXPLO5_V6.03. All of these compounds were calculated to have better oxygen balance (-36 to -52%) as compared to that of trinitrotoluene (-74%). Most of the nitrate ester derivatives were found to exhibit low impact sensitivities, high densities, good thermal stabilities, and promising detonation properties, and PN 3 was observed to be a superior candidate in terms of its energetic characteristics. Hence, the experimental and theoretical outcomes strongly reflect that the present approach of developing dendritic high energetic materials bearing green explosive characteristics might be a potential pathway for designing and synthesizing green explosives with desired characteristics.

Dynamic light scattering study of peanut agglutinin: size, shape and urea denaturation.

Peanut agglutinin (PNA)is a homotetrameric protein with a unique open quaternary structure. PNA shows non-two state profile in chaotrope induced denaturation. It passes through a monomeric molten globule like state before complete denaturation (Reddy et al 1999). This denaturation profile is associated with the change in hydrodynamic radius of the native protein. Though the molten globule-like state is monomeric in nature it expands in size due to partial denaturation. The size and shape of the native PNA as well as the change in hydrodynamic radius of the protein during denaturation has been studied by dynamic light scattering (DLS). The generation of two species is evident from the profile of hydrodynamic radii. This study also reveals the extent of compactness of the intermediate state.

Highly photostable zinc selective molecular marker bearing flexible pivotal unit: opto-fluorescence enhancement effect and imaging applications in living systems.

Novel molecular probes for imaging zinc in biological systems are gaining interest as they help in understanding the role of zinc in regulating various bio-events. In this regard, a new C2-symmetric molecular system has been developed and successfully applied as light-up material for signaling divalent zinc with green emission. The fluorescence enhancement was highly zinc specific and this newly developed probe bears a submicromolar detection capability. While probe and the ensemble -Zn(2+) exhibited remarkably high photostability, light-triggered fluorescence enhancement was observed in the case of -Zn(2+). The nature of the -Zn(2+) complex and the associated spectral shift are further supported by theoretical calculations. As the present probe absorbs in the visible region and emits in the green, it was preferred as a potential material for imaging zinc in biological systems including animal and plant cells such as pollen grains and fish egg cells. Such fluorescence imaging of zinc revealed the efficacy of the probe in detection and localization of zinc in various biological systems.

2,2,2-Trifluoroethanol-Induced structural change of peanut agglutinin at different pH: A comparative account.

Peanut Agglutinin (PNA) is a legume lectin with a unique open quarternary structure. It is a homotetrameric protein, the monomeric subunit of which is made up of 3 beta sheets. The structural change in this protein has been induced by 2,2,2-trifluoroethanol (TFE) at two different pH. At neutral pH, PNA exists as a homotetramer, while at pH 2.5, it is known to dissociate to a dimer. The effect of TFE has been studied at both the pH by intrinsic tryptophan fluorescence, far and near UV Circular Dichroism, ANS binding and dynamic light scattering. At low pH, 15% TFE is found to induce a molten globule like state that shows maximum ANS binding. Increasing concentration of TFE increases alpha helical content and the compactness of the protein. The compact PNA at higher concentration of TFE is structurally different from the native structure. The effect of TFE at neutral pH on PNA is somewhat different from that observed at low pH. TFE does not induce molten globule like state at this pH. The detailed study of the structural change of PNA by TFE has been presented.

Thermodynamic analysis of three state denaturation of Peanut Agglutinin.

Peanut Agglutinin (PNA) is a homotetrameric protein with a very unusual open quaternary structure. During denaturation, it first dissociates into a molten globule like state, which subsequently undergoes complete denaturation. Urea denaturation of PNA at neutral pH has been studied by intrinsic fluorescence spectroscopy and has been fitted to a three state model, A4 <=> 4I <=> 4U, to get all the relevant thermodynamic parameters. Urea denaturation leads to continuous red shift of wavelength maxima. The molten globule like state is formed in a short range of urea concentration. Refolding of the denatured PNA has been attempted by intrinsic fluorescence study. Refolding by instantaneous dilution shows the occurrence of the formation of an intermediate at a relatively rapid rate, within few seconds. The transition from PNA tetramer to molten globule like state is found to have a DeltaG value of approximately 33 kcal/mole while it is approximately 8 kcal/mole for the transition from molten globule like state to a completely denatured state. This in turn indicates that the tetramerization in PNA contributes significantly to the stability of the oligomer.

Spectral and kinetic characterization of 7,8-diaminopelargonic acid synthase from Mycobacterium tuberculosis.

The indispensability of biotin for crucial processes like lipid biosynthesis coupled to the absence of the biotin biosynthesis pathway in humans make the enzymes of this pathway, attractive targets for development of novel drugs against numerous pathogens including M. tuberculosis. We report the spectral and kinetic characterization of the Mycobacterium tuberculosis 7,8-Diaminopelargonic acid (DAPA) synthase, the second enzyme of the biotin biosynthesis pathway. In contrast to the E. coli enzyme, no quinonoid intermediate was detected during the steady state reaction between the enzyme and S-adenosyl-L-methionine (SAM). The second order rate constant for this half of the reaction was determined to be 1.75 +/- 0.11 M-1s-1. The Km values for 7-keto-8-aminopelargonic acid (KAPA) and SAM are 2.83 microM and 308.28 microM, respectively whereas the Vmax and kcat values for the enzyme are 0.02074 micromoles/min/ml and 0.003 s-1, respectively. Our initial studies pave the way for further detailed mechanistic and kinetic characterization of the enzyme.

Broad substrate stereospecificity of the Mycobacterium tuberculosis 7-keto-8-aminopelargonic acid synthase: Spectroscopic and kinetic studies.

Biotin is an essential enzyme cofactor required for carboxylation and transcarboxylation reactions. The absence of the biotin biosynthesis pathway in humans suggests that it can be an attractive target for the development of novel drugs against a number of pathogens. 7-Keto-8-aminopelargonic acid (KAPA) synthase (EC 2.3.1.47), the enzyme catalyzing the first committed step in the biotin biosynthesis pathway, is believed to exhibit high substrate stereospecificity. A comparative kinetic characterization of the interaction of the mycobacterium tuberculosis KAPA synthase with both L- AND D-alanine was carried out to investigate the basis of the substrate stereospecificity exhibited by the enzyme. The formation of the external aldimine with D-alanine (k = 82.63 m(-1) s(-1)) is approximately 5 times slower than that with L-alanine (k = 399.4 m(-1) s(-1)). In addition to formation of the external aldimine, formation of substrate quinonoid was also observed upon addition of pimeloyl-CoA to the preformed d-alanine external aldimine complex. However, the formation of this intermediate was extremely slow compared with the substrate quinonoid with L-alanine and pimeloyl-CoA (k = 16.9 x 10(4) m(-1) s(-1)). Contrary to earlier reports, these results clearly show that D-alanine is not a competitive inhibitor but a substrate for the enzyme and thereby demonstrate the broad substrate stereospecificity of the M. tuberculosis KAPA synthase. Further, d-KAPA, the product of the reaction utilizing D-alanine inhibits both KAPA synthase (Ki = 114.83 microm) as well as 7,8-diaminopelargonic acid synthase (IC50 = 43.9 microm), the next enzyme of the pathway.