RMSF.

<p>Per residue backbone RMSF of the first three eigenvectors for f298L (top) and f325L (bottom) simulations.</p

Per residue RMSD.

<p>Conformational changes within each domain based on per residue backbone RMSD calculated for two simulations by fitting to the backbone of corresponding domain (A) N-domain (B) C-domain.</p

Structural and dynamical insight into thermally induced functional inactivation of firefly luciferase

<div><p>Luciferase is the key component of light production in bioluminescence process. Extensive and advantageous application of this enzyme in biotechnology is restricted due to its low thermal stability. Here we report the effect of heating up above T_m on the structure and dynamical properties of luciferase enzyme compared to temperature at 298 K. In this way we demonstrate that the number of hydrogen bonds between N- and C-domain is increased for the free enzyme at 325 K. Increased inter domain hydrogen bonds by three at 325 K suggests that inter domain contact is strengthened. The appearance of simultaneous strong salt bridge and hydrogen bond between K529 and D422 and increased existence probability between R533 and E389 could mechanistically explain stronger contact between N- and C-domain. Mutagenesis studies demonstrated the importance of K529 and D422 experimentally. Also the significant reduction in SASA for experimentally important residues K529, D422 and T343 which are involved in active site region was observed. Principle component analysis (PCA) in our study shows that the dynamical behavior of the enzyme is changed upon heating up which mainly originated from the change of motion modes and associated extent of those motions with respect to 298 K. These findings could explain why heating up of the enzyme or thermal fluctuation of protein conformation reduces luciferase activity in course of time as a possible mechanism of thermal functional inactivation. According to these results we proposed two strategies to improve thermal stability of functional luciferase.</p></div

Hydrogen bond statistics.

<p>Hydrogen bond statistics.</p

Solvent accessible surface area.

<p>Solvent accessible surface area.</p

The projection of simulation trajectory on the first three eigenvectors and corresponding probability distributions.

<p>Also two extreme structures along with conformations of the linear interpolation between them are shown. (A) f298L. (B) f325L.</p

Time relaxation of enzyme structure obtained by backbone RMSD analysis.

<p>The Comparative diagram for various calculations of Backbone RMSD by regarding N-domain, C-domain and the whole protein as the reference fitting structure for (A) f298 and (B) f325. Comparisons between two simulations RMSDs calculated relative to (C) N-domain (D) C-domain and (E) whole protein backbone.</p

Eigenvalues.

<p>The eigenvalues of the first 20 eigenvectors in descending order for f298L and f325L MD simulations. The inset shows the cumulative contribution of eigenvectors.</p

Detection of Early Stage Apoptotic Cells Based on Label-Free Cytochrome c Assay Using Bioconjugated Metal Nanoclusters as Fluorescent Probes

Cytochrome <i>c</i> (Cyt <i>c</i>) is an important biomarker in cell lysates for the early stage of apoptosis or anticancer agents. Here, two novel label-free fluorescence assays based on hemoglobin-stabilized gold nanoclusters (Hb/AuNCs) and aptamer-stabilized silver nanoclusters (DNA/AgNCs) for analysis of Cyt <i>c</i> are presented. The heme group of the protein induces sensitive sensing platforms accompanied by the decreased fluorescence of both metal nanoclusters. The quenching processes observed found to be based on the fluorescence resonance energy transfer mechanism from Hb/AuNCs to Cyt <i>c</i> and photoinduced electron transfer from DNA/AgNCs to the aptamer-Cyt <i>c</i> complex. The linear range for Cyt <i>c</i> was found to be 0–10 μM for Hb/AuNCs and from 0 to 1 μM for DNA/AgNCs, with limits of detection of ∼15 nM. On the basis of strong binding affinity of DNA aptamers for their target proteins, the DNA/AgNCs probe was successfully applied to the quantitative determination of Cyt <i>c</i> in cell lysates, which opens a new avenue to early diagnostics and drug screening with high sensitivity. Compared to the conventional Western blot method, the presented assays are low cost, easy to prepare the fluorescent probes, and sensitive, while overall time for the detection and quantitation of Cyt <i>c</i> from isolated mitochondria is only 20 min. The proposed method for Cyt <i>c</i> detection may also be useful for the study of those materials that cause mitochondrial dysfunction and apoptotic cell death

Photoluminescence Mechanisms of Dual-Emission Fluorescent Silver Nanoclusters Fabricated by Human Hemoglobin Template: From Oxidation- and Aggregation-Induced Emission Enhancement to Targeted Drug Delivery and Cell Imaging

A novel fundamental understanding of the features of mechanism for the synthesis of luminescent silver nanoclusters (AgNCs) in human hemoglobin (Hb) as capping/reducing agents is presented based upon simultaneous size transition and fluorescence enhancement phenomena  The interesting features consist of both NC core oxidation and aggregation-induced emission (AIE) attributed to ligand-to-metal charge transfer (LMCT) or ligand-to-metal–metal charge transfer (LMMCT) from Ag­(I)-Hb complexes (through oxygen, nitrogen, and sulfur atoms of Hb residues donation to the Ag­(I) ions) forming Ag(0)@Ag­(I)–Hb core–shell NCs, the origin and consequence being a dual emission/single excitation nanosystem with large stocks shift and high quantum yield obtained even at high temperature which is a challenging subject,  is not reported until now. The bioconjugation of hyaluronic acid (HA) onto surfaces of an Hb layer (HA/AgNCs) produced a biocompatible platform with a doxorubicin drug (DOX) as DOX/HA/AgNCs for specific imaging and delivery of DOX via an efficient targeting of CD44-overexpressing cancer cells, which lead to an increased inhibition of tumor cell growth. Additionally, the cell viability analysis illustrated that the developed nanocarriers significantly enhanced the DOX uptake in HeLa cancer cells compared to HUVEC and HNCF-PI 52 normal cells allowing a selective cytotoxicity to HeLa cells. The suggested LMCT/LMMCT mechanism for an emission source combined with such attractive properties as a simple one-pot, nontoxic, synthesis route, long lifetime, large Stocks shift, excellent aqueous stability and photostability, and easy functionalization capability with good cell viability provided the possibility for a AgNCs nanoprobe for use to better understand the nucleation and growth mechanisms via computational modeling techniques (e.g., DFT study) and also for fabrication of new nanoprobes for developing multifunctional applications in the biobased chemical and electrochemical fields and in in vivo research