Molecular analysis of changes in DNA binding affinity and bending extent induced by the mutations on the aromatic amino residues in Cren7.

Proteins from Crenarchaeal organisms exhibit remarkable thermal stability. The aromatic amino acids in Cren7, a Crenarchaeal protein, regulate protein stability and further modulate DNA binding and its compaction. Specific aromatic amino acids were mutated, and using spectroscopic and theoretical approaches, we have examined the structure, DNA binding affinity, and DNA bending ability of mutants. and compared with wild-type (WT) Cren7. The reverse titration profiles were analysed by a noncooperativeMcGhee-von Hippel model to estimate affinity constant (Ka) and site size (n) associated with binding to the DNA. Biolayer interferometry (BLI) measurements showed that the binding affinity decreased at higher salt concentrations. For theoretical analysis of extent of DNA bending, radius of gyration and bending angle were compared for WT and mutants. Time evolution of order parameters based on translational and rotational motion of tryptophan residue (W26) was used for qualitative detection of stacking interactions between W26 of Cren7 and DNA nucleobases. It was observed that orientation of W26 in F41A favored formation of a new lone pair-lone pair interaction between DNA and Cren7. Consequently, in thermostable proteins, the aromatic residues at the terminus maintain structural stability, whereas the residues at the core optimize the degree of DNA bending and compaction.

Molecular contacts in the Cren7-DNA complex: A quantitative investigation for electrostatic interaction.

The molecular association of proteins with nucleic acids leading to the formation of macromolecular complexes is a crucial step in several biological processes. Stabilization of these complexes involves electrostatic interactions between ion pairs (salt bridges) of nucleic acid phosphates and protein side chains. The crenarchaeal DNA binding protein, Cren7 plays a key role in the regulation of chromosomal structure and gene expression in eukaryotic extremophiles. However, the molecular contacts that occur at the interface of protein-DNA complexes and their contribution to the electrostatic interaction have not been fully elucidated. This work presents a quantitative description of the mechanism of the electrostatic interaction between the protein and DNA. We have identified a few residues located at the Cren7-DNA interface that could potentially be responsible for the interaction. Structural studies using circular dichroism indicate mutation of these surface residues minimally affect their structure and stability. The binding affinity of these mutants for the DNA duplexes was examined from reverse titration, biolayer interferometry, and fluorescence anisotropy measurements with calf thymus DNA, polynucleotides, and small DNA oligonucleotides. The resulting kinetic parameters highlight a difference in electrostatic interactions potentials exhibited by residues positioned at different locations of the protein-DNA interface. Computational studies attribute this difference to their surrounding atmosphere and energetic stabilization parameters. The biophysical approach described here can be extended for other proteins that play a crucial role in DNA bending and compaction, to properly evaluate the role of specific residues on the mechanisms of DNA binding.

Structural and thermodynamic insights into the Cren7 mediated DNA organization in Crenarchaeota.

Archaea have histone homologues and chromatin proteins to organize their DNA into a compact form. This allows them to survive in extreme climates. Cren7 is one such chromatin protein conserved in Crenarchaeota. When Cren7 binds to model natural DNA, calf thymus DNA (CTD, 58% AT content) and polynucleotides under adverse solution conditions (high temperature, ionic strength), CD bands at 275-290 nm shift to higher wavelengths indicating structural changes in DNA. It formed a strong complex with CTD and poly(dA-dT)·poly(dA-dT), via a combination of electrostatic and non-electrostatic interactions. A low binding enthalpy indicated that the process was driven by entropy. The interaction was independent of the nature of the anions present in the solution. On studying the variation in protein affinity with salt concentration, it was estimated that the electrostatic interaction at the interface involves 3 pairs of ions at the protein-DNA interface. The affinity and binding site size decreased on changing the pH of the solution (between pH 6 and 8), but temperature did not result in such effects. Cren7 bound to 10 bp of DNA, increasing its flexibility and thermal stability by more than 30 °C. Increasing the amount of Cren7 produces cooperative structural transitions in DNAs without any similar transition in the protein. These crucial binding parameters, energetics, and structural changes decipher the mystery of Cren7 mediated DNA organization in Crenarchaeota.

Critical insights into the interactions of heat shock protein 70 with phospholipids.

Heat shock proteins (Hsps) stabilize the newly synthesized polypeptide chains preventing them from aggregation. They contribute to systemic response under stress and thus behave as signaling molecules. Hsp70 has been detected on the surface of stressed cells. It translocates to the extracellular environment through the plasma membrane without causing cell death. But the interaction of the protein with the membrane leading to the export process remains elusive. Hsp70 has a tendency to generate channels within lipid bilayers, and this has been a driving force for studying protein-lipid interactions. Transport of these proteins across the membrane paves their pathways for performing the desired function. We have attempted to characterize how the interaction of Hsp70 with negatively charged phospholipids affects the structure of lipids. This study will help in explaining the transport mechanism of proteins that are devoid of defined signaling pathways. The interaction of amino acids of Hsp70 with the head and tail group leads to the rearrangement of the hydration layer in contact with the bilayers. Critical analysis of the results obtained from small-angle X-ray scattering along with QCM-D provides valuable insights to analyze the effect of Hsp70 adsorption on an anionic POPS lipid bilayer.

Insecticide resistance and molecular characterization of knockdown resistance (kdr) in Culex quinquefasciatus mosquitoes in Sri Lanka.

Resistance to pyrethroids (PY) and organophosphate (OP) insecticides is widespread among populations of Culex quinquefasciatus, the major vector of lymphatic filariasis (LF). The present study was designed to detect the L1014F kdr (knockdown resistant) mutation among Cx. quinquefasciatus populations in the filarial belt of Sri Lanka. Mosquitoes were reared from field-caught larvae from seven localities where LF is endemic. Susceptibility status of Cx. quinquefasciatus to adulticides, 0.05% deltamethrin, 0.75% permethrin, 5% malathion, and the larvicide temephos was determined using the standard WHO susceptibility tests. A fragment of vgsc gene was amplified and sequenced to identify the responsible kdr mutations. The susceptibility test results revealed less than 90% mortalities for 0.05% deltamethrin, and 0.75% permethrin and temephos. For 5% malathion, all study sites except Maharagama revealed greater than 90% mortality. The L1014F kdr mutation was observed in all studied populations. Although the overall microfilaria rate is less than 1% in the country, there is a high risk of re-emergence of LF in Sri Lanka due to abundant Cx. quinquefasciatus mosquitoes, increased resistant status to currently used insecticides, imported LF cases, higher rates of microfilaria among neighboring countries, and the advancement of tourism.