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.

Elastic compliance of fibrillar assemblies in type I collagen.

Fibrillary assemblies of Type I collagen find important applications in tissue engineering and as matrices for biophysical studies. The mechanical and structural properties of these structures are governed by factors such as protein concentration, temperature, pH and ionic strength. This study reports on an impedance based analysis of the elastic compliance of fibrillary assemblies of Type I collagen using quartz crystal microbalance with dissipation (QCM-D) at a fundamental frequency of 5 MHz and overtones (n = 3,5,7,9,11). Here, In situ partial fibrillation of the adsorbing collagen followed by its fibrillary assemblies on hydrophilic gold coated quartz surface have been crosslinked using Gallic acid (GA), Chromium (III) gallate (Cr-GA), Catechin (Cat), Tetrakis(hydroxymethyl)phosphonium sulfate (THPS) and Oxazolidine (Ox). This approach allows direct comparison of how viscoelastic properties track the structural evolution of the fiber and network length scales. The collagen crosslinking shows significant positive impact on the protein's mechanical behaviour and on the type of crosslinking agents used. The elastic modulus increases as collagen 

Colloids versus solution state adsorption of proteins: Interaction of Myoglobin with supported lipid bilayers.

This study examines adsorption of Myoglobin (Mb) in solution and as colloid on supported lipid bilayers of neutral phospholipids and a mixture of neutral+cationic lipids formed on gold coated quartz in a Quartz crystal microbalance (QCM). Results indicate that thin adsorbed films of Mb in solution and as colloids, show atleast 3 steps in the interaction with the bilayers: i) An initial strain of a viscoelastic film ii) Entrained water that moves in and out of the adsorbed film and iii) The coupled load from the bulk liquid which increases the strain of the film. These three components constitute an effective viscoelastic film which is rigidly coupled to the QCM. Grazing incidence XRD (GIXD) shows that the bilayer head group remains nearly undisturbed for Mb solution with pure (neutral) and (neutral+cationic) mixtures, whereas for the colloids there is an increase in head group thickness with neutral and decrease in the case of mixture. Unsaturation in the alkyl tails in the neutral lipid resulting in flexible disordered bilayers and more entrained water in the cationic system results in these changes. The sensitivity of QCM-D, makes it useful to study real-time monitoring of bilayer structural robustness cytotoxicity, drug delivery and lipid self-assembly.

Initiating fibro-proliferation through interfacial interactions of myoglobin colloids with collagen in solution.

This work examines fibro-proliferation through interaction of myoglobin (Mb), a globular protein with collagen, an extracellular matrix fibrous protein. Designed colloids of Mb at pH 4.5 and 7.5 have been mixed with collagen solution at pH 7.5 and 4.5 in different concentrations altering their surface charges. For the Mb colloids, 100-200nm sizes have been measured from Transmission electron micrographs and zeta sizer. CD spectra shows a shift to beta sheet like structure for the protein in the colloids. Interaction at Mb/Collagen interface studied using Dilational rheology, Quartz crystal microbalance with dissipation and Differential Scanning calorimetry show that the perturbation is not only by the charge compensation arising from the difference in pH of the colloids and collagen, but also by the organized assembly of collagen at that particular pH. Results demonstrate that positive Mb colloids at pH 4.5, having more% of entrained water stabilize the collagen fibrils (pH 7.5) around them. Ensuing dehydration leads to effective cross-linking and inherently anisotropic growth of fibrils/fibres of collagen. In the case of Mb colloids at pH 7.5, the fibril formation seems to supersede the clustering of Mb suggesting that the fibro-proliferation is both pH and hydrophilic-hydrophobic balance dependent at the interface.

Combination of driselase and lysing enzyme in one molar potassium chloride is effective for the production of protoplasts from germinated conidia of Fusarium verticillioides.

Various cell wall degrading enzymes and the protoplasting media were evaluated for the production of protoplast in Fusarium verticillioides. Among the various enzymes tested, driselase at 12.5 mg/ml in 1 M KCl protoplasting medium produced the maximum number of protoplast. Next to driselase, lysing enzyme at 10 mg/ml in 1.2 M MgSO4 protoplasting medium was found to be the second best enzyme for the production of protoplast. More interestingly, the combined use of driselase @ 12.5 mg/ml and lysing enzyme @ 10 mg/ml in 1 M KCl exhibited the additive effect on protoplast formation. Germinated conidia of F. verticillioides are the most susceptible fungal material for protoplast production. The use of sucrose at 1.2 M in the regeneration medium supported the maximum regeneration of protoplast. From the present study, we recommend driselase (12.5 mg/ml) and lysing enzyme (10 mg/ml) in 1 M KCl protoplasting medium and germinated conidia of F. verticillioides for the maximum production of protoplasts and 1.2 M sucrose is the best osmoticum for the regeneration of protoplasts.