Natural protein-based electrospun nanofibers for advanced healthcare applications: progress and challenges.

Electrospinning is an electrostatic fiber fabrication technique that operates by the application of a strong electric field on polymer solution or melts. It is used to fabricate fibers whose size lies in the range of few microns to the nanometer range. Historic development of electrospinning has evinced attention due to its outstanding attributes such as small diameter, excellent pore inter-connectivity, high porosity, and high surface-to-volume ratio. This review aims to highlight the theory behind electrospinning and the machine setup with a detailed discussion about the processing parameters. It discusses the latest innovations in natural protein-based electrospun nanofibers for health care applications. Various plant- and animal-based proteins have been discussed with detailed sample preparation and corresponding processing parameters. The usage of these electrospun nanofibers in regenerative medicine and drug delivery has also been discussed. Some technical innovations in electrospinning techniques such as emulsion electrospinning and coaxial electrospinning have been highlighted. Coaxial electrospun core-shell nanofibers have the potential to be utilized as an advanced nano-architecture for sustained release targeted delivery as well as for regenerative medicine. Healthcare applications of nanofibers formed via emulsion and coaxial electrospinning have been discussed briefly. Electrospun nanofibers have still much scope for commercialization on large scale. Some of the available wound-dressing materials have been discussed in brief.

Multiple putative methemoglobin reductases in C. reinhardtii may support enzymatic functions for its multiple hemoglobins.

The ubiquitous nature of hemoglobins, their presence in multiple forms and low cellular expression in organisms suggests alternative physiological functions of hemoglobins in addition to oxygen transport and storage. Previous research has proposed enzymatic function of hemoglobins such as nitric oxide dioxygenase, nitrite reductase and hydroxylamine reductase. In all these enzymatic functions, active ferrous form of hemoglobin is converted to ferric form and reconversion of ferric to ferrous through reduction partners is under active investigation. The model alga C. reinhardtii contains multiple globins and is thus expected to have multiple putative methemoglobin reductases to augment the physiological functions of the novel hemoglobins. In this regard, three putative methemoglobin reductases and three algal hemoglobins were characterized. Our results signify that the identified putative methemoglobin reductases can reduce algal methemoglobins in a nonspecific manner under in vitro conditions. Enzyme kinetics of two putative methemoglobin reductases with methemoglobins as substrates and in silico analysis support interaction between the hemoglobins and the two reduction partners as also observed in vitro. Our investigation on algal methemoglobin reductases underpins the valuable chemistry of nitric oxide with the newly discovered hemoglobins to ensure their physiological relevance, with multiple hemoglobins probably necessitating the presence of multiple reductases.

Time-dependent study of graphene oxide-trypsin adsorption interface and visualization of nano-protein corona.

Understanding of interactions of nanomaterials with biomolecules (especially proteins) is of great importance to the area of nanobiotechnology. Graphene and its derivative such as graphene oxide (GO), are two-dimensional (2-D) nanomaterials with remarkable physical and chemical properties and have been broadly explored in biotechnology and biomedical application. Here, we have reported the nature of adsorption of trypsin on the GO surface, considering its biomedical implications. A simple incubation of trypsin on GO surface exhibits varying resistance to autolysis. The structural morphology of trypsin on the GO surface was studied by using atomic force microscopy (AFM), circular dichroism (CD), fluorescence, and total internal reflection fluorescence (TIRF) microscopies. Results suggest that the trypsin follows the Freundlich Isotherm. By the Langmuir model, the maximum adsorption capacity was found to be 100 mg/g. From protein assay results we have concluded that the native trypsin exhibits the highest catalytic efficiency (33.97*104 L mol-1 min-1) in comparison to other Trp-GO constructs. We have further visualized morphological change on GO-trypsin interface throughout the adsorption process by taking samples at definite time intervals, which suggests that the interaction of trypsin with GO is an example of the soft corona. Our findings may be implicated in enzyme engineering as well as enzyme-based bio-sensing applications.

RNA targeting by an anthracycline drug: spectroscopic and in silico evaluation of epirubicin interaction with tRNA.

Anthracyclines are putative anticancer agents used to treat a wide range of cancers. Among these anthracyclines, epirubicin is derived from the doxorubicin by the subtle difference in the orientation of C4-hydroxyl group at sugar molecule. Epirubicin has great significance as it has propitious anticancer potential with lesser cardiotoxicity and faster elimination from the body. The present study is done to understand the molecular aspect of epirubicin binding to tRNA. We have used various spectroscopic techniques like Fourier transform infrared spectroscopy (FTIR), absorption spectroscopy and circular dichroism to illustrate the binding sites, the extent of binding and conformational changes associated with tRNA after interacting with epirubicin. From infrared studies, we infer that epirubicin interacts with guanine and uracil bases of tRNA. Results obtained from infrared and CD studies suggest that epirubicin complexation with tRNA does not result in any conformational change in tRNA structure. Binding constant (2.1 × 103 M-1) calculated from the absorbance data illustrates that epirubicin has a weak interaction with tRNA molecule. These spectroscopic results like the binding site of epirubicin and binding energy of epirubicin-tRNA complex were also verified by the molecular docking. Results of the present study provide information that aids in the development of efficient RNA targeted drugs from the existing drugs by certain chemical modification in their structure resulting in lesser side effects and better efficacy.Communicated by Ramaswamy H. Sarma.

Penta- and hexa-coordinate ferric hemoglobins display distinct pH titration profiles measured by Soret peak shifts.

Hemoglobins with diverse characteristics have been identified in all kingdoms of life. Their ubiquitous presence indicates that these proteins play important roles in physiology, though function for all hemoglobins are not yet established with certainty. Their physiological role may depend on their ability to bind ligands, which in turn is dictated by their heme chemistry. However, we have an incomplete understanding of the mechanism of ligand binding for these newly discovered hemoglobins and the measurement of their kinetic parameters depend on their coordination at the heme iron. To gain insights into their functional role, it is important to categorize the new hemoglobins into either penta- or hexa-coordinated varieties. We demonstrate that simple pH titration and absorbance measurements can determine the coordination state of heme iron atom in ferric hemoglobins, thus providing unambiguous information about the classification of new globins. This method is rapid, sensitive and requires low concentration of protein. Penta- and hexa-coordinate hemoglobins displayed distinct pH titration profiles as observed in a variety of hemoglobins. The pentacoordinate distal histidine mutant proteins of hexacoordinate hemoglobins and ligand-bound hexacoordinate forms of pentacoordinate hemoglobins reverse the pH titration profiles, thus validating the sensitivity of this spectroscopic technique.

Correction: Heat, Acid and Chemically Induced Unfolding Pathways, Conformational Stability and Structure-Function Relationship in Wheat α-Amylase.

[This corrects the article DOI: 10.1371/journal.pone.0129203.].

Heat, Acid and Chemically Induced Unfolding Pathways, Conformational Stability and Structure-Function Relationship in Wheat α-Amylase.

Wheat α-amylase, a multi-domain protein with immense industrial applications, belongs to α+ß class of proteins with native molecular mass of 32 kDa. In the present study, the pathways leading to denaturation and the relevant unfolded states of this multi-domain, robust enzyme from wheat were discerned under the influence of temperature, pH and chemical denaturants. The structural and functional aspects along with thermodynamic parameters for α-amylase unfolding were probed and analyzed using fluorescence, circular dichroism and enzyme assay methods. The enzyme exhibited remarkable stability up to 70°C with tendency to aggregate at higher temperature. Acid induced unfolding was also incomplete with respect to the structural content of the enzyme. Strong ANS binding at pH 2.0 suggested the existence of a partially unfolded intermediate state. The enzyme was structurally and functionally stable in the pH range 4.0-9.0 with 88% recovery of hydrolytic activity. Careful examination of biophysical properties of intermediate states populated in urea and GdHCl induced denaturation suggests that α-amylase unfolding undergoes irreversible and non-coincidental cooperative transitions, as opposed to previous reports of two-state unfolding. Our investigation highlights several structural features of the enzyme in relation to its catalytic activity. Since, α-amylase has been comprehensively exploited for use in a range of starch-based industries, in addition to its physiological significance in plants and animals, knowledge regarding its stability and folding aspects will promote its biotechnological applications.

Spectroscopic and molecular docking studies on chlorambucil interaction with DNA.

Chlorambucil (CMB) is an anticancer drug used for the treatment of variety of cancers. Structural and conformational changes associated with DNA after binding with CMB were explored using spectroscopic techniques to get insight into the mechanism of action of CMB at molecular level. Different molar ratios of CMB-DNA complex were prepared with constant DNA concentration under physiological conditions. FTIR spectroscopy, UV-visible spectroscopy, CD spectroscopy and molecular docking studies were employed to determine the binding site and binding constant of CMB with DNA. The results show CMB binds DNA through nitrogenous bases (thymine, guanine and cytosine). The binding constant was calculated to be 1.3 × 10³ M⁻¹, which suggests weak binding of CMB with DNA double helix. FTIR and CD results show that CMB do not disturb native B-conformation of DNA and it continues to remain in its B conformation even at higher concentrations of CMB. The molecular docking results are in corroboration with our experimental results and provides structural insight into the interaction site.

Structural insight of dopamine ß-hydroxylase, a drug target for complex traits, and functional significance of exonic single nucleotide polymorphisms.

BACKGROUND: Human dopamine ß-hydroxylase (DBH) is an important therapeutic target for complex traits. Several single nucleotide polymorphisms (SNPs) have also been identified in DBH with potential adverse physiological effect. However, difficulty in obtaining diffractable crystals and lack of a suitable template for modeling the protein has ensured that neither crystallographic three-dimensional structure nor computational model for the enzyme is available to aid rational drug design, prediction of functional significance of SNPs or analytical protein engineering. PRINCIPAL FINDINGS: Adequate biochemical information regarding human DBH, structural coordinates for peptidylglycine alpha-hydroxylating monooxygenase and computational data from a partial model of rat DBH were used along with logical manual intervention in a novel way to build an in silico model of human DBH. The model provides structural insight into the active site, metal coordination, subunit interface, substrate recognition and inhibitor binding. It reveals that DOMON domain potentially promotes tetramerization, while substrate dopamine and a potential therapeutic inhibitor nepicastat are stabilized in the active site through multiple hydrogen bonding. Functional significance of several exonic SNPs could be described from a structural analysis of the model. The model confirms that SNP resulting in Ala318Ser or Leu317Pro mutation may not influence enzyme activity, while Gly482Arg might actually do so being in the proximity of the active site. Arg549Cys may cause abnormal oligomerization through non-native disulfide bond formation. Other SNPs like Glu181, Glu250, Lys239 and Asp290 could potentially inhibit tetramerization thus affecting function. CONCLUSIONS: The first three-dimensional model of full-length human DBH protein was obtained in a novel manner with a set of experimental data as guideline for consistency of in silico prediction. Preliminary physicochemical tests validated the model. The model confirms, rationalizes and provides structural basis for several biochemical data and claims testable hypotheses regarding function. It provides a reasonable template for drug design as well.