Inoka Deshapriya - Academia.edu (original) (raw)

Papers by Inoka Deshapriya

for their tremendous support, dedicated effort and kindness, beyond measure. Special thank to Mik... more for their tremendous support, dedicated effort and kindness, beyond measure. Special thank to Mike and Vamsi for countless hours and efforts they spent in training me and for all their assistance through out my early years. I have been tremendously fortunate to work with my current lab mates, Marc Novak,

A variety of inorganic and biological nanoparticles are synthesized by a novel precipitation appr... more A variety of inorganic and biological nanoparticles are synthesized by a novel precipitation approach. XRD patterns of the samples indicated amorphous nature of the particles. The particles were characterized using a battery of methods which include SEM, DLS, FTIR and gel electrophoresis methods. Enzymes loaded onto the particles indicated excellent retention of the biomolecular structure, and often activities of the bound enzymes were comparable to those of the free enzymes. The enzyme loaded nanoparticles are stable over several months and indicated little or no precipitation. Differential scanning calorimetric methods showed that the enzymes bound to the particles undergo thermal denaturation at temperatures comparable to those of the corresponding free enzymes, These and other details of our investigations will be presented.

ACS applied materials & interfaces, Jan 25, 2014

Controlling the properties of enzymes bound to solid surfaces in a rational manner is a grand cha... more Controlling the properties of enzymes bound to solid surfaces in a rational manner is a grand challenge. Here we show that preadsorption of cationized bovine serum albumin (cBSA) to α-Zr(IV) phosphate (α-ZrP) nanosheets promotes enzyme binding in a predictable manner, and surprisingly, the enzyme binding is linearly proportional to the number of residues present in the enzyme or its volume, providing a powerful, new predictable tool. The cBSA loaded α-ZrP (denoted as bZrP) was tested for the binding of pepsin, glucose oxidase (GOX), tyrosinase, catalase, myoglobin and laccase where the number of residues increased from the lowest value of ∼153 to the highest value of 2024. Loading depended linearly on the number of residues, rather than enzyme charge or its isoelectric point. No such correlation was seen for the binding of these enzymes to α-ZrP nanosheets without the preadsorption of cBSA, under similar conditions of pH and buffer. Enzyme binding to bZrP was supported by centrifuga...

Bioconjugate Chemistry, 2015

A simple and effective method for synthesizing highly fluorescent, protein-based nanoparticles (P... more A simple and effective method for synthesizing highly fluorescent, protein-based nanoparticles (Prodots) and their facile uptake into the cytoplasm of cells is described here. Prodots made from bovine serum albumin (nBSA), glucose oxidase (nGO), horseradish peroxidase (nHRP), catalase (nCatalase), and lipase (nLipase) were found to be 15-50 nm wide and have been characterized by gel electrophoresis, transmission electron microscopy (TEM), circular dichroism (CD), fluorescence spectroscopy, dynamic light scattering (DLS), and optical microscopic methods. Data showed that the secondary structure of the protein in Prodots is retained to a significant extent and specific activities of nGO, nHRP, nCatalase, and nLipase were 80%, 70%, 65%, and 50% of their respective unmodified enzyme activities. Calorimetric studies indicated that the denaturation temperatures of nGO and nBSA increased while those of other Prodots remained nearly unchanged, and accelerated storage half-lives of Prodots at 60 °C increased by 4- to 8-fold. Exposure of nGO and nBSA+ nGO to cells indicated rapid uptake within 1-3 h, accompanied by significant blebbing of the plasma membrane, but no uptake has been noted in the absence of nGO. The presence of nGO/glucose in the media facilitated the uptake, and hydrogen peroxide induced membrane permeability could be responsible for this rapid uptake of Prodots. In control studies, FITC alone did not enter the cell, BSA-FITC was not internalized even in the presence of nGO, and there has been no uptake of nBSA-FITC in the absence of nGO. These are the very first examples of very rapid cellular uptake of fluorescent nanoparticles into cells, particularly nanoparticles made from pure proteins. The current approach is a simple and efficient method for the preparation of bioactive, fluorescent protein nanoparticles of controllable size for cellular imaging, and cell uptake is under the control of two separate chemical triggers.

Photochemical & Photobiological Sciences, 2014

Ribosomes are molecular machines that function in polyribosome complexes to translate genetic inf... more Ribosomes are molecular machines that function in polyribosome complexes to translate genetic information, guide the synthesis of polypeptides, and modulate the folding of nascent proteins. Here, we report a surprising function for polyribosomes as a result of a systematic examination of the assembly of a large ribonucleoprotein complex, the vault particle. Structural and functional evidence points to a model of vault assembly whereby the polyribosome acts like a 3D nanoprinter to direct the ordered translation and assembly of the multi-subunit vault homopolymer, a process which we refer to as polyribosome templating. Structure-based mutagenesis and cell-free in vitro expression studies further demonstrated the critical importance of the polyribosome in vault assembly. Polyribosome templating prevents chaos by ensuring efficiency and order in the production of large homopolymeric protein structures in the crowded cellular environment and might explain the origin of many polyribosome-associated molecular assemblies inside the cell.

Langmuir, 2013

Specific approaches to the rational design of nanobio interfaces for enzyme and protein binding t... more Specific approaches to the rational design of nanobio interfaces for enzyme and protein binding to nanomaterials are vital for engineering advanced, functional nanobiomaterials for biocatalysis, sensing, and biomedical applications. This feature article presents an overview of our recent discoveries on structural, functional, and mechanistic details of how enzymes interact with inorganic nanomaterials and how they can be controlled in a systematic manner using α-Zr(IV)phosphate (α-ZrP) as a model system. The interactions of a number of enzymes having a wide array of surface charges, sizes, and functional groups are investigated. Interactions are carefully controlled to screen unfavorable repulsions and enhance favorable interactions for high affinity, structure retention, and activity preservation. In specific cases, catalytic activities and substrate selectivities are improved over those of the pristine enzymes, and two examples of high activity near the boiling point of water have been demonstrated. Isothermal titration calorimetric studies indicated that enzyme binding is coupled to ion sequestration or release to or from the nanobio interface, and binding is controlled in a rational manner. We learned that (1) bound enzyme stabilities are improved by lowering the entropy of the denatured state; (2) maximal loadings are obtained by matching charge footprints of the enzyme and the nanomaterial surface; (3) binding affinities are improved by ion sequestration at the nanobio interface; and (4) maximal enzyme structure retention is obtained by biophilizing the nanobio interface with protein glues. The chemical and physical manipulations of the nanobio interface are significant not only for understanding the complex behaviors of enzymes at biological interfaces but also for desiging better functional nanobiomaterials for a wide variety of practical applications.

Langmuir, 2013

Previously, an ion-coupled protein binding (ICPB) model was proposed to explain the thermodynamic... more Previously, an ion-coupled protein binding (ICPB) model was proposed to explain the thermodynamics of protein binding to negatively charged α-Zr(IV) phosphate (α-ZrP). This model is tested here using glucose oxidase (GO) and met-hemoglobin (Hb) and several cations (Zr(IV), Cr(III), Au(III), Al(III), Ca(II), Mg(II), Zn(II), Ni(II), Na(I), and H(I)). The binding constant of GO with α-ZrP was increased ∼380-fold by the addition of either 1 mM Zr(IV) or 1 mM Ca(II), and affinities followed the trend Zr(IV) ≃ Ca(II) > Cr(III) > Mg(II) ≫ H(I) > Na(I). Binding studies could not be conducted with Au(III), Al(III), Zn(II), Cu(II), and Ni(II), as these precipitated both proteins. Zr(IV) increased Hb binding constant to α-ZrP by 43-fold, and affinity enhancements followed the trend Zr(IV) > H(I) > Mg(II) > Na(I) > Ca(II) > Cr(III). Zeta potential studies clearly showed metal ion binding to α-ZrP and affinities followed the trend, Zr(IV) ≫ Cr(III) > Zn(II) > Ni(II) > Mg(II) > Ca(II) > Au(III) > Na(I) > H(I). Electron microscopy showed highly ordered structures of protein/metal/α-ZrP intercalates on micrometer length scales, and protein intercalation was also confirmed by powder X-ray diffraction. Specific activities of GO/Zr(IV)/α-ZrP and Hb/Zr(IV)/α-ZrP ternary complexes were 2.0 × 10(-3) and 6.5 × 10(-4) M(-1) s(-1), respectively. While activities of all GO/cation/α-ZrP samples were comparable, those of Hb/cation/α-ZrP followed the trend Mg(II) > Na(I) > H(I) > Cr(III) > Ca(II) ≃ Zr(IV). Metal ions enhanced protein binding by orders of magnitude, as predicted by the ICPB model, and binding enhancements depended on charge as well as the phosphophilicity/oxophilicity of the cation.

Journal of Nano Research, 2010

A new, simple, and versatile method was developed to prepare protein nanoparticles, for the first... more A new, simple, and versatile method was developed to prepare protein nanoparticles, for the first time, and the approach was extended to prepare organic, inorganic, and biological nanomaterials. For example, nanoparticles of met-hemoglobin and glucose oxidase are readily prepared by contacting a fine spray of aqueous solutions of the proteins to an organic solvent such as methanol or acetonitrile. The protein nanoparticles suspended in organic solvents retained their secondary structure and biological activities to a significant extent. Using this approach, we also successfully prepared nanoparticles of transition metal complexes, organic molecules, nucleic acids, inorganic polymers, and organic polymers. Particle size depended on reagent concentrations, pH and the solvent used, and particle sizes have been controlled from 20 to 200 nm by adjusting these parameters. In each case, particle sizes and size distributions were determined by dynamic light scattering and the data have been...

Journal of Materials Chemistry, 2012

ABSTRACT Stabilization of proteins against thermal deactivation is a major challenge, and a simpl... more ABSTRACT Stabilization of proteins against thermal deactivation is a major challenge, and a simple, facile, novel chemical approach is described here to overcome this hurdle. We report here, for the first time, the successful synthesis of ultrastable protein nanoparticles consisting of met-hemoglobin (Hb) conjugated with low molecular weight polyacrylic acid (PAA, Mw 8000) to form discrete nanoparticles. Hb–PAA nanoparticles were not deactivated when subjected to prolonged thermal treatment such as steam sterilization conditions (122 °C, 40 minutes, 17–20 psi), while the unprotected Hb lost most of its activity when subjected to the same heating conditions. Several Hb–PAA derivatives which resist thermal inactivation, in a similar manner, are produced and characterized. Interestingly, the highest activity retention, after the above thermal treatment, was 100% for the untreated samples. This resistance to heat is attributed to the enhanced thermodynamic stability of the Hb–PAA conjugate and improved re-folding of the denatured state to the native form, facilitated by PAA conjugation to Hb. This is a unique approach to stabilize Hb against thermal inactivation, and it is a major breakthrough in the production of stable Hb-based nanomaterials that can be safely sterilized in an autoclave for biomedical/in vivo applications.

for their tremendous support, dedicated effort and kindness, beyond measure. Special thank to Mik... more for their tremendous support, dedicated effort and kindness, beyond measure. Special thank to Mike and Vamsi for countless hours and efforts they spent in training me and for all their assistance through out my early years. I have been tremendously fortunate to work with my current lab mates, Marc Novak,

A variety of inorganic and biological nanoparticles are synthesized by a novel precipitation appr... more A variety of inorganic and biological nanoparticles are synthesized by a novel precipitation approach. XRD patterns of the samples indicated amorphous nature of the particles. The particles were characterized using a battery of methods which include SEM, DLS, FTIR and gel electrophoresis methods. Enzymes loaded onto the particles indicated excellent retention of the biomolecular structure, and often activities of the bound enzymes were comparable to those of the free enzymes. The enzyme loaded nanoparticles are stable over several months and indicated little or no precipitation. Differential scanning calorimetric methods showed that the enzymes bound to the particles undergo thermal denaturation at temperatures comparable to those of the corresponding free enzymes, These and other details of our investigations will be presented.

ACS applied materials & interfaces, Jan 25, 2014

Controlling the properties of enzymes bound to solid surfaces in a rational manner is a grand cha... more Controlling the properties of enzymes bound to solid surfaces in a rational manner is a grand challenge. Here we show that preadsorption of cationized bovine serum albumin (cBSA) to α-Zr(IV) phosphate (α-ZrP) nanosheets promotes enzyme binding in a predictable manner, and surprisingly, the enzyme binding is linearly proportional to the number of residues present in the enzyme or its volume, providing a powerful, new predictable tool. The cBSA loaded α-ZrP (denoted as bZrP) was tested for the binding of pepsin, glucose oxidase (GOX), tyrosinase, catalase, myoglobin and laccase where the number of residues increased from the lowest value of ∼153 to the highest value of 2024. Loading depended linearly on the number of residues, rather than enzyme charge or its isoelectric point. No such correlation was seen for the binding of these enzymes to α-ZrP nanosheets without the preadsorption of cBSA, under similar conditions of pH and buffer. Enzyme binding to bZrP was supported by centrifuga...

Bioconjugate Chemistry, 2015

A simple and effective method for synthesizing highly fluorescent, protein-based nanoparticles (P... more A simple and effective method for synthesizing highly fluorescent, protein-based nanoparticles (Prodots) and their facile uptake into the cytoplasm of cells is described here. Prodots made from bovine serum albumin (nBSA), glucose oxidase (nGO), horseradish peroxidase (nHRP), catalase (nCatalase), and lipase (nLipase) were found to be 15-50 nm wide and have been characterized by gel electrophoresis, transmission electron microscopy (TEM), circular dichroism (CD), fluorescence spectroscopy, dynamic light scattering (DLS), and optical microscopic methods. Data showed that the secondary structure of the protein in Prodots is retained to a significant extent and specific activities of nGO, nHRP, nCatalase, and nLipase were 80%, 70%, 65%, and 50% of their respective unmodified enzyme activities. Calorimetric studies indicated that the denaturation temperatures of nGO and nBSA increased while those of other Prodots remained nearly unchanged, and accelerated storage half-lives of Prodots at 60 °C increased by 4- to 8-fold. Exposure of nGO and nBSA+ nGO to cells indicated rapid uptake within 1-3 h, accompanied by significant blebbing of the plasma membrane, but no uptake has been noted in the absence of nGO. The presence of nGO/glucose in the media facilitated the uptake, and hydrogen peroxide induced membrane permeability could be responsible for this rapid uptake of Prodots. In control studies, FITC alone did not enter the cell, BSA-FITC was not internalized even in the presence of nGO, and there has been no uptake of nBSA-FITC in the absence of nGO. These are the very first examples of very rapid cellular uptake of fluorescent nanoparticles into cells, particularly nanoparticles made from pure proteins. The current approach is a simple and efficient method for the preparation of bioactive, fluorescent protein nanoparticles of controllable size for cellular imaging, and cell uptake is under the control of two separate chemical triggers.

Photochemical & Photobiological Sciences, 2014

Ribosomes are molecular machines that function in polyribosome complexes to translate genetic inf... more Ribosomes are molecular machines that function in polyribosome complexes to translate genetic information, guide the synthesis of polypeptides, and modulate the folding of nascent proteins. Here, we report a surprising function for polyribosomes as a result of a systematic examination of the assembly of a large ribonucleoprotein complex, the vault particle. Structural and functional evidence points to a model of vault assembly whereby the polyribosome acts like a 3D nanoprinter to direct the ordered translation and assembly of the multi-subunit vault homopolymer, a process which we refer to as polyribosome templating. Structure-based mutagenesis and cell-free in vitro expression studies further demonstrated the critical importance of the polyribosome in vault assembly. Polyribosome templating prevents chaos by ensuring efficiency and order in the production of large homopolymeric protein structures in the crowded cellular environment and might explain the origin of many polyribosome-associated molecular assemblies inside the cell.

Langmuir, 2013

Specific approaches to the rational design of nanobio interfaces for enzyme and protein binding t... more Specific approaches to the rational design of nanobio interfaces for enzyme and protein binding to nanomaterials are vital for engineering advanced, functional nanobiomaterials for biocatalysis, sensing, and biomedical applications. This feature article presents an overview of our recent discoveries on structural, functional, and mechanistic details of how enzymes interact with inorganic nanomaterials and how they can be controlled in a systematic manner using α-Zr(IV)phosphate (α-ZrP) as a model system. The interactions of a number of enzymes having a wide array of surface charges, sizes, and functional groups are investigated. Interactions are carefully controlled to screen unfavorable repulsions and enhance favorable interactions for high affinity, structure retention, and activity preservation. In specific cases, catalytic activities and substrate selectivities are improved over those of the pristine enzymes, and two examples of high activity near the boiling point of water have been demonstrated. Isothermal titration calorimetric studies indicated that enzyme binding is coupled to ion sequestration or release to or from the nanobio interface, and binding is controlled in a rational manner. We learned that (1) bound enzyme stabilities are improved by lowering the entropy of the denatured state; (2) maximal loadings are obtained by matching charge footprints of the enzyme and the nanomaterial surface; (3) binding affinities are improved by ion sequestration at the nanobio interface; and (4) maximal enzyme structure retention is obtained by biophilizing the nanobio interface with protein glues. The chemical and physical manipulations of the nanobio interface are significant not only for understanding the complex behaviors of enzymes at biological interfaces but also for desiging better functional nanobiomaterials for a wide variety of practical applications.

Langmuir, 2013

Previously, an ion-coupled protein binding (ICPB) model was proposed to explain the thermodynamic... more Previously, an ion-coupled protein binding (ICPB) model was proposed to explain the thermodynamics of protein binding to negatively charged α-Zr(IV) phosphate (α-ZrP). This model is tested here using glucose oxidase (GO) and met-hemoglobin (Hb) and several cations (Zr(IV), Cr(III), Au(III), Al(III), Ca(II), Mg(II), Zn(II), Ni(II), Na(I), and H(I)). The binding constant of GO with α-ZrP was increased ∼380-fold by the addition of either 1 mM Zr(IV) or 1 mM Ca(II), and affinities followed the trend Zr(IV) ≃ Ca(II) > Cr(III) > Mg(II) ≫ H(I) > Na(I). Binding studies could not be conducted with Au(III), Al(III), Zn(II), Cu(II), and Ni(II), as these precipitated both proteins. Zr(IV) increased Hb binding constant to α-ZrP by 43-fold, and affinity enhancements followed the trend Zr(IV) > H(I) > Mg(II) > Na(I) > Ca(II) > Cr(III). Zeta potential studies clearly showed metal ion binding to α-ZrP and affinities followed the trend, Zr(IV) ≫ Cr(III) > Zn(II) > Ni(II) > Mg(II) > Ca(II) > Au(III) > Na(I) > H(I). Electron microscopy showed highly ordered structures of protein/metal/α-ZrP intercalates on micrometer length scales, and protein intercalation was also confirmed by powder X-ray diffraction. Specific activities of GO/Zr(IV)/α-ZrP and Hb/Zr(IV)/α-ZrP ternary complexes were 2.0 × 10(-3) and 6.5 × 10(-4) M(-1) s(-1), respectively. While activities of all GO/cation/α-ZrP samples were comparable, those of Hb/cation/α-ZrP followed the trend Mg(II) > Na(I) > H(I) > Cr(III) > Ca(II) ≃ Zr(IV). Metal ions enhanced protein binding by orders of magnitude, as predicted by the ICPB model, and binding enhancements depended on charge as well as the phosphophilicity/oxophilicity of the cation.

Journal of Nano Research, 2010

A new, simple, and versatile method was developed to prepare protein nanoparticles, for the first... more A new, simple, and versatile method was developed to prepare protein nanoparticles, for the first time, and the approach was extended to prepare organic, inorganic, and biological nanomaterials. For example, nanoparticles of met-hemoglobin and glucose oxidase are readily prepared by contacting a fine spray of aqueous solutions of the proteins to an organic solvent such as methanol or acetonitrile. The protein nanoparticles suspended in organic solvents retained their secondary structure and biological activities to a significant extent. Using this approach, we also successfully prepared nanoparticles of transition metal complexes, organic molecules, nucleic acids, inorganic polymers, and organic polymers. Particle size depended on reagent concentrations, pH and the solvent used, and particle sizes have been controlled from 20 to 200 nm by adjusting these parameters. In each case, particle sizes and size distributions were determined by dynamic light scattering and the data have been...

Journal of Materials Chemistry, 2012

ABSTRACT Stabilization of proteins against thermal deactivation is a major challenge, and a simpl... more ABSTRACT Stabilization of proteins against thermal deactivation is a major challenge, and a simple, facile, novel chemical approach is described here to overcome this hurdle. We report here, for the first time, the successful synthesis of ultrastable protein nanoparticles consisting of met-hemoglobin (Hb) conjugated with low molecular weight polyacrylic acid (PAA, Mw 8000) to form discrete nanoparticles. Hb–PAA nanoparticles were not deactivated when subjected to prolonged thermal treatment such as steam sterilization conditions (122 °C, 40 minutes, 17–20 psi), while the unprotected Hb lost most of its activity when subjected to the same heating conditions. Several Hb–PAA derivatives which resist thermal inactivation, in a similar manner, are produced and characterized. Interestingly, the highest activity retention, after the above thermal treatment, was 100% for the untreated samples. This resistance to heat is attributed to the enhanced thermodynamic stability of the Hb–PAA conjugate and improved re-folding of the denatured state to the native form, facilitated by PAA conjugation to Hb. This is a unique approach to stabilize Hb against thermal inactivation, and it is a major breakthrough in the production of stable Hb-based nanomaterials that can be safely sterilized in an autoclave for biomedical/in vivo applications.