Papers by Marina Kasimova
PloS one, 2015
PEGylation is a strategy used by the pharmaceutical industry to prolong systemic circulation of p... more PEGylation is a strategy used by the pharmaceutical industry to prolong systemic circulation of protein drugs, whereas formulation excipients are used for stabilization of proteins during storage. Here we investigate the role of PEGylation in protein stabilization by formulation excipients that preferentially interact with the protein. The model protein hen egg white lysozyme was doubly PEGylated on two lysines with 5 kDa linear PEGs (mPEG-succinimidyl valerate, MW 5000) and studied in the absence and presence of preferentially excluded sucrose and preferentially bound guanine hydrochloride. Structural characterization by far- and near-UV circular dichroism spectroscopy was supplemented by investigation of protein thermal stability with the use of differential scanning calorimetry, far and near-UV circular dichroism and fluorescence spectroscopy. It was found that PEGylated lysozyme was stabilized by the preferentially excluded excipient and destabilized by the preferentially bound ...
Biochemistry, 2009
The protein alpha-crystallin, a member of the small heat shock protein family, has the ability to... more The protein alpha-crystallin, a member of the small heat shock protein family, has the ability to prevent aggregation of partially denatured proteins, an effect demonstrated both in vivo and in vitro. In this work, we have probed the apparent thermal destabilization of bovine insulin by alpha-crystallin, using differential scanning calorimetry, near-UV circular dichroism, and intrinsic fluorescence spectroscopy. The thermal denaturation of insulin, followed by differential scanning calorimetry, is greatly affected by the presence of alpha-crystallin. Even at a ratio of alpha-crystallin subunit to insulin monomers as low as 1:10, a significant decrease in the transition temperature and a change in the shape of the transition are evident. These changes are detected for both zinc-free (mainly dimeric) and zinc-containing (predominantly hexameric) insulin. The transition temperatures measured by near-UV circular dichroism are consistent with the calorimetry results; however, no changes in the spectra of insulin occur below the transition temperature in the presence of alpha-crystallin. The intrinsic fluorescence of alpha-crystallin indicates association with insulin above 40 degrees C. On the basis of this, we conclude that alpha-crystallin promotes the dissociation of insulin oligomers to a lower-association state species with a lower thermal stability. Furthermore, we propose that the dissociation of insulin is caused by the ability of alpha-crystallin to bind to the insulin self-association surfaces and thus stabilize insulin dimers and monomers.
Biochemistry, 1995
The binding of the fusion-inhibiting peptide Z-D-Phe-L-Phe-Gly to unilamellar lipid vesicles of d... more The binding of the fusion-inhibiting peptide Z-D-Phe-L-Phe-Gly to unilamellar lipid vesicles of dioleoylphosphatidylcholine (DOPC) was investigated by isothermal titration calorimetry (ITC). The peptide Z-D-Phe-L-Phe-Gly is known to inhibit fusion of myxo- and paramyxoviruses with cells as well as cell-cell and vesicle-vesicle fusion in model systems. Calorimetric titrations conducted over a range of temperatures permitted characterization of the thermodynamics of the interaction of Z-D-Phe-L-Phe-Gly with model DOPC lipid membranes. Simultaneous global analysis of 15 ITC binding curves acquired at four different temperatures allowed determination of the equilibrium site association constant (K), stoichiometry of binding (n), binding enthalpy change (delta H), and heat capacity change of binding (delta Cp) in a single set of experiments. The binding affinity and enthalpy change per mole of DOPC bound at 25 degrees C was log K = 2.463 +/- 0.075 and delta H = -1.07 +/- 0.12 kcal/mol DOPC while the binding heat capacity change per mole of DOPC bound was delta Cp = -20.3 +/- 2.8 cal/(K.mol DOPC) with a temperature dependence (from 10-45 degrees C) of d(delta Cp)/dT = 0.37 +/- 0.18 cal/(K2.mol DOPC). A temperature-independent binding stoichiometry was determined to be n = 5.56 +/- 0.33 DOPC molecules per Z-D-Phe-L-Phe-Gly. A comparison of these results with previous peptide-lipid binding studies is discussed as is their relevance to a current model of the interaction of fusion-inhibiting peptides with phospholipid membranes.
Journal of Molecular Biology, May 3, 2002
N NMR relaxation parameters and amide 1 H/ 2 H-exchange rates have been used to characterize the ... more N NMR relaxation parameters and amide 1 H/ 2 H-exchange rates have been used to characterize the structural flexibility of human growth hormone (rhGH) at neutral and acidic pH. Our results show that the rigidity of the molecule is strongly affected by the solution conditions. At pH 7.0 the backbone dynamics parameters of rhGH are uniform along the polypeptide chain and their values are similar to those of other folded proteins. In contrast, at pH 2.7 the overall backbone flexibility increases substantially compared to neutral pH and the average order parameter approaches the lower limit expected for a folded protein. However, a significant variation of the backbone dynamics through the molecule indicates that under acidic conditions the mobility of the residues becomes more dependent on their location within the secondary structure units. In particular, the order parameters of certain loop regions decrease dramatically and become comparable to those found in unfolded proteins. Furthermore, the HN-exchange rates at low pH reveal that the residues most protected from exchange are clustered at one end of the helical bundle, forming a stable nucleus. We suggest that this nucleus maintains the overall fold of the protein under destabilizing conditions. We therefore conclude that the acid state of rhGH consists of a structurally conserved, but dynamically more flexible helical core surrounded by an aura of highly mobile, unstructured loops. However, in spite of its prominent flexibility the acid state of rhGH cannot be considered a "molten globule" state because of its high stability. It appears from our work that under certain conditions, a protein can tolerate a considerable increase in flexibility of its backbone, along with an increased penetration of water into its core, while still maintaining a stable folded conformation.
Modern Magnetic Resonance, 2006
ABSTRACT The power of NMR as a method for characterizing the solution state of proteins stems fro... more ABSTRACT The power of NMR as a method for characterizing the solution state of proteins stems from the highly specific way that NMR reports on protein structure and dynamics. This is due to the fact that NMR parameters, such as chemical shifts, relaxation rates and exchange-rate parameters report specifically about the local environment around the investigated nuclei. This obviously requires that the relevant NMR resonances have been assigned to the individual nuclei of the protein structures, which with present day techniques for isotope labeling and multidimensional NMR methods is possible for proteins with molecular weights higher than 30 kDa. After the resonances have been assigned, the overall three-dimensional structure of a protein can be determined from distance and angular constraints derived from additional spectral information.
International Journal of Pharmaceutics, 2011
Dendrimers are attractive vehicles for nucleic acid delivery due to monodispersity and ease of ch... more Dendrimers are attractive vehicles for nucleic acid delivery due to monodispersity and ease of chemical design. The purpose of this study was to elucidate the self-assembly process between small interfering RNA (siRNA) and different generation poly(amidoamine) dendrimers and to characterize the resulting structures. The generation 4 (G4) and G7 displayed equal efficiencies for dendriplex aggregate formation, whereas G1 lacked this ability. Nanoparticle tracking analysis and dynamic light scattering showed reduced average size and increased polydispersity at higher dendrimer concentration. The nanoparticle tracking analysis indicated that electrostatic complexation results in an equilibrium between differently sized complex aggregates, where the centre of mass depends on the siRNA:dendrimer ratio. Isothermal titration calorimetric data suggested a simple binding for G1, whereas a biphasic binding was evident for G4 and G7 with an initial exothermic binding and a secondary endothermic formation of larger dendriplex aggregates, followed by agglomeration. The initial binding became increasingly exothermic as the generation increased, and the values were closely predicted by molecular dynamics simulations, which also demonstrated a generation dependent differences in the entropy of binding. The flexible G1 displayed the highest entropic penalty followed by the rigid G7, making the intermediate G4 the most suitable for dendriplex formation, showing favorable charge density for siRNA binding.
Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues Ii, 2004
ABSTRACT Without exceptions, in all living cells NADH is a key metabolite linking a large number ... more ABSTRACT Without exceptions, in all living cells NADH is a key metabolite linking a large number of metabolic pathways. Flux rates through such pathways are an essential component in the understanding of the functioning of living cells. Knowledge about the way these fluxes depend on the concentrations of the metabolites involved (including NADH/NAD+) allows calculation of these fluxes. Therefore, a method to determine the concentration of free NADH is necessary. A distinction between the free and protein-bound NADH can be made on the basis of fluorescence emission spectra and fluorescence lifetimes. A method for such measurements using a microscopic set-up for time-gated fluorescence spectroscopy has been introduced by Schneckenburger and co-workers (Paul RJ, Schneckenburger H. Naturwissenschaften 83, pp. 32-35, 1996). We further improve this method by first characterizing NADH binding to model proteins by isothermal titration calorimetry and fluorescence. This allows a precise calculation of bound and free NADH and their respective spectra. An analysis of experimental data is advanced by applying two-component deconvolution and subsequent fitting. Using this method we can detect a significant proportion of free NADH in isolated potato tuber mitochondria respiring malate. Taken together these improvements allow a more accurate characterization of the NADH turnover in biological systems.
THE PLANT CELL ONLINE, 2006
The reduced coenzyme NADH plays a central role in mitochondrial respiratory metabolism. However, ... more The reduced coenzyme NADH plays a central role in mitochondrial respiratory metabolism. However, reports on the amount of free NADH in mitochondria are sparse and contradictory. We first determined the emission spectrum of NADH bound to proteins using isothermal titration calorimetry combined with fluorescence spectroscopy. The NADH content of actively respiring mitochondria (from potato tubers [Solanum tuberosum cv Bintje]) in different metabolic states was then measured by spectral decomposition analysis of fluorescence emission spectra. Most of the mitochondrial NADH is bound to proteins, and the amount is low in state 3 (substrate þ ADP present) and high in state 2 (only substrate present) and state 4 (substrate þ ATP). By contrast, the amount of free NADH is low but relatively constant, even increasing a little in state 3. Using modeling, we show that these results can be explained by a 2.5-to 3-fold weaker average binding of NADH to mitochondrial protein in state 3 compared with state 4. This indicates that there is a specific mechanism for free NADH homeostasis and that the concentration of free NADH in the mitochondrial matrix per se does not play a regulatory role in mitochondrial metabolism. These findings have far-reaching consequences for the interpretation of cellular metabolism.
Langmuir, 2012
Enzymatically stable cell-penetrating α-peptide/β-peptoid peptidomimetics constitute promising dr... more Enzymatically stable cell-penetrating α-peptide/β-peptoid peptidomimetics constitute promising drug delivery vehicles for the transport of therapeutic biomacromolecules across membrane barriers. The aim of the present study was to elucidate the mechanism of peptidomimetic-lipid bilayer interactions. A series of peptidomimetics consisting of alternating cationic and hydrophobic residues displaying variation in length and N-terminal end group were applied to fluid-phase, anionic lipid bilayers, and their interaction was investigated using isothermal titration calorimetry (ITC) and ellipsometry. Titration of lipid vesicles into solutions of peptidomimetics resulted in exothermic adsorption processes, and the interaction of all studied peptidomimetics with anionic lipid membranes was found to be enthalpy-driven. The enthalpy and Gibbs free energy (ΔG) proved more favorable with increasing chain length. However, not all charges contribute equally to the interaction, as evidenced by the charge-normalized ΔG being inversely correlated to the sequence length. Ellipsometry data suggested that the hydrophobic residues also played an important role in the interaction process. Furthermore, ΔG extracted from ellipsometry data showed good agreement with that obtained with ITC. To further elucidate their interaction with biological membranes, quantitative uptake and cellular distribution were studied in proliferating HeLa cells by flow cytometry and confocal microscopy. The cellular uptake of carboxyfluorescein-labeled peptidomimetics showed a similar ranking as that obtained from the adsorbed amount, and binding energy to model membranes demonstrated that the initial interaction with the membrane is of key importance for the cellular uptake.
Journal of Molecular Biology, 1998
The structural stability of recombinant human growth hormone (rhGH) has been studied by different... more The structural stability of recombinant human growth hormone (rhGH) has been studied by differential scanning calorimetry, circular dichroism and by following the tyrosine and histidine chemical shifts in the 1H NMR spectrum. These studies demonstrate that the folding/unfolding equilibrium of rhGH involves a partially folded dimeric intermediate. The formation of this dimeric intermediate is a reversible process. At acid pH (pH 3) the conformational equilibrium is reversible even at high protein concentrations (10 mg/ml). At neutral pH reversibility is observed only at low protein concentrations (<0.5 mg/ml). The free energy of this intermediate conformation is only approximately 3 kcal/mol apart from the native state indicating that the conformational equilibrium can be effectively modulated by changes in solvent composition or physical conditions. According to the spectroscopic and thermodynamic results, the formation of the dimeric intermediate occurs without a major loss in helical content and is driven by the formation of substantial hydrophobic contacts between two partially folded molecules. A thermodynamic model that accounts quantitatively for the experimental data has been developed. These studies demonstrate that partially folded conformations of certain proteins are able to form stoichiometric complexes, and that the formation of these complexes provide a significant source of stabilizing Gibbs energy for conformational states that, otherwise, will be characterized by extremely unfavorable free energies.
Journal of Molecular Biology, 2002
N NMR relaxation parameters and amide 1 H/ 2 H-exchange rates have been used to characterize the ... more N NMR relaxation parameters and amide 1 H/ 2 H-exchange rates have been used to characterize the structural flexibility of human growth hormone (rhGH) at neutral and acidic pH. Our results show that the rigidity of the molecule is strongly affected by the solution conditions. At pH 7.0 the backbone dynamics parameters of rhGH are uniform along the polypeptide chain and their values are similar to those of other folded proteins. In contrast, at pH 2.7 the overall backbone flexibility increases substantially compared to neutral pH and the average order parameter approaches the lower limit expected for a folded protein. However, a significant variation of the backbone dynamics through the molecule indicates that under acidic conditions the mobility of the residues becomes more dependent on their location within the secondary structure units. In particular, the order parameters of certain loop regions decrease dramatically and become comparable to those found in unfolded proteins. Furthermore, the HN-exchange rates at low pH reveal that the residues most protected from exchange are clustered at one end of the helical bundle, forming a stable nucleus. We suggest that this nucleus maintains the overall fold of the protein under destabilizing conditions. We therefore conclude that the acid state of rhGH consists of a structurally conserved, but dynamically more flexible helical core surrounded by an aura of highly mobile, unstructured loops. However, in spite of its prominent flexibility the acid state of rhGH cannot be considered a "molten globule" state because of its high stability. It appears from our work that under certain conditions, a protein can tolerate a considerable increase in flexibility of its backbone, along with an increased penetration of water into its core, while still maintaining a stable folded conformation.
Journal of Controlled Release, 1998
We have previously reported on the biological activity of members of a library of low molecular w... more We have previously reported on the biological activity of members of a library of low molecular weight compounds (carriers) that enable the oral delivery of proteins (Milstein, Proceedings of the 1995 Miami Bio/Technology Winter Symposium on Protein Engineering and Structural Biology, IRL Press at Oxford University Press, 1995, p. 13; Leone-Bay et al., J. Med. Chem. 38 (1995) 4263-4269; Leone-Bay et al., J. Med. Chem. 39 (1996) 2571-2578; [1-3]). When rats or primates are orally administered a solution of carrier and either recombinant human alpha-interferon (rhIFN), insulin or recombinant human growth hormone (rhGH) significant serum concentrations of the proteins are detectable. The transport activity of these compounds is positively correlated with their structural effects on the protein molecules. Direct measurement of the interaction of these carrier molecules with the proteins indicates that they reversibly destabilize the native state of the molecule favoring a partially unfolded conformation. Apparently these intermediate protein conformations are transport competent and are able to be absorbed through the intestinal tissue and into the bloodstream. Since the measured binding of the carriers to the partially unfolded proteins is relatively weak (Kb = 100 M(-1)) and the systemic activity of the proteins appears to be unaffected, the changes in the structure of the proteins are manifestly reversible.
Drug Discovery Today, 2010
H.M. Nielsen). Biomacromolecules as proteins and nucleic acids are promising drug candidates. How... more H.M. Nielsen). Biomacromolecules as proteins and nucleic acids are promising drug candidates. However, one problem with biomacromolecules is that they usually have to pass the cell membrane to exert their effect. Utilization of cell penetrating peptides (CPPs) might be a way to transport biomacromolecules across the cell membrane. It is becoming increasingly evident that CPP uptake pathways may vary depending on the physico-chemical properties of the CPP and the cargo they deliver, the specific cell types and the specific experimental conditions. Nevertheless, the interaction between CPPs and membrane is the very first step of the internalization. Analysis of the CPPs interaction with liposomes is expected to provide information about the CPPs interaction with the cell membrane. We have performed a thermodynamic characterization and spectroscopic of the binding between a series of novel CPPs and anionic liposomes. Recently, we described a new class of CPPs, which seem to show superior biological effect compared to the well described CPPs. The molecular design of these alpha-peptide-beta-peptoid chimeras is based on alternating repeats of (-amino acids and (-peptoid residues. The rationale was to benefit from the structure-promoting effects and lipophilicity from the unnatural chiral (peptoid residues, and the (-amino acid residues providing cationic properties and hydrogen bonding possibilities. The chimeras are very stable toward proteolysis, non-hemolytic, possess antibacterial activity and promising cell-penetrating potential. Interpretation of the data obtained in ITC-experiments showed that an increased number of basic residues in the novel CPPs sequence resulted in a more favorable interaction with the anionic liposomes. Additional experiments revealed that a hydrophobic interaction was a part of the binding. From CD spectra it was concluded, that no major structural changes occurred in the novel CPPs when they were in the presence of anionic liposomes. The initial electrostatic attraction in CPPs internalization mechanism was confirmed by comparing Gibbs free energy ((G) with the number of basic residues. Furthermore, it is proposed that the hydrophobic interaction registered could be between hydrophobic groups on the novel CPP and the hydrophobic region of the liposome. Another possibility could be simultaneously increased lipid-lipid interaction in the hydrophobic region of the liposome. In conclusion, when comparing the novel CPPs with results obtained for the well described CPP penetratin it seems, that the binding to anionic liposomes is more favorable for all novel CPPs investigated.
Biomacromolecules, 2010
... Linda B. Jensen , Kell Mortensen , Giovanni M. Pavan § , Marina R. Kasimova , Ditte K. ... more ... Linda B. Jensen , Kell Mortensen , Giovanni M. Pavan § , Marina R. Kasimova , Ditte K. Jensen , Veronika Gadzhyeva , Hanne M. Nielsen , and Camilla Foged* . Department of Pharmaceutics and Analytical Chemistry ...
Biomacromolecules, 2011
Chitosan is a biocompatible easily degradable polysaccharide, which, because of its positive char... more Chitosan is a biocompatible easily degradable polysaccharide, which, because of its positive charge, is able to interact favorably with deprotonated carboxyl groups of proteins. The strength of these chargeÀcharge interactions is generally low, resulting in poor colloidal stability of the complexes. To investigate if other noncovalent forces contribute to stabilizing such systems, we have selected R-lactalbumin, βlactoglobulin, β-casein, and human growth hormone, characterized by a common acidic pI value (∼5) that ensures their overall negative charge at physiological pH. Binding energetics between chitosan and proteins was studied by isothermal titration calorimetry, whereas the thermal stability was assessed by differential scanning calorimetry. Our data show that colloidal stability of the particles depends on protein identity as well as temperature, indicating the involvement of nonelectrostatic interactions (e.g., hydrophobic effect) as driving forces for the complex formation. This suggests that chitosanÀprotein drug delivery systems can be improved through preparation process optimization with regard to temperature.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2012
Cell-penetrating peptides (CPPs) provide a promising approach for enhancing intracellular deliver... more Cell-penetrating peptides (CPPs) provide a promising approach for enhancing intracellular delivery of therapeutic biomacromolecules by increasing transport through membrane barriers. Here, proteolytically stable cell-penetrating peptidomimetics with α-peptide/β-peptoid backbone were studied to evaluate the effect of α-chirality in the β-peptoid residues and the presence of guanidinium groups in the α-amino acid residues on membrane interaction. The molecular properties of the peptidomimetics in solution (surface and intramolecular hydrogen bonding, aqueous diffusion rate and molecular size) were studied along with their adsorption to lipid bilayers, cellular uptake, and toxicity. The surface hydrogen bonding ability of the peptidomimetics reflected their adsorbed amounts onto lipid bilayers as well as with their cellular uptake, indicating the importance of hydrogen bonding for their membrane interaction and cellular uptake. Ellipsometry studies further demonstrated that the presence of chiral centers in the β-peptoid residues promotes a higher adsorption to anionic lipid bilayers, whereas circular dichroism results showed that α-chirality influences their overall mean residue ellipticity. The presence of guanidinium groups and α-chiral β-peptoid residues was also found to have a significant positive effect on uptake in living cells. Together, the findings provide an improved understanding on the behavior of cell-penetrating peptidomimetics in the presence of lipid bilayers and live cells.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2010
The modified ISCOMs, so-called Posintro™ nanoparticles, provide an opportunity for altering the s... more The modified ISCOMs, so-called Posintro™ nanoparticles, provide an opportunity for altering the surface charge of the particles, which influences their affinity for the negatively charged antigen sites, cell membranes and lipids in the skin. Hypothetically, this increases the passage of the ISCOMs (or their components) and their load through the stratum corneum. The subsequent increase in the uptake by the antigen-presenting cells results in enhanced transcutaneous immunization. To understand the nature of penetration of Posintro™ nanoparticles into the intercorneocyte space of the stratum corneum, the interaction between the nanoparticles and lipid model systems in form of liposomes and/or supported lipid bilayer was studied. As a lipid model we used Stratum Corneum Lipid (SCL), a mixture similar in composition to the lipids of the intercorneocyte space. By Förster Resonance Energy Transfer (FRET), Atomic Force Microscopy (AFM), Electrochemical Impedance Spectroscopy (EIS) and cryo-Transmission Electron Microscopy (cryo-TEM) it was shown that application of nanoparticles to the SCL bilayers results in lipid disturbance. Investigation of this interaction by means of Isothermal Titration Calorimetry (ITC) confirmed existence of an enthalpically unfavorable reaction. All these methods demonstrated that the strength of electrostatic repulsion between the negatively charged SCL and the nanoparticles affected their interaction, as decreasing the negative charge of the Posintro™ nanoparticles leads to enhanced disruption of lipid organization.
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Papers by Marina Kasimova