Nanomedical Relevance of the Intermolecular Interaction Dynamics-Examples from Lysozymes and Insulins.

Insulin and lysozyme share the common features of being prone to aggregate and having biomedical importance. Encapsulating lysozyme and insulin in micellar nanoparticles probably would prevent aggregation and facilitate oral drug delivery. Despite the vivid structural knowledge of lysozyme and insulin, the environment-dependent oligomerization (dimer, trimer, and multimer) and associated structural dynamics remain elusive. The knowledge of the intra- and intermolecular interaction profiles has cardinal importance for the design of encapsulation protocols. We have employed various biophysical methods such as NMR spectroscopy, X-ray crystallography, Thioflavin T fluorescence, and atomic force microscopy in conjugation with molecular modeling to improve the understanding of interaction dynamics during homo-oligomerization of lysozyme (human and hen egg) and insulin (porcine, human, and glargine). The results obtained depict the atomistic intra- and intermolecular interaction details of the homo-oligomerization and confirm the propensity to form fibrils. Taken together, the data accumulated and knowledge gained will further facilitate nanoparticle design and production with insulin or lysozyme-related protein encapsulation.

Inhibition of lysozyme amyloidogenesis by phospholipids. Focus on long-chain dimyristoylphosphocholine.

BACKGROUND: Protein amyloid aggregation is an important pathological feature of a group of different degenerative human diseases called amyloidosis. We tested effect of two phospholipids, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) on amyloid aggregation of hen egg white (HEW) lysozyme in vitro. METHODS: Effect of phospholipids was investigated using spectroscopic techniques (fluorescence and CD spectroscopy), atomic force microscopy and image analysis. RESULTS: Phospholipids DMPC and DHPC are able dose-dependently inhibit lysozyme fibril formation. The length of the phospholipid tails and different structural arrangement of the phospholipid molecules affect inhibitory activity; long-chain DMPC inhibits fibrillization more efficiently. Interestingly, interference of DMPC with lysozyme amyloid fibrils has no effect on their morphology or amount. CONCLUSIONS: Phospholipid molecules have significant effect on lysozyme amyloid fibrillization. We suggest that inhibitory activity is due to the interference of phospholipids with lysozyme leading to the blocking of the intermolecular protein interactions important for formation of the cross-ß structure within the core of the fibrils. The higher inhibitory activity of DMPC is probably due to adsorption of protein molecules on the liposome surfaces which caused decrease of species needed for fibrillization. Interaction of the phospholipids with formed fibrils is not sufficient enough to interrupt the bonds in ß-sheets which are required for destroying of amyloid fibrils. GENERAL SIGNIFICANCE: The obtained results contribute to a better understanding of the effect of phospholipids on amyloid fibrillization of the lysozyme. The data suggest that DMPC and DHPC phospholipids represent agents able to modulate lysozyme amyloid aggregation.

Lysozyme stability and amyloid fibrillization dependence on Hofmeister anions in acidic pH.

We have explored an effect of Hofmeister anions, Na2SO4, NaCl, NaBr, NaNO3, NaSCN and NaClO4, on stability and amyloid fibrillization of hen egg white lysozyme at pH 2.7. The stability of the protein was analyzed by differential scanning calorimetry. The Hofmeister effect of the anions was assessed by the parameter dT trs/d[anion] (T trs, transition temperature). We show that dT trs/d[anion] correlates with anion surface tension effects and anion partition coefficients indicating direct interactions between anions and lysozyme. The kinetic of amyloid fibrillization of lysozyme was followed by Thioflavin T (ThT) fluorescence. Negative correlation between dT trs/d[anion] and the nucleation rate of fibrillization in the presence of monovalent anions indicates specific effect of anions on fibrillization rate of lysozyme. The efficiency of monovalent anions to accelerate fibrillization correlates with inverse Hofmeister series. The far-UV circular dichroism spectroscopy and atomic force microscopy findings show that conformational properties of fibrils depend on fibrillization rate. In the presence of sodium chloride, lysozyme forms typical fibrils with elongated structure and with the secondary structure of the ß-sheet. On the other hand, in the presence of both chaotropic perchlorate and kosmotropic sulfate anions, the fibrils form clusters with secondary structure of ß-turn. Moreover, the acceleration of fibril formation is accompanied by decreased amount of the formed fibrils as indicated by ThT fluorescence. Taken together, our study shows Hofmeister effect of monovalent anions on: (1) lysozyme stability; (2) ability to accelerate nucleation phase of lysozyme fibrillization; (3) amount, and (4) conformational properties of the formed fibrils.

The circular dichroism and differential scanning calorimetry study of the properties of DNA aptamer dimers.

We have applied circular dichroism (CD), temperature-gradient gel electrophoresis (TGGE) and differential scanning calorimetry (DSC) to study the properties of novel bioengineered DNA aptamer dimers sensitive to fibrinogen (F) and heparin (H) binding sites of thrombin and compared them with canonical single stranded aptamer sensitive to fibrinogen binding site of thrombin (Fibri). The homodimer (FF) and heterodimer (FH) aptamers were constructed based on hybridization of their supported parts. CD results showed that both FF and FH dimers form stable guanine quadruplexes in the presence of potassium ions like those in Fibri. The thermal stability of aptamer dimers was slightly lower compared to those of canonical aptamers, but sufficient for practical applications. Both FF and FH aptamer dimers exhibited a potassium-dependent inhibitory effect on thrombin-mediated fibrin gel formation, which was on average two-fold higher than those of canonical single stranded Fibri aptamers.

A circular dichroism study of the stability of guanine quadruplexes of thrombin DNA aptamers at presence of K+ and Na+ ions.

The effect of K+ and Na+ on the properties of DNA aptamers that selective binds to fibrinogen (FIBRI) and heparin (HEPA) exosites of human alpha-thrombin was studied by circular dichroism (CD). The complexes of FIBRI-K+ were slightly more stable than HEPA-K+. However, lower stability was observed for HEPA-K+ at presence of Na+ in comparison with FIBRI-K+. The analysis of CD melting curves suggests differences in thermal stability of both aptamers at presence of K+. The melting temperatures (Tm) and changes in van't Hoff enthalpy for HEPA-K+ complexes were lower in comparison with those for FIBRI-K+. With increasing HEPA concentration the Tm value increased, but Tm did not change with increasing FIBRI concentration. This suggests formation of HEPA aggregates, while FIBRI aptamers are in monomeric form.

Specific volume and compressibility of human serum albumin-polyanion complexes.

The ultrasound velocimetry, densitometry, and differential scanning calorimetry have been used to study the formation of the complexes between human serum albumin (HSA) and polyanions heparin (HEP) and/or dextran sulfate (DS). The values of the ultrasound velocity and specific volume allowed us to determine the specific adiabatic compressibility, phi(K)/beta(0), which reflects the degree of volume compressibility of the complexes. We showed that in the presence of HEP and DS the adiabatic compressibility of HSA decreases with increasing concentration of polyanions. HEP more strongly interacts with HSA than DS. pH of electrolyte in the range 4.7-8.5 weakly affects the adiabatic compressibility. Changes of compressibility of HSA can be caused by increase of the hydration due to the formation of the HSA-polyanion complexes and due to partial unfolding of HSA. The HSA-polyanion interaction resulted in decrease of phase transition temperature of the protein. This evidences about protein destabilization in the presence of polyanions.