Biomedical subjects

Onofrio Annunziata

Publications and source records attributed to Onofrio Annunziata.

8 recordsLinked to original sources

PubMed2006

The effect of salt stoichiometry on protein-salt interactions determined by ternary diffusion in aqueous solutions.

We report the four diffusion coefficients for the lysozyme-MgCl2-water ternary system at 25 degrees C and pH 4.5. The comparison with previous results for the lysozyme-NaCl-water ternary system is used to examine the effect of salt stoichiometry on the transport properties of lysozyme-salt aqueous mixtures. We find that the two cross-diffusion coefficients are very sensitive to salt stoichiometry. One of the cross-diffusion coefficients is examined in terms of common-ion, excluded-volume, and protein-preferential hydration effects. We use the four ternary diffusion coefficients to extract chemical-potential cross-derivatives and protein-preferential interaction coefficients. These thermodynamic data characterize the protein-salt thermodynamic interactions. We demonstrate the presence of the common-ion effect (Donnan effect) by analyzing the dependence of the preferential-interaction coefficient not only with respect to salt concentration but also with respect to salt stoichiometry. We conclude that the common-ion effect and the protein-preferential hydration are both important for describing the lysozyme-MgCl2 thermodynamic interaction.

Diffusion↗

PubMed2006

The effect of salt on protein chemical potential determined by ternary diffusion in aqueous solutions.

We use accurate thermodynamic derivatives extracted from high-precision measurements of the four volume-fixed diffusion coefficients in ternary solutions of lysozyme chloride in aqueous NaCl, NH4Cl, and KCl at pH 4.5 and 25 degrees C to (a) assess the relative contributions of the common-ion and nonideality effects to the protein chemical potential as a function of salt concentration, (b) compare the behavior of the protein chemical potential for the three salts, which we found to be consistent with the Hofmeister series, and (c) discuss our thermodynamic data in relation to the dependence of the protein solubility on salt concentration. The four diffusion coefficients are reported at 0.6 mM lysozyme chloride and 0.25, 0.5, 0.9, 1.2, and 1.5 M KCl and extend into the protein-supersaturated region. The chemical potential cross-derivatives are extracted from diffusion data using the Onsager reciprocal relation and the equality of molal cross-derivatives of solute chemical potentials. They are compared to those calculated previously from diffusion data for lysozyme in aqueous NaCl and NH4Cl. We estimate the effective charge on the diffusing lysozyme cation at the experimental concentrations. Our diffusion measurements on the three salts allowed us to analyze and interpret the four diffusion coefficients for charged proteins in the presence of 1:1 electrolytes. Our results may provide guidance to the understanding of protein crystallization.

Ammonium Chloride↗

PubMed2005

Protein diffusion coefficients determined by macroscopic-gradient Rayleigh interferometry and dynamic light scattering.

Dynamic light scattering (DLS) is extensively used for measuring macromolecule diffusion coefficients. Contrary to classical techniques based on macroscopic concentration gradients, DLS probes microscopic fluctuations in concentration. DLS accuracy and its concordance with macroscopic-gradient techniques remains an outstanding important issue. We measured lysozyme diffusion coefficients in aqueous salt using both DLS and Rayleigh interferometry, a highly accurate macroscopic-gradient technique. The precision of our results is unprecedented. We find that our DLS values were systematically 2% higher than interferometry values. We believe that our interferometric measurements have produced the most accurate diffusion data ever reported for a protein, providing a new standard for quality control of DLS measurements. Furthermore, by interferometry, we have determined the whole diffusion coefficient matrix required for rigorously describing lysozyme-salt coupled diffusion. For the first time, we experimentally demonstrate that DLS does not provide the protein diffusion coefficient but one eigenvalue of the diffusion coefficient matrix.

Diffusion↗

PubMed2005

Decrease in protein solubility and cataract formation caused by the Pro23 to Thr mutation in human gamma D-crystallin.

The P23T mutation in the human gammaD-crystallin gene has in recent years been associated with a number of well known cataract phenotypes. To understand the molecular mechanism of lens opacity caused by this mutation, we expressed human gammaD-crystallin (HGD), the P23T mutant, and other related mutant proteins in Escherichia coli and compared the structures and thermodynamic properties of these proteins in vitro. The results show that the cataract-causing mutation P23T does not exhibit any significant structural change relative to the native protein. However, in marked contrast to the native protein, the mutant shows a dramatically lowered solubility. The reduced solubility results from the association of the P23T mutant to form a new condensed phase that contains clusters of the mutant protein. The monomer-cluster equilibrium is represented by a solubility curve in the phase diagram. When the solubility limit is exceeded, the mutant protein forms the condensed phase after a nucleation time of 10-20 min. We found that the solubility of the P23T mutant exhibits an inverse dependence on temperature, i.e., the protein clusters are increasingly soluble as the temperature of the solution decreases. The solubility of P23T can be substantially altered by the introduction of specific mutations at or in the immediate vicinity of residue 23. We examined the mutants P23S, P23V, P23TInsP24, and P23TN24K and found that the latter two mutations can restore the solubility of the P23T mutant. These findings may help develop a strategy for the rational design of small molecule inhibitors of this type of condensed phase.

Amino Acid Sequence↗

PubMed2005

Oligomerization and phase transitions in aqueous solutions of native and truncated human beta B1-crystallin.

Human betaB1-crystallin is a major eye-lens protein that undergoes in vivo truncation at the N-terminus with aging. By studying native betaB1 and truncated betaB1DeltaN41, which mimics an age-related in vivo truncation, we have determined quantitatively the effect of truncation on the oligomerization and phase transition properties of betaB1 aqueous solutions. The oligomerization studies show that the energy of attraction between the betaB1DeltaN41 proteins is about 10% greater than that of the betaB1 proteins. We have found that betaB1DeltaN41 aqueous solutions undergo two distinct types of phase transitions. The first phase transition involves an initial formation of thin rodlike assemblies, which then evolve to form crystals. The induction time for the formation of rodlike assemblies is sensitive to oligomerization. The second phase transition can be described as liquid-liquid phase separation (LLPS) accompanied by gelation within the protein-rich phase. We refer to this process as heterogeneous gelation. These two phase transitions are not observed in the case of betaB1 aqueous solutions. However, upon the addition of poly(ethylene glycol) (PEG), we observe heterogeneous gelation also for betaB1. Our PEG experiments allow us to estimate the difference in phase separation temperatures between betaB1 and betaB1DeltaN41. This difference is consistent with the increase in energy of attraction found in our oligomerization studies. Our work suggests that truncation is a cataractogenic modification since it favors protein condensation and the consequent formation of light scattering elements, and highlights the importance of the N-terminus of betaB1 in maintaining lens transparency.

Circular Dichroism↗

PubMed2003

Observation of liquid-liquid phase separation for eye lens gammaS-crystallin.

gammaS-crystallin (gammaS) is an important human and bovine eye lens protein involved in maintaining the transparency of the eye. By adding small amounts of polyethylene glycol (PEG) to the binary aqueous bovine gammaS solutions, we have observed liquid-liquid phase separation (LLPS) at -8 degrees C and revealed that, in the binary gammaS-water system, this phase transition would occur at -28 degrees C. We have measured both the effect of PEG concentration on the LLPS temperature and proteinPEG partitioning between the two liquid coexisting phases. We use our measurements of proteinPEG partitioning to determine the nature and the magnitude of the gammaS-PEG interactions and to quantitatively assess the effectiveness of PEG as a crystallizing agent for gammaS. We use our measurements of LLPS temperature as a function of protein and PEG concentration to successfully determine the location of the critical point for the binary gammaS-water system. This phase transition cannot be observed in the absence of PEG because it is inaccessible due to the freezing of the system. Our findings indicate that the effective interactions between gammaS molecules in the binary gammaS-water system are attractive. We compare the magnitude of the attraction found for gammaS with the results obtained for the other gamma-crystallins for which the critical temperature is located above the freezing point of the system. This work suggests that PEG can be used to reveal the existence of LLPS for a much wider range of binary protein-water systems than known previously.

Animals↗

PubMed2002

Effect of polyethylene glycol on the liquid-liquid phase transition in aqueous protein solutions.

We have studied the effect of polyethylene glycol (PEG) on the liquid-liquid phase separation (LLPS) of aqueous solutions of bovine gammaD-crystallin (gammaD), a protein in the eye lens. We observe that the phase separation temperature increases with both PEG concentration and PEG molecular weight. PEG partitioning, which is the difference between the PEG concentration in the two coexisting phases, has been measured experimentally and observed to increase with PEG molecular weight. The measurements of both LLPS temperature and PEG partitioning in the ternary gammaD-PEG-water systems are used to successfully predict the location of the liquid-liquid phase boundary of the binary gammaD-water system. We show that our LLPS measurements can be also used to estimate the protein solubility as a function of the concentration of crystallizing agents. Moreover, the slope of the tie-lines and the dependence of LLPS temperature on polymer concentration provide a powerful and sensitive check of the validity of excluded volume models. Finally, we show that the increase of the LLPS temperature with PEG concentration is due to attractive protein-protein interactions.

Animals↗

PubMed2002

Mathematical model for diffusion of a protein and a precipitant about a growing protein crystal in microgravity.

Equations are presented that model diffusion of a protein to the surface of a growing crystal in a convection-free environment. The equations apply to crystal growth solutions that contain both a protein and a protein precipitant. The solutions are assumed ternary and the equations include all four diffusion coefficients necessary for the full description of the diffusion process. The four diffusion coefficients are assumed constant. Effects of crystal/solution moving boundary and the effect of a protein adsorption barrier at the crystal interface are included. The equations were applied to the system lysozyme chloride + NaCl + H2O, which has served as the primary model system for the study of crystal growth of proteins and for which there are now published ternary diffusion coefficients. Calculated results with and without the inclusion of cross-term diffusion coefficients are compared.

Chemical Precipitation↗