M.U. Palma

Spinodal lines and Flory-Huggins free-energies for solutions of human hemoglobins HbS and HbA.

Gelation of deoxygenated solutions of sickle-cell human Hemoglobin (HbS) is of high theoretical interest and it has serious pathological consequences. For this reason HbS is probably the most studied protein capable of self-organization. This notwithstanding, the location in the T, c plane of the region of thermodynamic instability of solutions of deoxy-HbS (as bounded by the spinodal line and as distinct from the gelation region) has remained unknown, along with related values of Flory-Huggins enthalpies and entropies. In the present work this information is derived from experiments for the two cases of (deoxy) HbS and of human adult hemoglobin (HbA). Experiments also show critical exponents having mean-field values, which validates a Flory-Huggins approach. Altogether, the present work offers a quantitative understanding of the thermodynamic effects of the genetic HbA----HbS mutation and it opens the way to similar quantitative evaluations of contributions of pH, salts, cosolutes, and single peptides (even for nongelling hemoglobins), and of potential therapeutic strategies.

SELF-ASSEMBLY OF BIOPOLYMERIC STRUCTURES BELOW THE THRESHOLD OF RANDOM CROSS-LINK PERCOLATION

Self-assembly of extended structures via cross-linking of individual biomolecules often occurs in solutions at concentrations well below the estimated threshold for random cross-link percolation. This requires solute-solute correlations. Here we study bovine serum albumin. Its unfolding causes the appearance of an instability region of the sol, not observed for native bovine serum albumin. As a consequence, spinodal demixing of the sol is observed. The thermodynamic phase transition corresponding to this demixing is the determinative symmetry-breaking step allowing the subsequent occurrence of (correlated) cross-linking and its progress up to the topological phase transition of gelation. The occurrence of this sequence is of marked interest to theories of spontaneous symmetry-breaking leading to morphogenesis, as well as to percolation theories. The present results extend the validity of conclusions drawn from our previous studies of other systems, by showing in one single case, system features that we have hitherto observed separately in different systems. Time-resolved experimental observations of the present type also bring kinetic and diffusional processes and solute-solvent interactions into the picture of cross-link percolation.

Spontaneous symmetry-breaking pathways: time-resolved study of agarose gelation

Abstract Extensive time-resolved studies of self-assembly of agarose gels, performed with the use of a variety of techniques allowed identification of the initial break of symmetry and the actual path leading to self-assembly at concentrations well below the random percolation threshold. The overall process is seen to occur through the following sequence: (i) break of symmetry in the sol, causing the spontaneous generation of mesoscopic polymer-rich and solvent-rich regions; (ii) percolation, or nearly percolation [see (iv) below], of polymer-rich regions through the sample, still in the sol state; (iii) start of polymer cross-linking within polymer-rich regions; (iv) progress of cross-link percolation, channeled along the pathways of polymer-rich regions. The analogous role of either permanent or transient demixing of the sol in providing preferential paths for cross-links and promoting gelation at moderate and low concentrations has been established also in a variety of other biopolymeric systems.

Irreversible formation of intermediate BSA oligomers requires and induces conformational changes.

Understanding the relation between protein conformational changes and aggregation, and the physical mechanisms leading to such processes, is of primary importance, due to its direct relation to a vast class of severe pathologies. Growing evidence also suggests that oligomeric intermediates, which may occur early in the aggregation pathway, can be themselves pathogenic. The possible cytotoxicity of oligomers of non‐disease‐associated proteins adds generality to such suggestion and to the interest of studies of oligomer formation. Here we study the early stages of aggregation of Bovine Serum Albumin (BSA), a non pathogenic protein which has proved to be a useful model system. Dynamic light scattering and circular dichroism measurements in kinetic experiments following step‐wise temperature rises, show that the “intermediate” form, which initiates large‐scale aggregation, is the result of structural and conformational changes and concurrent formation of oligomers, of average size in the range of 100–200 Å. Two distinct thresholds are observed. Beyond the first one oligomerization starts and causes partial irreversibility of conformational changes. Beyond the second threshold, additional secondary structural changes occurring in proteins being recruited progress on the same time scale of oligomerization. The concurrent behavior causes a mutual stabilization of oligomerization, and of structural and conformational changes, evidenced by a progressive increase of their irreversibility. This process interaction appears to be pivotal in producing irreversible oligomers. Proteins 2004;9999:000–000.

Time scale of protein aggregation dictated by liquid-liquid demixing

The growing impact of protein aggregation pathologies, together with the current high need for extensive information on protein structures are focusing much interest on the physics underlying the nucleation and growth of protein aggregates and crystals. Sickle Cell Hemoglobin (HbS), a point‐mutant form of normal human Hemoglobin (HbA), is the first recognized and best‐studied case of pathologically aggregating protein. Here we reanalyze kinetic data on nucleation of deoxy‐HbS aggregates by referring them to the (concentration‐dependent) temperature Ts characterizing the occurrence of the phase transition of liquid‐liquid demixing (LLD) of the solution. In this way, and by appropriate scaling of kinetic data at different concentrations, so as to normalize their spans, the apparently disparate sets of data are seen to fall on a master curve. Expressing the master curve vs. the parameter ϵ = (T − Ts) / Ts, familiar from phase transition theory, allows eliciting the role of anomalously large concentration fluctuations associated with the LLD phase transition and also allows decoupling quantitatively the role of such fluctuations from that of microscopic, inter‐protein interactions leading to nucleation. Referring to ϵ shows how in a narrow temperature span, that is at T≈Ts, nucleation kinetics can undergo orders‐of‐magnitude changes, unexpected in terms of ordinary chemical kinetics. The same is true for similarly small changes of other parameters (pH, salts, precipitants), capable of altering Ts and consequently ϵ. This offers the rationale for understanding how apparently minor changes of parameters can dramatically affect protein aggregation and related diseases. Proteins 2003;51:147–153.

The role of pH on instability and aggregation of sickle hemoglobin solutions.

Understanding the physical basis of protein aggregation covers strong physical and biomedical interests. Sickle hemoglobin (HbS) is a point‐mutant form of normal human adult hemoglobin (HbA). It is responsible for the first identified “molecular disease,” as its propensity to aggregation is responsible for sickle cell disease. At moderately higher than physiological pH value, this propensity is inhibited: The rate of aggregate nucleation becomes exceedingly small and solubility after polymerization increases. These order‐of‐magnitude effects on polymer nucleation rates and concurrent relatively modest changes of solubility after polymerization are here shown to be related to both pH‐induced changes of location and shape of the liquid–liquid demixing (LLD) region. This allows establishment of a self‐consistent contact between the thermodynamics of the solution as such (i.e., the LLD region), the kinetics of fiber nucleation, the theory of percolation, and the thermodynamics of gelation. The observed pH‐induced changes are largely attributable to strong perturbations of hydrophobic hydration configurations and related free energy by electric charges. Similar mechanisms of effective control of aggregate nucleation rates by means of agents such as cosolutes, pH, salts, and additives, shifting the LLD and associated regions of anomalous fluctuations, promise to be relevant to the whole field of protein aggregation pathologies. Proteins 2004;00:000–000.

Co-solute control of the self-assembly of a biopolymeric supramolecular structure

Abstract We report time-resolved experiments on the effects of modulation of hydrophobic interactions by addition of co-solutes on the non-nucleate (spinodal) decomposition of the aqueous sol of a biostructural polysaccharide. Decomposition is known to trigger in this system the self-assembly of an extended supramolecular structure and to provide a canvas for the latter. The present experiments show how the canvas and the final stability of the decomposed system are affected in a non-trivial way by co-solutes, thus demonstrating a simple and cell-free mechanism capable of controlling the assembly, stability and mesoscopic structure of supramolecular order.

Physics and biophysics of solvent induced forces: hydrophobic interactions and context-dependent hydration

Abstract Solvent induced forces (SIFs) among solutes derive from solvent structural modification due to solutes, and consequent thermodynamic drive towards minimization of related free energy costs. The role of SIFs in biomolecular conformation and function is appreciated by observing that typical SIF values fall within the 20–200 pN interval, and that proteins are stable by only a few kcal mol–1 (1 kcal mol–1 corresponds to 70 pN Å). Here we study SIFs, in systems of increasing complexity, using Molecular Dynamics (MD) simulations which give time- and space-resolved details on the biologically significant scale of single protein residues and sidechains. Of particular biological relevance among our results are a strong modulability of hydrophobic SIFs by electric charges and the dependence of this modulability upon charge sign. More generally, the present results extend our understanding of the recently reported strong context-dependence of SIFs and the related potential of mean force (PMF). This context-dependence can be strong enough to propagate (by relay action) along a composite solute, and to reverse SIFs acting on a given element, relative to expectations based on its specific character (hydrophobic/ philic, charged). High specificity such as that of SIFs highlighted by the present results is of course central to biological function. Biological implications of the present results cover issues such as biomolecular functional interactions and folding (including chaperoning and pathological conformational changes), coagulation, molecular recognition, effects of phosphorylation and more.

Biomolecular-Solvent Stereodynamic Coupling Probed by Deuteration

Thermodynamic interpretation of experiments with isotopically perturbed solvent supports the view that solvent stereodynamics is directly relevant to thermodynamic stability of biomolecules. According with the current understanding of the structure of the aqueous solvent, in any stereodynamic configuration of the latter, connectivity pathways are identifiable for their topologic and order properties. Perturbing the solvent by isotopic substitution or, e.g., by addition of co-solvents, can therefore be viewed as reinforcing or otherwise perturbing these topologic structures. This microscopic model readily visualizes thermodynamic interpretation. In conclusion, the topologic stereodynamic structures of connectivity pathways in the solvent, as modified by interaction with solutes, acquire a specific thermodynamic and biological significance, and the problem of thermodynamic and functional stability of biomolecules is seen in its full pertinent phase space.

Solvent-Induced Free Energy Landscape and Solute-Solvent Dynamic Coupling in a Multielement Solute

Molecular dynamics simulations using a simple multielement model solute with internal degrees of freedom and accounting for solvent-induced interactions to all orders in explicit water are reported. The potential energy landscape of the solute is flat in vacuo. However, the sole untruncated solvent-induced interactions between apolar (hydrophobic) and charged elements generate a rich landscape of potential of mean force exhibiting typical features of protein landscapes. Despite the simplicity of our solute, the depth of minima in this landscape is not far in size from free energies that stabilize protein conformations. Dynamical coupling between configurational switching of the system and hydration reconfiguration is also elicited. Switching is seen to occur on a time scale two orders of magnitude longer than that of the reconfiguration time of the solute taken alone, or that of the unperturbed solvent. Qualitatively, these results are unaffected by a different choice of the water-water interaction potential. They show that already at an elementary level, solvent-induced interactions alone, when fully accounted for, can be responsible for configurational and dynamical features essential to protein folding and function.