The role of water in hemoglobin function and stability

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…

Physical Origin and Biological Significance of Solvent Induced Forces

The notion of solvent-induced forces (SIFs), as distinct from solute-solute forces acting through the solvent, is illustrated in terms of: i) the microscopic space-and time-resolved view provided by Molecular Dynamics Simulations; ii) the standard statistical-mechanical formulation, and iii) the inherent structures of water. It is shown that the origin of SIFs is in the non-additivity of the effects of solute-perturbation on the H-bond network in the solvent. This nonadditivity does not require non-additivity of water-water and solute-water interaction potentials. Two experimental studies, illustrating different aspects of SIFs are discussed in detail. One is the case of Human Adult Hemoglo…

A Unified Theory of Liquid-Liquid Demixing and Polymer Formation Kinetics

Sickle hemoglobin is a natural hemoglobin mutation with a hydrophobic replacement of a charged aminoacid on the molecular surface. This leads to aggregation into rigid helical structures (“polymerization”), the underlying cause of sickle cell disease. It has also been shown that polymerization occurs in close correspondence with the phase transition of liquid-liquid demixing , or with the critically diverging fluctuations of local concentration occurring in its proximity. Due to this correspondence, polymerization kinetics remarkably appear to exhibit, with respect to demixing temperature, the same universal scaling features shown by amplitudes and lifetimes of fluctuations occurring in pro…

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. Th…

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 exponen…

Protein aggregation/crystallization and minor structural changes: universal versus specific aspects.

AbstractProtein association covers wide interests in biophysics, protein science, and biotechnologies, and it is often viewed as governed by conformation details. More recently, the existence of a universal physical principle governing aggregation/crystallization processes has been suggested by a series of experiments and shown to be linked to the universal scaling properties of concentration fluctuations occurring in the proximity of a phase transition (spinodal demixing in the specific case). Such properties have provided a quantitative basis for capturing kinetic association data on a universal master curve, ruled by the normalized distance of the state of the system from its instability…

Photon scattering as a probe of microviscosity and channel size in gels such as sickle haemoglobin.

The aggregation of sickle-cell haemoglobin (HbS) is one of the most physiologically important and widely studied macromolecular gelation processes. Both the thermodynamics and kinetics of the process are important in determining the pathological consequences of deoxygenation of the red cells (and both must be understood if a rational strategy is to be developed for pharmacological intervention). We describe here a new and versatile technique for the study of the structure and formation of the HbS aggregates, that should be widely applicable to gel systems generally. We use laser autocorrelation spectroscopy to observe the diffusion of monodisperse polystyrene latex spheres in the interstice…

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 sc…

Lysozyme crystallization rates controlled by anomalous fluctuations

Abstract Nucleation of protein aggregates and crystals is a process activated by statistical fluctuations of concentration. Nucleation rates may change by several orders of magnitude upon apparently minor changes in the multidimensional space of parameters (temperature, pH, protein concentration, salt type and concentrations, additives). We use available data on hen egg lysozyme crystal induction times in different solution conditions. We measure by static and dynamic light scattering the amplitudes and lifetimes of anomalously ample and long-lived fluctuations occurring in proximity of the liquid–liquid demixing region of the given lysozyme solutions. This allows determining the related sp…

Introduction to the microsymposium and on the modulation of thermodynamic parameters relative to biomolecule-solvent interactions

Abstract As a result of introducing a macromolecule in a solvent, changes in the structures of both solute and solvent are generally found. In this specific meaning we refer here to ‘relaxation phenomena’. The discussion will either concern or keep in a view the case of biomolecules and to which extent these changes are involved in their thermodynamic stability or functional structure: Dr. Ben-Naim will discuss hydrophobic interactions and structural changes in the solvent; Dr. Wen will focus on thermodynamic properties of aqueous solutions on hydrophobic solutes; Dr. Cordone, Dr. Fornili and myself shall discuss results of experiments concerning solutes on several types(proteins, polynucle…

Spinodal demixing, percolation and gelation of biostructural polymers

We present a variety of new experiments which concern the self-assembly of a polymeric network from homogeneous solutions of Agarose, a representative biostructural polysaccharide used for previous studies at our laboratories. They allow deriving a semi-quantitative phase diagram in the T, C plane. The diagram includes both the spinodal and gelation lines. Below a value of about 2% w/v, concentration is not sufficient for direct gelation; however, quenching of the sol from high temperatures to below the spinodal line initiates the spinodal demixing. The latter generates two sets of regions having respectively, higher- and lower-than-average polymer concentrations. In the higher-concentratio…

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…

Solvent-induced free energy landscape and solute-solvent dynamic coupling in a multielement solute

AbstractMolecular 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 syst…

Protein crystallization: universal thermodynamic vs. specific effects of PEG

The interest of nucleation of protein crystals and aggregates (including oligomerization) spans from basic physics theory all the way to biophysics, nanophysics, clinical sciences, biotechnologies, food technologies and polymer–solvent interactions. Understanding nucleation within a theoretical framework capable of providing quantitative predictions and control of nucleation rates, or even the very occurrence of crystallization, is a long-sought goal of remarkable relevance to each of the above fields. A large amount of work has been aimed at such goal, but success has been so far rather limited. Work at our laboratory has more recently highlighted a direct link between nucleation rates and…

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

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 A). 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 …

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 scalin…

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 str…

Interaction of explicit solvent with hydrophobic/philic/charged residues of a protein: Residue character vs. conformational context

Molecular dynamics simulations of model solutes in explicit molecular water have recently elicited novel aspects of the strong nonpair additivity of the potential of mean force (PMF) and related solvent-induced forces (SIFs) and hydration. Here we present the results of the same type of work on SIFs acting on bovine pancreatic trypsin inhibitor (BPTI) at single residue/sidechain resolution. In this system, nonpair additivity and the consequent dependence of SIFs on the protein conformational context are sufficiently strong to overturn SIFs on some individual residues, relative to expectations based on their individual characters. This finding calls for a revisitation and offers a richer and…

Enthalpic and entropic contributions of water molecules to the functional T → R transition of human hemoglobin in solution

Generalized solvent-mediated forces contribute to free energy at the functional T → R transition of human hemoglobin A (HbA). Their contribution is here sorted out quantitatively in both its enthalpic and entropic parts, along with the average number of water molecules involved. The latter (about 75 waters in average) must be considered together with HbA as one statistically defined functional unit for oxygen transport. Their configurations are expected to undergo frequent structural rearrangements. Lifetimes of statistically relevant configurations do not need to (although, of course, they may) exceed by more than a factor 5 the normal H-bond lifetimes of the pure solvent. Compared to the …