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We compare the role of a conformational switch and that of a point mutation in the thermodynamic stability of a protein solution and in the consequent propensity toward aggregation. We study sickle-cell hemoglobin (HbS), the beta6 Glu-Val point mutant of adult human hemoglobin (HbA), in its R (CO-liganded) conformation, and compare its aggregation properties to those of both HbS and HbA in their T (unliganded) conformation. Static and dynamic light scattering measurements performed for various hemoglobin concentrations showed critical divergences with mean field exponents as temperature was increased. This allowed determining spinodal data points T(S)(c) by extrapolation. These points were fitted to theoretical expressions of the T(S)(c) spinodal line, which delimits the region where the homogeneous solution becomes thermodynamically unstable against demixing in two sets of denser and dilute mesoscopic domains, while remaining still liquid. Fitting provided model-free numerical values of enthalpy and entropy parameters measuring the stability of solutions against demixing, namely, 93.2 kJ/mol and 314 J/ degrees K-mol, respectively. Aggregation was observed also for R-HbS, but in amorphous form and above physiological temperatures close to the spinodal, consistent with the role played in nucleation by anomalous fluctuations governed by the parameter epsilon = (T - T(S))/T(S). Fourier transform infrared (FTIR) and optical spectroscopy showed that aggregation is neither preceded nor followed by denaturation. Transient multiple interprotein contacts occur in the denser liquid domains for R-HbS, T-HbS, and T-HbA. The distinct effects of their specific nature and configurations, and those of desolvation on the demixing and aggregation thermodynamics, and on the aggregate structure are highlighted.

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. © 2003 Wiley-Liss, Inc.

We discuss features of the effect of solvent on protein folding andaggregation, highlighting the physics related to the particulate nature and the peculiar structure of the aqueous solvent, and the biological significance of interactions between solvent and proteins. To this purpose we use a generalized energy landscape of extended dimensionality. A closer look at the properties of solvent induced interactions and forces proves useful for understanding the physical grounds of `ad hoc' interactions and for devising realistic ways of accounting for solvent effects. The solvent has long been known to be a crucially important part of biological systems, and times appear mature for it to be adequately accounted for in the protein folding problem. Use of the extended dimensionality energy landscape helpseliciting the possibility of coupling among conformational changes and aggregation, such as proved by experimental data in the literature.

This work concerns the aggregation properties of (Gly-Val-Gly-Val-Pro)(251) rec, a polypentapeptide reflecting a highly conserved repetitive unit of the bioelastomer, elastin. On raising the temperature of aqueous solutions above 25 degrees C, this polypeptide was already known to undergo concurrent conformational changes (hydrophobic folding), phase separation, and self-assembly with formation of aggregated three-stranded filaments composed of dynamic polypeptide helices, called beta-spirals. Aggregates obtained from the solution can be shaped into bands that acquire entropic elastic properties upon gamma-irradiation and can perform a variety of energy conversions. Previous studies have shown that aggregation is prompted by the (diverging) critical fluctuations of concentration occurring in the solution, in vicinity of its spinodal line. Here, we present combined circular dicroism (CD) and light scattering experiments, and independent fittings of experimental data to the theoretical spinodal and binodal (coexistence) lines. Results show the following logical and causal sequence of processes: (a) Smooth and progressive conformational changes promoted by concentration fluctuations occurring as temperature is raised "pull down" (in the temperature scale) the instability region of the solution. (b) This further promotes critical fluctuations. (c) The related locally high concentration prompts a further substantial conformational change ending in triple-helix formation and coacervation. (d) This intertwining of processes, covering different length scales (from that of individual peptides to the mesoscopic one of demixed regions), is related to the fact that solvent-induced interactions play a strong role over the entire scale span. These results concur with other recent ones in pointing out that process interactions over many length-scales probably reflect a frequent if not ubiquitous pattern in protein aggregation. This may be highly relevant to the desirable deep understanding of such phenomenon, whose interests cover many fields.

We study by molecular-dynamics simulations the effect of electric charges of either sign on hydrophobic interactions and on the dynamics of hydration water, using explicit water and very simplified solutes. Results show that the presence of a charged solute can disrupt the "hydrophobic contact bond" between two apolar solutes nearby, by forcing them towards a different configuration. As a consequence of different structural changes of the solvent caused by charges of opposite sign, the effect is markedly charge-sign-dependent. Analogous weaker effects appear to be induced by the presence of one additional apolar element. The dynamics of hydration water around each solute is also seen to be strongly influenced by the presence of other (charged or uncharged) nearby solutes. Comparison between our results on hydration water dynamics around charged solutes and available experimental data allows sorting out the effects of solute charge sign and size. Our results also offer a plain interpretation of the equivalence of the effects on water structure due to solute ions and to high pressures. These results reflect at a basic paradigmatic level the immensely more complex cases of well-known phenomena such as salting-in and salting-out, and of protein conformational changes caused, e.g., by the arrival of a charged or of an apolar group (phosphorilation or methylation). As it will be discussed, they help in the direction of Delbruck's desirable "progress towards a radical physical explanation" for this class of phenomena.

A strong interest is currently focused on protein self-association and deposit. This usually involves conformational changes of the entire protein or of a fragment. It can occur even at low concentrations and is responsible for pathologies such as systemic amyloidosis, Alzheimer's and Prion diseases, and other neurodegenerative pathologies. Readily available proteins, exhibiting at low concentration self-association properties related to conformational changes, offer very convenient model systems capable of providing insight into this class of problems. Here we report experiments on bovine serum albumin, showing that the process of conformational change of this protein towards an intermediate form required for coagulation occurs simultaneously and interacts with two more processes: mesoscopic demixing of the solution and protein cross-linking. This pathway of three interacting processes allows coagulation even at very low concentrations, and it has been recently observed also in the case of a nonpeptidic polymer. It could therefore be a fairly common feature in polymer coagulation/gelation. Proteins 1999;37:116-120.

We report studies of the interplay among processes of molecular conformational changes, spinodal demixing of the solution, and molecular crosslinking involved in the physical gelation of a biopolysaccharide-water system. Multiple interactions and kinetic competition among these processes were studied under largely different absolute and relative values of their individual rates by appropriate choices of the quenching temperature at constant polymer concentration. Quenching temperature strongly affects the rate of growth but not the final value of the fractal dimension of the gel. Kinetic competition plays a central role in determining the final conformation of individual molecules and the structure and properties of the final gel. This behavior highlights the frustrated nature of the system, and the need of bringing kinetics sharply into focus in gelation theories. General aspects of the present findings and, specifically, the interplay of molecular conformation changes, solution demixing, and molecular crosslinking extend the relevance of these studies to the fast growing field of amyloid condensation and Prion diseases.

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 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.

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.

We discuss features of the effect of solvent on protein folding andaggregation, highlighting the physics related to the particulate nature and the peculiar structure of the aqueous solvent, and the biological significance of interactions between solvent and proteins. To this purpose we use a generalized energy landscape of extended dimensionality. A closer look at the properties of solvent induced interactions and forces proves useful for understanding the physical grounds of `ad hoc' interactions and for devising realistic ways of accounting for solvent effects. The solvent has long been known to be a crucially important part of biological systems, and times appear mature for it to be adequately accounted for in the protein folding problem. Use of the extended dimensionality energy landscape helpseliciting the possibility of coupling among conformational changes and aggregation, such as proved by experimental data in the literature.

Experimental data are presented concerning a large transient viscosity surge occurring in the course of spinodal demixing of agarose aqueous solutions. The study includes the effects of water perturbation by minor proportions of compatible cosolutes. Three observations are noteworthy. One concerns an upward or downward shift of the spinodal temperature, caused by cosolutes, which agrees with their expected modulation of solvent-induced forces. The second concerns the time of appearance of the viscosity surge. This is observed to follow a critical law, with an exponent independent of polymer concentration and solvent perturbation. The third concerns the inverse-power-law dependence of the viscosity peak value on shear. When the shear is scaled with an appropriate relaxation time, related to the overall interdomain structure generated by spinodal demixing, all data fall on a master curve, independent of polymer concentration, quenching depth, and solvent perturbation. This allows assigning the observed viscosity behavior to distortion and rupture of the overall interdomain structure generated by spinodal demixing.

A molecular dynamics statistical mechanical simulation study of solvent‐induced forces (SIFs) acting between two fixed model solutes in a bath of 727 water molecules is reported. Simulations were run up to 500 ps on a multiprocessor system, involving 60 T800‐20 transputers. Results are discussed within the statistical mechanical approach of inherent structures. It is shown that SIFs are associated with the occurrence of nonpermanent, but longer‐lived statistically relevant solvent configurations promoted by solutes. Their size and sign are shown to be critically dependent on the type of solute, solute–solute distance, and mutual orientation. These features give SIFs the specificity character crucially required for functional interactions and for functional conformation and dynamics of biomolecules.

Solvent-induced forces (SIFs) have been for a long time erroneously related to the presence of more or less uniform “hydration shells”, consisting of more or less permanently bound water molecules. As such misconceptions fade away, it becomes clear that hydration should be viewed statistically and in terms of topological rather than geometric order, and that minor changes (due to solutes) in the structural-rearrangement times of the solvent can reflect major effects in SIFs. Recent work on SIFs at our laboratory, here critically summarized, shows: i) Precisely localized average intramolecular SIFs adjusting the operation of a protein to just what is physiologically required. ii) The involvement of SIFs in the thermodynamic stability of biomelecular solutions and “soups” and related fluctuation behaviour. Sustained, undamped fluctuations occurring in the instability region, and also subcritical ones, occurring close to (yet, not in) the instability region, have been shown to be capable of triggering important concentration- dependent biomolecular processes, iii) The involvement of large numbers of water molecules (of the order of 100) in intra- and interbiomolecular SIFs. This dramatically expands the dimensionality and extension of the functional phase space and helps understand for the first time the extraordinary thermodynamic functional stability of biomolecules.

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 bare protein, the “functional unit” here evidenced for the first time involves a larger, higher dimensionality region of the phase space. The study allows a microscopic view of solvent-mediated generalized forces and evidences their qualitative and quantitative relevance to biomacromolecular function and stability.

Density data for aqueous solutions of monohydric alcohols down to supercooling are presented and combined with data concerning viscosity of the same systems, and with available data on pure water. Two conceptually different families of longer‐lived, high‐connectivity H‐bond structures can be sorted out in the irregular, frequently restructured network of H bonds: spontaneous structures, as in pure water, and alcohol‐induced structures. Molar volumes for both are obtained and compared, allowing microscopic conclusions which agree quantitatively with available thermodynamic data. For the three alcohols studied, the well‐known negative excess entropy of mixing shows a strict proportionality to the fraction of water molecules sorted out in our study as taking statistically part in alcohol‐promoted cages. The remarkable proportionality extends to all alcohols, all concentrations, and all temperatures studied. Apparent (and expected) geometric distortions of alcohol‐promoted cages do not affect this proportiona...

We report a first set of results of Molecular Dynamics simulations of phase separation in a two-dimensional system of identical particles bearing attachment sites at their surface. Morphology of regions of aggregation so obtained appear similar to images of biologically self-assembled structures. Also, they evidence the relevance of the region of thermodynamic instability and of the presence and range of interaction of specific attachment sites. Particles of our system are apt to represent indifferently, though grossly, globular proteins or rigid cells, bearing specific and fixed adhesion sites at their surface. Results illustrate some basic aspects of the origin of the morphology of extended biomolecular/cellular structures, self-organized from initially homogeneous solutions.

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 the universal scaling properties of concentration fluctuations occurring in the vicinity of a phase transition. The phase transition here concerned is that of non nucleated liquid–liquid demixing of the solution. This novel universality feature allows viewing nucleation processes within one and the same frame, and to capture all normalized nucleation rates on one and the same “master curve” for different proteins, as a function of one parameter only. The quantitative value of the latter is the result of the joint, non additive effects of protein composition, conformation and state (e.g. oligomers), as well as of the temperature of non nucleated liquid–liquid demixing of the solution at the given protein concentration and at the given conditions of the solution. The present work was undertaken for the purpose of ascertaining if (and if so, in what way) the universality feature can allow the quantitative prediction of nucleation rates changes caused by the addition to the solvent of additives empirically known for their strong effect on such rates, as well as the very occurrence of crystallization. To this purpose we have used PEG (polyethylene glycol), which is perhaps the most familiar and most-used additive, and have measured by static and dynamic light scattering the properties of concentration fluctuation of the system as a function of temperature, for various PEG concentration and polymerisation degrees. Experiments have shown that the action of PEG can in no way be accounted for in terms of changes of specific local contacts or of a one-to-one chaperone-like action. Instead, the effect of PEG is seen to be due to alteration of the thermodynamic properties of the solution. This leaves unchanged the universality features and consequently also the validity and predictive power of the master curve in the various conditions.

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-concentration regions the functional polymer-polymer interaction (that is, self-assembly) is favoured. In fact, as in the course of demixing the point representative of higher-concentration regions reaches the region below the gelation line, gelation is allowed and indeed observed to occur. This evidences the possibly more general role of spinodal demixing (extended to multi-component systems) as a pathway for specific, local and biofunctional enhancements of concentrations. At very low concentrations (e.g. 10−4 w/v) the kinetics of demixing is still observed to occur, but the set of higher-concentration regions is no longer percolative. In these conditions, gelation occurs only within each individual region, while the specimen remains free-running. This illustrates a novel aspect of the notion of non-gelling concentrations.

Protein 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 region. Here we report new data on lysozyme crystal nucleation. They strengthen the evidence in favor of universality and show that the system enters the region of universal behavior in a stepwise manner as a result of minor conformation changes. Results also show that the link between conformation details and universal behavior is actuated by interactions mediated by the solvent. Outside the region of universal behavior, nucleation rates become unpredictable and undetectably long.

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 spinodal temperatures T S and ɛ = ( T – T S ) / T S . Experimental induction times appear to depend solely upon ɛ over many orders of magnitude. This is quantitatively accounted for in terms of an extended two-stage nucleation model, which jointly takes into consideration amplitudes, lifetimes and scaling properties of anomalous fluctuations. One and the same relation describes quantitatively and equally well the present case of lysozyme crystallization (the best studied case of protein crystallization) and that of sickle hemoglobin fiber formation (the best studied case of protein fiber formation). Comparison with other recent models shows that taking into account lifetimes of anomalous fluctuations allows capturing the essence of the observed behavior.

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 A. 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.

This work concerns the aggregation properties of (Gly-Val-Gly-Val-Pro)(251) rec, a polypentapeptide reflecting a highly conserved repetitive unit of the bioelastomer, elastin. On raising the temperature of aqueous solutions above 25 degrees C, this polypeptide was already known to undergo concurrent conformational changes (hydrophobic folding), phase separation, and self-assembly with formation of aggregated three-stranded filaments composed of dynamic polypeptide helices, called beta-spirals. Aggregates obtained from the solution can be shaped into bands that acquire entropic elastic properties upon gamma-irradiation and can perform a variety of energy conversions. Previous studies have shown that aggregation is prompted by the (diverging) critical fluctuations of concentration occurring in the solution, in vicinity of its spinodal line. Here, we present combined circular dicroism (CD) and light scattering experiments, and independent fittings of experimental data to the theoretical spinodal and binodal (coexistence) lines. Results show the following logical and causal sequence of processes: (a) Smooth and progressive conformational changes promoted by concentration fluctuations occurring as temperature is raised "pull down" (in the temperature scale) the instability region of the solution. (b) This further promotes critical fluctuations. (c) The related locally high concentration prompts a further substantial conformational change ending in triple-helix formation and coacervation. (d) This intertwining of processes, covering different length scales (from that of individual peptides to the mesoscopic one of demixed regions), is related to the fact that solvent-induced interactions play a strong role over the entire scale span. These results concur with other recent ones in pointing out that process interactions over many length-scales probably reflect a frequent if not ubiquitous pattern in protein aggregation. This may be highly relevant to the desirable deep understanding of such phenomenon, whose interests cover many fields.

In this Molecular Dynamics study we use a supersimplified multi-element model solute in explicit solvent. Its configurational space is two-dimensional and its potential energy landscape is flat in vacuo. We study how the landscape is affected by solvent induced interactions, when accounted for to all orders, with no use of Kirkwood’s approximation or similar ones. A landscape of potential of mean force (PMF) possessing distinctive protein like features of realistic size is generated by the solvent. Building on a previous study (San Biagio et al., Biophys. J. 1999, in press), effects of different symmetries, geometries and charges on the PMF and effects due to the dynamical solute-solvent configurational coupling are evidenced.

It has recently been recognized that pathological protein coagulation is responsible for lethal pathologies as diverse as amyloidosis, Alzheimer and TSE. Understanding the coagulation mechanisms is therefore stirring great interest. In previous studies we have shown that on profoundly different systems coagulation is the result of a strong interaction between two processes on different length scales (mesoscopic and microscopic). Here we report experiments on bovine serum albumin (BSA) showing that the overall mechanism is the result of at least 3 distinct and strongly intertwined processes, on both length scales: molecular conformational changes, solution demixing and intermolecular crosslinking. This mechanism involves the statistical mechanics of protein-solvent interaction, its relation to the protein’s landscape of configurational free energy and to the solution’s thermodynamic stability, and its relation to the topological problem of crosslink-percolation, responsible for coagulation.

A strong interest is currently focused on protein self-association and deposit. This usually involves conformational changes of the entire protein or of a fragment. It can occur even at low concentrations and is responsible for pathologies such as systemic amyloidosis, Alzheimer's and Prion diseases, and other neurodegenerative pathologies. Readily available proteins, exhibiting at low concentration self-association properties related to conformational changes, offer very convenient model systems capable of providing insight into this class of problems. Here we report experiments on bovine serum albumin, showing that the process of conformational change of this protein towards an intermediate form required for coagulation occurs simultaneously and interacts with two more processes: mesoscopic demixing of the solution and protein cross-linking. This pathway of three interacting processes allows coagulation even at very low concentrations, and it has been recently observed also in the case of a nonpeptidic polymer. It could therefore be a fairly common feature in polymer coagulation/gelation. Proteins 1999;37:116-120.

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 Hemoglobin, where the SIFs contribution to the functional conformational transition has been quantitatively determined. In this case, SIFs have the localized and specific character required for biomolecular stability and function. The large number of water molecules involved in these SIFs provides the additional and philosophically important result of a great expansion of the phase space relevant to the thermodynamic probability of functional conformations. Another case is the gelation of Bovine Serum Albumin. Here SIFs have a mean-field behaviour, and contribute to the first break of symmetry in the sol, towards self-assembly. In this way SIFs contribute establishing solute-solute correlations and generating preferential paths, which channel the percolation of crosslinks along polymer-rich regions. By this symmetry-breaking mechanism, SIFs can allow gelation at very low polymer concentrations and, more in general, they can be highly relevant to morphogenesis. Experiments on non-additivity of effects of solvent perturbation promoted by alcohols and by osmolytes are also presented. Results allow tracing the origin of the effects of cosolutes in modulating SIFs and solvent-mediated interactions.

The IR absorption spectrum of water and NaClO4 solutions in the 1.45 μ region was studied as a function of temperature and salt concentration. Band profile synthesis with Gaussian components shows the existence of three such components. The temperature and concentration dependence of their intensities, peak frequencies, and widths is analyzed and discussed.

A biological system usually operates under nonstable values of relevant parameters, such as pH, temperature, and ionic strength. The question therefore poses whether such fluctations do affect its relevant processes. Experimental studies on the role of random temperature fluctuations on functional encounters of biostructural polymer molecules, and consequent self-assembly of supramolecular structures, have evidenced an additional, noise-induced order of these structures. This type of effect is the result of nonlinearity in physical systems, and the case of a biosystem is especially interesting. As recent experiments have shown, spinodal decomposition resulting from thermodynamic instability may favor the onset of the supramolecular ordering process. If the random fluctuations of temperature are imposed in such conditions of thermodynamic instability, their ordering effect is further enhanced.

Results are reported on near-i.r. absorption spectra of H2O and D2O down to the temperature region of supercooled liquid. No anomalous behaviour is observed when the absorbance at any given frequency is studied as a function of temperature. A critical-exponent behaviour strikingly correlating with that exhibited by transport properties is instead observed when spectral deconvolution methods are used. Results are discussed in relation with current views on water structure, based on molecular-dynamics simulation results and connectivity calculations.

Hydrogels of a significant biostructural polymer (agarose) were studied turbidometrically in the course of their isothermal self-assembly. Supramolecular order was characterized by an order parameter and associated correlation length. This type of characterization, in general, may help more in the quantitative measurement of order in biostructures. The picture of processes leading to isothermal gelation appears much richer than expected, and it is probably of more general validity, as it agrees with predictions based on the statistical model of site-bond correlated percolation. At the higher temperatures a spinodal decomposition occurs well before, and possibly triggers [P. L. San Biagio, et al. (1986) Biopolymers25, 2255–2269], the start of gelation. In this case, the spatial modulation of concentration, as well as topological features of the gel, appear to reflect those of the spinodally decomposed sol. When an upper limit is approached for the temperature of isothermal gelation, both the order parameter and the correlation length exhibit a critical-like behavior. At lower temperatures, the sequence of processes and the resulting features of the gel appear dictated by the (temperature- and concentration-dependent) interplay of spinodal decomposition and gelation processes and kinetics. The present results are believed to provide a basis for a deeper understanding of the relation between the processes of gelation and of spinodal decomposition, and between their time scales.

The weakly absorbed 1.2-μ combination band of water was accurately studied as a function of temperature, in both its profile and first derivative, to obtain information on the bulk properties of liquid water. Arrhenius plots of integrated component intensity ratios showed well aligned experimental points, except for a neatly defined break occurring in the 30 to 40°C temperature interval for pure water A similar break, shifted by some 18°C towards lower temperature, was found in the case of 1M NaClO4 aqueous solutions. The breaks are tentatively assumed to result from abrupt though subtle changes involving low-frequency modes. This tentative, assumption appears independent of specific models and assignments and, although in agreement with available neutron-scattering and NMR data, needs more direct verification.

In order to measure the anisotropy in paramagnetic resonance absorption in single crystals, a method has been devised which does not require initial knowledge of the directions of the principal magnetic axes, and by which it is possible to determine the principal values together with the directions of the axes. It is shown that under certain assumptions the characteristic surface ofg is an ellipsoid, and that thus, with a proper choice of the axes, the following equation holds:g=g x α2+g y β2+ +g z γ2, whereα, β, γ stand for the direction cosines ofH. The method has been applied for completing previous measurements, thus rendering their interpretation possible.

After an introduction to the problem, an analysis is made of the output of different types of phase detectors for any given input. Expressions are given for the frequencies present in the output (for any given input) and for the related amplitudes and phases. Such expressions agree with and analytically define the well known result that a noise figure close to unity may be reached using phase-detection.

The vibronic absorption spectrum of the Ni++ ion in an electric field of trigonal (nearly cubic) symmetry at temperatures down to 4°K is reported and analysed in terms of ligand field and vibronic theory. The positions, widths, shapes and strengths of the bands are satisfactorily explained, together with their associated fine structures. In particular, the complex structure of the red band is well explained in detail as a consequence of the mutual perturbation of the 1E and 3T4 levels, which strongly modifies the vibronic coupling and results in two fairly narrow bands, each showing resolved vibrational structure, superposed on a broad band. A Trees correction term, αL(L+1), helps the agreement between observed and calculated electronic levels. The following parameters are found to give good agreement: Δ = 9100 cm−1, B=955 cm−1, C=3750 cm−1, α=80 cm−1, ζ = 600 cm−1.

The optical spectrum of the Fe2+ ion in an electric field of trigonal symmetry has been investigated down to liquid helium temperature. An account is given of the results, which suggest the operation of the dynamic Jahn-Teller effect and reveal the presence of a charge-transfer absorption and the action upon the latter of the low-symmetry component of the electric field. A structure which is present on the low-energy side of the main vibronic band, has been proved to be due to the crystal lattice rather than to the magnetic ions.

An electron spin resonance spectrum, due to the formation of cupric ions during illumination, has been observed in crystals of silver chloride containing 0–1 mol %, of cuprous chloride, which have been quenched to−183[ddot]c immediately following irradiation. The spectrum fades rapidly if the crystals are kept at any temperature above −140[ddot]c after the end of illumination. An interpretation of this behaviour is given which is based upon the Mitchell theory of the photochemical process in crystals of silver halides.

Data on paramagnetic resonance absorption, on the distance between the levels responsible for optical absorption, on polarized light absorption and on the structure deduced with X-rays are given which, consistently in agreement with theoretical considerations, confirm the hypothesis previously advanced of the existence of a double covalent bond in the vanadyl group of a vanadylsulphate.

Dielectric losses have been measured in hexammine nickel chloride crystals, which contain a system of protons (${\mathrm{H}}^{+}$ ions) that is responsible for a motional phase transition. The losses are well approximated by Debye curves, both above and below the transition temperature. Since this transition is not accompanied by the onset of a dipolar moment, long-range forces may be neglected, as in the similar but more complex case of hydrogen-bonded ferroelectrics, and the present results may shed light on the dynamical model of this class of substances.

We report results of molecular dynamics (MD) simulations of composite model solutes in explicit molecular water solvent, eliciting novel aspects of the recently demonstrated, strong many-body character of hydration. Our solutes consist of identical apolar (hydrophobic) elements in fixed configurations. Results show that the many-body character of PMF is sufficiently strong to cause 1) a remarkable extension of the range of hydrophobic interactions between pairs of solute elements, up to distances large enough to rule out pairwise interactions of any type, and 2) a SIF that drives one of the hydrophobic solute elements toward the solvent rather than away from it. These findings complement recent data concerning SIFs on a protein at single-residue resolution and on model systems. They illustrate new important consequences of the collective character of hydration and of PMF and reveal new aspects of hydrophobic interactions and, in general, of SIFs. Their relevance to protein recognition, conformation, function, and folding and to the observed slight yet significant nonadditivity of functional effects of distant point mutations in proteins is discussed. These results point out the functional role of the configurational and dynamical states (and related statistical weights) corresponding to the complex configurational energy landscape of the two interacting systems: biomolecule + water.

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.

This is a short review of experiments performed at our Laboratory on the self-assembly of supramolecular structures in vitro. The experiments show that fluctuations of a relevant parameter driven from outside, as well as spontaneous fluctuations of local concentration occurring under or near thermodynamic instability, and non-linear interactions between these two types of fluctuations, can trigger or enhance the self-assembly of supramolecular order. The pattern generated by spontaneous fluctuations is capable of setting the canvas for the resulting self-assembled structure. Experiments performed so far concern two diverse systems: a polysaccharide widely found in nature, which makes firm aqueous biostructural gels (agarose), and a synthetic model system of bioelastomers, the polypentapeptide (Val-Pro- Gly-Val-Gly)n which represents the most significant sequence of the bioelastomeric protein, elastin. Work on the latter system was and is being done in collaboration with D.W. Urry’s group of Birmingham, AL.

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.