G. Careri

Protein hydration and function.

Publisher Summary Hydration can be considered a process of adding water incrementally to dry protein, until a level of hydration is reached beyond which further addition of water produces no change and only dilutes the protein. The hydration process has several stages and an end point, reflected similarly in different types of measurements. Time-average measurements appear to have a common pattern and most closely fit a single picture of the process. Dynamic measurements sometimes show a dependence on hydration level that lies outside the common pattern of the time-average results. The chapter summarizes the literature that bears on the protein hydration process and the hydration shell, categorized by type of measurement, and provides picture of the hydration process and the hydration shell and an assessment of the ways in which the hydration shell may modulate enzyme and other protein functions. The percolation model is discussed that focuses on the long-range connectivity established at a threshold coverage of a surface or volume with conducting or otherwise functional elements.

Water and globular proteins

Abstract Dynamic, thermodynamic and structural studies of the hydration of globular proteins indicate how the macromolecule-water interface can influence folding, enzymatic activity and other biological properties.

COOPERATIVE CHARGE FLUCTUATIONS BY MIGRATING PROTONS IN GLOBULAR PROTEINS

A review of the hydrogen bonded network on the protein surface shows the presence of a charged complex system with parallel and competitive interactions, including ionizable side-chains, migrating protons, bound water and nearby backbone peptides. This system displays cooperative effects of dynamical nature, reviewed for lysozyme as a case. By increasing the water coverage of the protein powder, the bound water cluster exhibits a percolative transition, detectable by the onset of large water-assisted displacements of migrating protons, with a parallel emergence of protein mobility and biological function. By lowering the temperature, migrating protons exhibit a glassy dielectric relaxation in the low frequency range, pointing to a frustration by competing interactions similar to that observed in spin glasses and fragile glass forming liquids. The observation of these dissipative processes implies the occurrence of spontaneous charge fluctuations. A simplified model of the protein surface, where conformational and ionizable side-chain fluctuations are averaged out, is used to discuss the statistical physics of these cooperative effects. Some biological implications of this dynamical cooperativity for enzymatic activity are briefly suggested at the end.

Dielectric properties of Artemia cysts at low water contents. Evidence for a percolative transition

Cellular cysts of the crustacean Artemia provide a useful model for studies on water-dependent mechanisms in cellular function because they can undergo reversible cycles of dehydration-rehydration. We explored their dielectric behavior over the frequency range of 10 kHz to 1 MHz, at water contents between near zero and 0.5 g H2O/g dry weight (g/g). The dc conductivity and static dielectric permittivity were evaluated from electrostatic analysis of data obtained with a three-layered capacitor. Below cyst hydrations of 0.05 g/g, negligible dielectric response was observed at all frequencies. Between 0.05 and 0.25 g/g the permittivity increased sharply then reached a near plateau up to cyst hydrations close to 0.35 g/g, above which a second abrupt increase occurred. Values for the dielectric loss (tan delta) exhibited frequency-dependent peaks over the hydration range of 0.05-0.3 g/g, followed by an abrupt increase near 0.35 g/g, an hydration at which metabolism is first initiated in this system. These hydration-dependent dielectric changes are compared with previous studies on the biology and physics of this system, and evaluated by a model involving percolative ionic (likely protonic) conduction. Percolative behavior is characterized by a sharp increase in conductivity at a critical threshold of hydration (hc) according to a power law in which the exponent, t, equals 1.65 for a three-dimensional infinite lattice. For the Artemia cyst, t = 1.64 above hc = 0.35 g/g, which is in excellent agreement with theory. These results are compared to similar studies on lysozyme which also exhibits percolative behavior connected with the onset of biological function.

Temperature dependence of dielectric relaxation in H2O and D2O ice. A dissipative quantum tunneling approach

We have measured the dielectric relaxation time of orientational defects for several H2O and D2O polycrystalline ice samples, in the temperature range 200–270 K, and over the frequency range 0.3–1000 kHz. Results are in good agreement with previous studies, and at T<240 K, departures from the familiar Arrhenius law have been observed. We show that these deviations from classical rate theory can be well described within the framework of dissipative quantum tunneling (DQT) theory, assuming impurity‐generated Bjerrum defects responsible for the observed dielectric relaxation process over the entire temperature range investigated. The temperature regions where quantum tunneling, crossover to thermal hopping, and quantum corrections to classical laws, respectively, prevail are clearly identified, and experimental data have been successfully fitted with theoretical predictions. Particularly significant is the perfect agreement, near the crossover temperature Tc, of all our different samples with a universal sca...

Proton tunneling in hydrated biological tissues near 200 K.

We measure the protonic conductivity in water clusters adsorbed on intact samples of viable biological samples (corn embryo and endosperm, Artemia cysts, and Typha pollen) below room temperature. In the low-temperature region, the conductivity increases with temperature as exp T6, in agreement with prediction by the theory of dissipative quantum tunneling. We detect the onset of this effect near 180 K, where a glass transition in the hydrated protein matrix is known to take place. Above 220 K other transitions are superimposed onto this simple behavior.

Onsager coupling in enzymes

BioSystems. 8 (1977) 185-186 @ ElsevierlNorth-Holland Scientific Publishers, Ltd. QNSAGER COUPLING IN ENZYMES * G. CARERI and E. GRATTON Zstituto di Fisica. Universitci di Roma, (Received Italy January 17th. 1977) In a conference at Coral Gables in Novem- ber 1973, on the occasion of the seventieth birthday of Lars Onsager, one of us, Careri (1974), proposed a mechanism of enzyme action grounded on the capability of the enzyme to correlate in time the statistical fluctuations of some macrovariables relevant for catalysis. This property can be expressed by a non-vanishing Qnsager matrix, and Lars Onsager was very pleased with this picture because the enzymes being catalysts must work reversibly around equilibriu said the “Onsager approximation” to hold in this case. As a result of Lars Onsager’s eneourage- ment, the statistical time events which occur in enzymes have been critically reviewed by Careri et al. (1975) and the presence of nano- second fluctuations in a dry lysozyme pow- der has been proved by oxygen quenching by Careri, .Gratton in the experiment ed). ‘This experimental and Weber (unpub f spontaneous fhc- fact implies the ex tuations also in the active site of the enzyme, and, together with the known stochastic nature of the solvation effects, allow us to propose here an oversimplified model of the enzymatic action using one term of the Onsager matrix only, as follows. dig all the biochemical consider- ut the stepwis energy barrier the system must receive some free energy from the surrounding bath, and let us assume that this can be accomplished by a fluctuation. Molecular considerations not to be reviewed here, led to the conclusion that the major process by which the free energy can be exchanged between the macro- molecule and the bath must be identified in the accommodation and release of the water molecules bound on the backbone amide groups which are accessible to the solvent. This bound water is known to amount up to ZO-30% in weight of the macromJecule itself. Since theginding of the water molecule to the amide group increases the polar char- acter of the peptide bond, and this induces a change in its conformation because of the increased planarity of the amide group, there is a possible propagation of these conforma- tional changes from the surface of the enzyme to its active site, where even subtle conforma- tional changes are essential to lower the free energy profile of the chemical reaction. Therefore a fluctuation of the bound water density at the surface can be coupled with a spont~eous confo~ati~~~ ~u~t~~t~on at the antive core, and this statistical cou can be expressed by a non-vanis coefficient, when the two stati ely that in order to o reaction coor * Invited paper. Dedicated to the ~e~~~ Onsager. ** Permanent address: Laboratorio Ricer&e onterotondo, Roma, Italy. of Lark di Base,

Magnetic susceptibility of lysozyme

Abstract We have measured the magnetic susceptibility of hydrated powder of lysozyme by the Faraday method at different values of the applied magnetic field, and of lysozyme aqueous solutions by a superconducting magnetometer at different temperature. We find lysozyme to behave as a normal diamagnetic substance, and not in the abnormal way detected by previous authors.

Dielectric relaxation of a proton glass in hydrated protein powders

Dielectric relaxation in the kHz to MHz range displayed by hydrated powders of the protein lysozyme at room temperature is mainly due to protons migrating between ionized side chains. Quite recently we have measured the frequency and temperature dependence of this relaxation, and we have detected the freezing to a broad distribution of relaxation times, typical of proton glasses. Below the freezing temperature of about 270 K the system is not ergodic, in analogy with the behavior of proton glass crystals made up by random mixtures of ferro- and antiferroelectric compounds.

Emergence of Biological Function in the Framework of a Percolation Model

Recent work from this laboratory has shown that hydrated intact biomaterials exhibit dielectric behaviour owing to proton conductivity, and that this behaviour can be described in the frame of percolation theory. Long range proton displacement appears only above the critical hydration for percolation h c (g water/g dry weight), when motion takes place on fluctuating clusters of hydrogen-bonded water molecules. The emergence of biological function, was found to coincide with the critical hydration for percolation h c , and the critical exponent of conductivity to agree with theoretical predictions.