An X-ray study of the pH property of frog sciatic nerve

Low-angle X-ray diffraction patterns have been recorded from frog sciatic nerve in pH solutions of 0.1–13.0. The normal X-ray pattern of frog sciatic nerve in Ringer's solution is maintained at pH 4.0–10.0. In acid pH, 2.5–4.0, and in alkaline pH, 10.0–11.0, the nerve myelin is in the partial swollen state. The partial swollen state and the normal state are reversible. Two physical states, the anomalous swollen state and the condensed state, at acid pH below 2.5 and the separated state at alkaline pH above 12.3 have been identified. These three physical states, the anomalous swollen state, the condensed state and the separated state, are reversible with each other on changing the pH solution but the normal state cannot be regained.     

The regulatory-adaptive state and evaluation of stress resistance in humans

The method of estimating human stress resistance by the dynamics of the regulatory-adaptive state is described. The regulatory-adaptive state has been evaluated by the parameters of cardio-respiratory synchronism in the normal state and in stress. Subjects with no changes in the regulatory-adaptive state or with its decrease by 5–6% have been included in the group with a high level of stress resistance. Subjects with a decrease of the regulatory-adaptive state by more than 50% in response to stress have been included in the low stress resistance group. The method has been tested in three models of stress: (1) examination (58 students); (2) parachute jumping (35 beginner parachute jumpers); (3) entrance to an emergency zone (30 rescuers). All three models are highly informative. Stress resistance levels have also been estimated by psycho-logical methods. The estimation of stress resistance by the dynamics of the regulatory-adaptive state allows characterizing the ability of a person to endure stress and can be included in the set of tests for staff recruitment in extreme professions.     

Analysis of bifurcations in continuous fermentation using recombinant microorganisms with delayed responses

When the feed rate to a fermenter is varied periodically in order to favor the growth of plasmid-containing cells, a transition may occur from the starting stationary state to another state. The resulting state may be constant or oscillatory. A generalised model based on the adaption times of plasmid-free and plasmid-harboring cells has been used. Analytical conditions have been derived for bifurcation from one nonoscillatory state to another or to an oscillatory state (Hopf bifurcation). The frequency of oscillation is shown to have an upper bound, which can be controlled by manipulating certain process parameters. The production of tryptophan synthetase by the plasmid pPLc23trpAl in E. coli is used as an example to determine the nature of the Hopf bifurcations.     

Structural changes and fluctuations of proteins: I. A statistical thermodynamic model

A general theory of the structural changes and fluctuations of proteins has been proposed based on statistical thermodynanic considerations at the chain level.The “structure” of protein was assumed to be characterized by the state of secondary bonds between unique pairs of specific sites on peptide chains. Every secondary bond changes between the bonded and unboned states by thermal agitation and the “structure” is continuously fluctuating. The free energy of the “structural state” that is defined by the fraction of secondary bonds in the bonded state has been expressed by the bond energy, the cooperative interaction between bonds, the mixing entropy of bonds, and the entropy of polypeptide chains. The most probable “structural state” can be simply determined by graphical analysis and the effect of temperature or solvent composition on it is discussed. The temperature dependence of the free energy, the probability distribution of structural states and the specific heat have been calculated for two examples of structural change.The theory predicts two different types of structural changes from the ordered to disordered state, a “structural transition” and a “gradual structural change” with rising temperature, In the “structural transition”, the probability distribution has two maxima in the temperature range of transition. In the “gradual structural change”, the probability distribution has only one maximum during the change.A considerable fraction of secondary bonds is in the unbonded state and is always fluctuating even in the ordered state at room temperature. Such structural fluctuations in a single protein molecule have been discussed quantitatively.The theory is extended to include small molecules which bind to the protein molecule and affect the structural state. The changes of structural state caused by specific and non-specific binding and allosteric effects are explained in a unified manner.     

Contribution of the 6-120 disulfide bond of alpha-lactalbumin to the stabilities of its native and molten globule states.

The unfolding and refolding of a derivative of alpha-lactalbumin, in which the disulfide bond between Cys6 and Cys120 is selectively reduced and S-carboxymethylated, are investigated by equilibrium and kinetic circular dichroism measurements. The native conformation of this derivative is known to be essentially identical to that of intact alpha-lactalbumin. The equilibrium unfolding of the derivative involves a stable intermediate, which is also similar to the molten globule state of the disulfide intact protein. The results of stopped-flow circular dichroism experiments show that the same intermediate is formed rapidly as a transient intermediate in kinetic refolding. The conformational stabilities for the native and intermediate states have been estimated and compared with the stabilities for the corresponding states of intact alpha-lactalbumin. The stabilization of the native state by the disulfide has been interpreted in terms of a decrease in chain entropy in the unfolded state and elimination of the strain imposed on the disulfide bond in the native state. The molten globule state is also stabilized by the disulfide bond, although the degree of stabilization of the molten globule state is smaller than of the native state. The results suggest that, in the molten globule state, some ordered structures are present within the loop moiety formed by the 6-120 disulfide.     

Hydrophobins, the fungal coat unravelled

Hydrophobins are among the most surface active molecules and self-assemble at any hydrophilic-hydrophobic interface into an amphipathic film. These small secreted proteins of about 100 amino acids can be used to make hydrophilic surfaces hydrophobic and hydrophobic surfaces hydrophilic. Although differences in the biophysical properties of hydrophobins have not yet been related to differences in primary structure it has been established that the N-terminal part, at least partly, determines wettability of the hydrophilic side of the assemblage, while the eight conserved cysteine residues that form four disulphide bridges prevent self-assembly of the hydrophobin in the absence of a hydrophilic-hydrophobic interface. Three conformations of class I hydrophobins have been identified: the monomeric state, which is soluble in water, the alpha-helical state, which is the result of self-assembly at a hydrophobic solid, and the beta-sheet state, which is formed during self-assembly at the water-air interface. Experimental evidence strongly indicates that the alpha-helical state is an intermediate and that the beta-sheet state is the end form of assembly. The latter state has a typical ultrastructure of a mosaic of 10 nm wide rodlets, which have been shown to resemble the amyloid fibrils.     

Kinetics of the pretransition of synthetic phospholipids. A calorimetric study

The pretransition in aqueous dispersions of two synthetic phospholipids (dimyristoylphosphatidylcholine and dipalmitoylphosphatidylcholine) has been examined in detail by differential scanning calorimetry. The transition from the high-temperature state (state above pretransition) to the low-temperature state (state below pretransition) is complex and appears to occur via some metastable states. In contrast, the kinetics of the transition from the low- to the high-temperature state is consistent with an activated two-state model. The observed hysteresis is shown to arise mainly from the kinetic nature of the pretransition.     

The great escape: do parasites break Dollo's law?

A long-held assumption in evolutionary studies is that a character that changes from a complex to a simple state is unlikely to return to the same complex state. The extreme version of this assumption has been codified as Dollo's law. Unfortunately, this paradigm has supported the idea that simple and complex traits are qualitatively different, when it is more sensible to suggest that there is a quantitative difference. Dollo's law has been the predominant paradigm in parasitology, where a move from a free-living state to parasitism has been considered a unidirectional pathway or 'one-way trip' because organisms lose the structures required to return to the free-living state. Several recent studies have suggested that complex structures can be regained from simple traits, and we suggest that this is also possible for parasites.     

Thermodynamic analysis of three state denaturation of Peanut Agglutinin

Peanut Agglutinin (PNA) is a homotetrameric protein with a very unusual open quaternary structure. During denaturation, it first dissociates into a molten globule like state, which subsequently undergoes complete denaturation. Urea denaturation of PNA at neutral pH has been studied by intrinsic fluorescence spectroscopy and has been fitted to a three state model, A4 <=> 4I <=> 4U, to get all the relevant thermodynamic parameters. Urea denaturation leads to continuous red shift of wavelength maxima. The molten globule like state is formed in a short range of urea concentration. Refolding of the denatured PNA has been attempted by intrinsic fluorescence study. Refolding by instantaneous dilution shows the occurrence of the formation of an intermediate at a relatively rapid rate, within few seconds. The transition from PNA tetramer to molten globule like state is found to have a DeltaG value of approximately 33 kcal/mole while it is approximately 8 kcal/mole for the transition from molten globule like state to a completely denatured state. This in turn indicates that the tetramerization in PNA contributes significantly to the stability of the oligomer.     

Structural studies of translating ribosomes. II. Comparative sedimentation analysis of pre- and post-translocation states

Using the solid-phase translation system technique where template poly(U) is covalently coupled to Sepharose through cleavable disulfide bridges translating monoribosomes carrying a polypeptide (polyPhe) of 10 to 20 amino acids long have been isolated. Both pre-translocation state and post-translocation state ribosomes have been obtained. It has been shown that the sedimentation coefficient of the pre-translocation state ribosomes exceeds that of the post-translocation state ribosomes by a magnitude of about 1S. This difference is independent on the sedimentation rate (hydrostatic pressure) in the range of 20 000 to 40 000 rev/min and, most likely, is not a direct contribution of the increase of the particle mass at the expense of an additional tRNA in the pre-translocation state ribosomes. Together with other data, this result suggests that translating ribosomes in the pre-translocation state are more compact than post-translocation state ribosomes.     

From metmyoglobin to deoxy myoglobin: relaxations of an intermediate state

Metmyoglobin has been reduced at low temperature (below 100 K) using x-rays or by excitation of tris(2,2′,bipyridine)ruthenium(II) chloride with visible light. Upon reduction, an intermediate state is formed where the structure of the protein is very similar to that of metmyoglobin with the water molecule still bound to the heme iron, but the iron is II low spin. The nature of the intermediate state has been investigated with optical spectroscopy. The Q_o and Q_v bands of the intermediate state are split, suggesting that the protoporphyrin is distorted. The intermediate state undergoes a relaxation observed by a shifting of the Soret band at temperatures above 80 K. Above 140 K, the protein begins to relax to the deoxy conformation. The relaxation kinetics of the protein have been monitored optically as a function of time and temperature from minutes to several hours and from 150 K to 190 K. By measuring the entire visible spectrum, we are able to distinguish between electron transfer processes and the protein relaxation from the intermediate state to deoxy myoglobin. The relaxation has been measured in both horse myoglobin and sperm whale myoglobin with the relaxation occurring on faster time scales in horse myoglobin. Both the reduction kinetics and the relaxation show non-exponential behavior. The reduction kinetics can be fit well to a stretched exponential. The structural relaxation from the intermediate state to the deoxy conformation shows a more complex, dynamical behavior and the reaction is most likely affected by the relaxation of the protein within the intermediate state. Received: 30 June 1997 / Accepted: 6 November 1997     

Criteria for the determination of the direction of character state changes

Phylogenetic reconstructions cannot be adequately assessed except in terms of probability models which represent the processes of change. An tionary tree model of bifurcating splits, together with a Brownian motion model of genetic drift have been shown to allow successful reconstruction of phylogeny from data relating to gene frequencies of blood groups from human populations. Changes in state cannot be dealt with by the Brownian motion model, and no adequate models have been proposed for character state changes from the many possible sources. So, while the tionary tree model is applicable to these problems too, only heuristic methods of analysis of the state data are available, and these are known to be unsatisfactory under certain conditions. The concepts of homology, polarity and homoplasy have been developed out of the attempt to describe the nature of morphological state data, which is unpredictably related to genetic state data. The experimental study of comparative functional morphology, at any developmental stage of an organism, is considered to be the only valid tool for investigating the resulting character state tree hypothesis. However, the speculative nature of such investigations is admitted.     

Molecular dynamics simulation of packing and mobility of lipids in bilayer membranes

A model of DSPC lipid membrane in gel and liquid-crystalline states has been developed. The parameters have been determined that enable one to calculate the molecular dynamics of lipid bilayers in the full-atromic approximation. The parameters of packing and mobility of lipid molecules for the liquid crystalline state of the bilayer have been calculated. The values agree well with experimental data. Based on the model of the liquid crystalline state of the membrane, a system in the gel-like state has been constructed. The model of the gel-like state reproduces well the packing of lipids in real bilayers, whereas the mobility of molecules in the gel-like state was found to be overestimated.     

Stabilizing selection and linkage disequilibrium

An approach to the investigation of the evolution of quantitative traits on the basis of analysis of two-locus marginal systems dynamics has been developed. It has been shown that under stabilizing selection the "quasi-stationary" state is quickly reached and maintained continuously. The "quasi-stationary" state is characterized by small changes in allele frequencies and by linkage disequilibrium that significantly decreases genotypic variance. Equations defining the role of linkage disequilibrium in the stationary state of mutation-selection balance are derived.     

Vibrational distribution of nitrogen molecules in the C3Πu state in a near-surface microwave plasma in nitrogen at pressures of 1–5 Torr

The radiation of the second positive nitrogen system has been used to study the spatial dependence of the vibrational distribution of nitrogen molecules in the C³Π_u state in the near-surface plasma layer of an electrode microwave discharge in nitrogen at pressures of 1–5 Torr. It has been shown that the vibrational distribution changes at a scale of 100 μm. It has been concluded that this state is populated owing to the electron impact from the ground state. The possibility of using the local approximation for the electron energy distribution function to explain the experimental results has been analyzed.     

Fluctuating state of the protein globule

It has been shown that bovine and human alpha-lactalbumins and carbonic anhydrase B can be transformed under different influence into a peculiar state possessing physical characteristics intermediate between those for the native and unfolded states. In this state a protein molecule is compact and has the secondary structure similar to that of the native molecule, but does not melt cooperatively at heating, has an anomalously fast H-D exchange and a more or less symmetrical average environment of aromatic and other side groups. A model of this "intermediate" state of protein molecule is proposed, according to which the intermediate state differs from the native one mainly by the substantial increase of protein structure fluctuations and the sharp decrease of van der Waals and other specific interactions. It has been shown that the transition from the native state to this intermediate state is the phase transition of the first order. The role of the intermediate state in protein folding is discussed.     

Conformational changes of Spo0F along the phosphotransfer pathway

Spo0F is a secondary messenger in the sporulation phosphorelay, and its structure has been characterized crystallographically in the apo-state, in the metal-bound state, and in an interacting state with a phosphotransferase. Additionally, the solution structure of the molecule has been characterized by nuclear magnetic resonance techniques in the unliganded state and in complex with beryllofluoride. Spo0F is a single-domain protein with a well-defined three-dimensional structure, but it is capable of adapting to specific conformations for catching and releasing the phosphoryl moiety. This commentary deals with the conformational fluctuations of the molecule as it moves from an apo-state to a metal-coordinated state, to a phosphorylated state, and then to a phosphoryl-transferring state.     

The cytochrome c fold can be attained from a compact apo state by occupancy of a nascent heme binding site

NMR techniques and 8-anilino-1-napthalenesulphonate (ANS) binding studies have been used to characterize the apo state of a variant of cytochrome c(552) from Hydrogenobacter thermophilus. In this variant the two cysteines that form covalent thioether linkages to the heme group have been replaced by alanine residues (C11A/C14A). CD studies show that the apo state contains approximately 14% helical secondary structure, and measurements of hydrodynamic radii using pulse field gradient NMR methods show that it is compact (R(h), 16.6 A). The apo state binds 1 mol of ANS/mol of protein, and a linear reduction in fluorescence enhancement is observed on adding aliquots of hemin to a solution of apo C11A/C14A cytochrome c(552) with ANS bound. These results suggest that the bound ANS is located in the heme binding pocket, which would therefore be at least partially formed in the apo state. Consistent with these characteristics, the formation of the holo state of the variant cytochrome c(552) from the apo state on the addition of heme has been demonstrated using NMR techniques. The properties of the apo state of C11A/C14A cytochrome c(552) reported here contrast strongly with those of mitochondrial cytochrome c whose apo state resembles a random coil under similar conditions.     

Membrane fusion in the rod outer segment disk membrane

The rod outer segment disk membrane of bovine retina has been isolated in a predominantly fused state. The physical and chemical properties of the membrane in the fused state are profoundly different from the corresponding properties of the same membrane in the unfused state. Exposure to light induces the transition of the disk membrane from the fused to the unfused state. Evidence is presented which suggests that the fusion-defusion cycle of the disk membrane is a primary event of photoexcitation and nerve stimulation.     

Dialysis cultivation of microorganisms as the appropriate model of population control in ecosystem research

Opportunities for application of methods of ecological control of anthropogenic impact on ecosystems have been considered. The biotic approach based on dialysis cultivation of microorganism populations has been suggested in order to evaluate the ecological state of living systems. Dialysis cultures are characterized with a high level of biomass accumulation and considerable extension of exponential and stationary growth phases. The cell state at dialysis cultivation corresponds to the state of the organisms in native nature conditions. The method for analysis of responses from microbe cells on external factors’ influence has been suggested. The method is based on the registration of alterations of time-spatial parameters in cell populations by layer-by-ayer noninvasive analysis using the spectroscopy of internal reflection.