The place of inactivated actin and its kinetic predecessor in actin folding-unfolding

The kinetics of actin unfolding induced by guanidine hydrochloride of different concentrations was studied. The parametric representation of the kinetic dependencies of tryptophan fluorescence intensity changes recorded at two wavelengths allowed us to detect and characterize a new essentially unfolded kinetic intermediate. Its characteristics suggested that this intermediate state is a premolten globule. It was shown that the equilibrium transition between inactivated and completely unfolded states is also a two-step process and proceeds via an essentially unfolded kinetic intermediate. The new kinetic pathway of actin unfolding--refolding was proposed. According to it, the founded essentially unfolded kinetic state is the on-pathway intermediate, while inactivated actin is the off-pathway misfolded state stabilized by aggregation of partially folded macromolecules of protein.     

Differences in internal dynamics of actin under different structural states detected by neutron scattering

F-actin, a helical polymer formed by polymerization of the monomers (G-actin), plays crucial roles in various aspects of cell motility. Flexibility of F-actin has been suggested to be important for such a variety of functions. Understanding the flexibility of F-actin requires characterization of a hierarchy of dynamical properties, from internal dynamics of the actin monomers through domain motions within the monomers and relative motions between the monomers within F-actin to large-scale motions of F-actin as a whole. As a first step toward this ultimate purpose, we carried out elastic incoherent neutron scattering experiments on powders of F-actin and G-actin hydrated with D₂O and characterized the internal dynamics of F-actin and G-actin. Well established techniques and analysis enabled the extraction of mean-square displacements and their temperature dependence in F-actin and in G-actin. An effective force constant analysis with a model consisting of three energy states showed that two dynamical transitions occur at ∼150 K and ∼245 K, the former of which corresponds to the onset of anharmonic motions and the latter of which couples with the transition of hydration water. It is shown that behavior of the mean-square displacements is different between G-actin and F-actin, such that G-actin is “softer” than F-actin. The differences in the internal dynamics are detected for the first time between the different structural states (the monomeric state and the polymerized state). The different behavior observed is ascribed to the differences in dynamical heterogeneity between F-actin and G-actin. Based on structural data, the assignment of the differences observed in the two samples to dynamics of specific loop regions involved in the polymerization of G-actin into F-actin is proposed.     

Theoretical study of a landscape of protein folding-unfolding pathways. Folding rates at midtransition

This paper presents a new method for calculating the folding-unfolding rates of globular proteins. The method is based on solution of kinetic equations for a network of folding-unfolding pathways of the proteins. The rates are calculated in the point of thermodynamic equilibrium between the native and completely unfolded states. The method has been applied to all the proteins listed by Jackson [Jackson, S. E. (1998) Folding Des. 3, R81-R91] and some peptides. Although the studied protein chains differ by more than 1 order of magnitude in size and exhibit two- as well as three-state kinetics in water, and their folding rates cover more than 11 orders of magnitude, the theoretical estimates are reasonable close to the experimentally measured folding rates in midtransition (the correlation coefficient being as high as 0.78). This means that the presented theory (having no adjustable parameters at all) is consistent with the experimental observations.     

Identification of the unstable neurophysin disulfide and localization to the hormone-binding site. Relationship to folding-unfolding pathways.

For unliganded neurophysin, the effects of reduction of a single disulfide and limited regeneration of activity following reduction have suggested metastable disulfide pairing relative to that of the neurophysin precursor. This metastability was confirmed in the present study by the demonstration of almost complete regeneration of activity from the reduced state in the presence of ligand peptides, conditions mimicking precursor folding. To assign the source of the metastability of the unliganded mature protein, the disulfide(s) most susceptible to reduction and the last to be reoxidized following complete reduction were identified. Partial reduction of the first disulfide followed by trapping of the generated thiols with [14C]iodoacetate gave a distribution of label consistent with identification of the unstable disulfide as the 10-54 bridge and rapid interchange of the Cys-10 thiol with other disulfides in the amino-terminal disulfide domain. The same thiol distribution was seen at the terminal stage of reoxidation following complete reduction, providing evidence that unfolding and folding pathways are the same at this stage. The results indicate that, in the absence of bound peptide, the state with correct pairing of the 10-54 bridge has no significant thermodynamic advantage over interchanged states of the amino-terminal domain. However, since the 10-54 bridge is located at the peptide-binding site, the correct pairing is directly stabilized by ligand peptides. Moreover, since the other three bridges of the amino domain are homologous to bridges in the carboxyl-terminal domain that do not appear to be unstable, the results allow the possibility that the 10-54 bridge, which is unique to the amino domain, destabilizes other disulfides in that domain.     

Changes in structural and functional properties of actin in denervated rabbit skeletal muscles

Amino acid composition of actin from white skeletal muscles of the rabbit was studied 14 and 50 days after denervation. The content of valine, histidine and tyrosine was shown to decrease and that of lysine and leucine to increase on the 14th day following denervation. The content of these amino acids was almost normalised on the 50th day. In case of actin obtained from the contralateral leg similar but less pronounced changes were revealed. The results obtained are discussed as to the role played by some of amino acids in actin polymerization.     

Phosphorylation of the minimal inhibitory region at the C-terminus of caldesmon alters its structural and actin binding properties

Caldesmon is an inhibitory protein believed to be involved in the regulation of thin filament activity in smooth muscles and is a major cytoplasmic substrate for MAP kinase. NMR spectroscopy shows that the actin binding properties of the minimal inhibitory region of caldesmon, residues 750-779, alter upon MAP kinase phosphorylation of Ser-759, a residue not involved in actin binding. This phosphorylation leads to markedly diminished actin affinity as a result of the loss of interaction at one of the two sites that bind to F-actin. The structural basis for the altered interaction is identified from the observation that phosphorylation destabilises a turn segment linking the two actin binding sites and thereby results in the randomisation of their relative disposition. This modulatory influence of Ser-759 phosphorylation is not merely a function of the bulkiness of the covalent modification since the stability of the turn region is observed to be sensitive to the ionisation state of the phosphate group. The data are discussed in the context of the inhibitory association of the C-terminal domain of caldesmon with F-actin.     

Evidence for structurally different attached states of myosin cross-bridges on actin during contraction of fish muscle.

Using data from fast time-resolved x-ray diffraction experiments on the synchrotrons at Daresbury and (Deutsches Elektronen Synchrotron [DESY]), it is shown that during contraction of fish muscle there are at least two distinct configurations of myosin cross-bridges on actin, that they appear to have different tension producing properties and that they probably differ in the axial tilt of the cross-bridges on actin. Evidence is presented for newly observed myosin-based layer lines in patterns from active fish muscle, together with intensity changes of the actin layer lines. On the equator, the 110 reflection changes much faster (time for 50% change t1/2 = 21 +/- 4 ms after activation) than the 100 reflection (t1/2 = 35 +/- 8 ms) and tension (t1/2 = 41 +/- 3 ms) during the rising phase of tetanic contractions. These and higher order reflections have been used to show the time course of mass attachment at actin during this rising phase. Mass arrival (t1/2 = 25 ms) precedes tension by approximately 15 ms. Analysis has been carried out to evaluate the effects of changes in sarcomere length during the tetanus. It is shown that any such effects are very small. Difference "equatorial" electron density maps between active muscle at a time when mass arrival at actin is just complete, but the tension is still rising, and at a later time well into the tension plateau, show that the structural difference between the lower and higher force states corresponds to mass movement consistent with axial swinging of heads from a nonstereospecific actin attached state (low force) to a more stereospecific (high force) state.     

Fluorescence properties of actin and analysis of the content of native actin in its preparations]

Changes of properties of actin preparations from rabbit skeletal muscles in the course of purification were studied. It is shown that independent on initial properties of actin preparations at successive polimerization, sedimentation and depolimerization cycles: 1) the quantity of protein in supernatant diminishes progressively, 2) intrinsic viscosity of F-actin increases, 3) the value of spectral parameter A = (I320/I365)296., which in characteristic of the fluorescence spectrum position of tryptophan residues, increases and approaches the extremal value, 4) the effect of short wave shift of the spectrum at actin polimerazation becomes more pronounced. The actin preparation with the extremal value of A = 2,6 (native actin) has [eta] = 8,8; deltaAg leads to f approximately 0,25; lambdamax=325 nm. Inactivation of actin results in the long-wave shift of fluorescence spectrum (lambdamax=337 nm, A = 1,30) suggesting the disturbance of exclusively compact globular structure of native protein macromolecular. The ratio is described which enables to use parameter A for the quick estimation of the content of native actin in its preparations. The technical simplicity of the measurement of parameter A enables to use it for the characterization of individual fractions in gelfiltration of actin preparations.     

Photofootprint of nucleosome core DNA in intact chromatin having different structural states

Recently, we reported that the distribution of ultraviolet light (u.v.) induced pyrimidine dimers in nucleosome core DNA has a striking 10.3(+/- 0.1) base periodicity and the regions of enhanced quantum yield map to positions where DNA strands are farthest from the core histone surface. Improvement of the mapping procedure has allowed us to analyze this distribution in more detail, and compare the distribution pattern for nucleosome cores from intact chromatin having different higher-order structures (from the 10 nm filament to the 30 nm fiber). At all levels of chromatin compaction, we observed the following. (1) The average periodicity in pyrimidine dimer yield is 10.3 bases. (2) The peak-to-peak spacing in this distribution is significantly different from 10.3 bases in the region covering three helix turns immediately 5' of the dyad axis. (3) There is a suppression of photoproduct formation in the region of the dyad axis, especially at position 84 from the 5' end. (4) The approximately 10 base ensembles have alternating peak intensities throughout core DNA. Furthermore, peak deconvolution analysis of the pyrimidine dimer pattern yielded a striking similarity in photoproduct yield for the different levels of chromatin compaction. Irradiation of isolated core DNA yields a much more random distribution of photoproducts, although a weak modulation pattern is observed (indicating that there is a non-random alignment of adjacent pyrimidines in our core DNA preparations). This pattern includes a depression in photoproduct yield near position 95, suggesting that the sequence in this region plays a role in nucleosome positioning. These results show that the u.v. photofootprint is a sensitive, diagnostic probe of core histone-DNA interactions in intact chromatin, and these interactions are not significantly altered by changes in the structural state of the chromatin fiber.     

New structural components detected in B. pertussis of different serotypes]

The authors present electron microscopic data on the study of morphological peculiarities of the strains belonging to various serological types. All the strains studied had a structure characteristic of Gram-negative bacteria. Two types intracellular membranes and intracytoplasmic crystal-like structures were revealed in a number of strains (No. 222, No. gZ353, No. 475), irrespective of the serological types. Dynamics of formation of the crystal-like structures in the bacterial cell was described. Marked changes occurred in the cells with the formation of crystal-like structures: cytoplasmic membrane became detached from the cell wall with the resultant contraction of the cytoplasm; large periplastic spaces formed.     

Physical-chemical properties of C-phycocyanin isolated from an acido-thermophilic eukaryote, Cyanidium caldarium.

C-Phycocyanin from an acido-thermophilic eukaryotic alga, Cyanidium caldarium, was characterized with respect to subunit structure, absorption spectrum and fluorescence properties and was found to be similar to C-phycocyanins from mesophilic sources. The pH-dependence of fluorescence polarization and the changes in sedimentation velocity as a function of pH, concentration and temperature indicate the presence of extremely large amounts of unusually stable 19S aggregates. It was not possible to disaggregate this phycocyanin completely to monomer under normal conditions. The amino acid composition is similar to that of phycocyanins from other thermophilic and halophilic sources. The isoelectric point of this C-phycocyanin was 5.11, an unusually high value. The properties of this C-phycocyanin suggest an increase in protein stability as its mode of adaptation to the environmental stress of high temperature.     

Biochemical and structural characterization of actin from Dictyostelium discoideum.

Actin has been purified from amoebae of Dictyostelium discoideum by a procedure which is notable in that proteolysis has been diminished to undetectable levels and "selective" purification steps have been avoided. The overall yield of this procedure is 5- to 10- fold greater than that of a previous report (Spudich, J. A. (1974) J. Biol. Chem. 249, 6013-6020). The detailed biochemical and structural properties of this new preparation (preparation B) have been compared to those of Dictyostelium actin prepared by the previous procedure (preparation A) as well as to rabbit skeletal muscle actin. Preparation B actin is similar to muscle actin in its molecular weight, ability to activate myosin, filament structure, and polymerization properties. Preparation B actin has the same molecular weight and isoelectric point as preparation A actin, which is more acidic than that of skeletal muscle actin. However, preparation B actin and muscle actin form longer filaments than preparation A actin, as judged by viscometry and electron microscopy.     

Structural connectivity in actin: effect of C-terminal modifications on the properties of actin.

In this study, we use fluorescent probes and proteolytic digestions to demonstrate structural coupling between distant regions of actin. We show that modifications of Cys-374 in the C-terminus of actin slow the rate of nucleotide exchange in the nucleotide cleft. Conformational coupling between the C-terminus and the DNasal loop in subdomain II is observed in proteolytic digestion experiments in which a new C-terminal cleavage site is exposed upon DNasel binding. The functional consequences of C-terminal modification are evident from S-1 ATPase activity and the in vitro motility experiments with modified actins. Pyrene actin, labeled at Cys-374, activates S-1 ATPase activity only half as well as control actin. This reduction is attributed to a lower Vmax value because the affinity of pyrene actin to S-1 is not significantly altered. The in vitro sliding velocity of pyrene actin is also decreased. However, IAEDANS labeling of actin (also at Cys-374) enhances the Vmax of acto-S-1 ATPase activity and the in vitro sliding velocity by approximately 25%. These results are discussed in terms of conformational coupling between distant regions in actin and the functional implications of the interactions of actin-binding proteins with the C-terminus of actin.     

Conformational properties of alpha-synuclein in its free and lipid-associated states

alpha-Synuclein (alphaS) is a presynaptic terminal protein that is believed to play an important role in the pathogenesis of Parkinson's disease (PD). We have used NMR spectroscopy to characterize the conformational properties of alphaS in solution as a free monomer and when bound to lipid vesicles and lipid-mimetic detergent micelles. Free wild-type alphaS is largely unfolded in solution, but exhibits a region with a preference for helical conformations that may be important in the aggregation of alphaS into fibrils. The N-terminal region of alphaS binds to synthetic lipid vesicles and detergent micelles in vitro and adopts a highly helical conformation, consistent with predictions based on sequence analysis. The C-terminal part of the protein does not associate with either vesicles or micelles, remaining free and unfolded. These results suggest that one function of alphaS may be to tether as of yet unidentified partners to lipid surfaces via interactions with its C-terminal tail.     

Structural and dynamic states of actin in the erythrocyte

Analysis of the nucleotide tightly associated with isolated erythrocyte cytoskeletons show it to be ADP, rather then ATP. This confirms that at least a major part of the erythrocyte actin is in the F-form. A re-evaluation of the stoichiometry of spectrin and actin in the erythrocyte (taking account of a gross difference between the color responses of the two proteins on staining of electrophoretic gels) leads to values of 1x10(5) and 5x10(5) for the number of molecules of spectrin tetramer and actin respectively per cell. It has been found possible to perform spectrophotometric DNAase I assays fro actin on lysed whole cells. The concentration of monomeric actin at 0 degrees C is approximately 16 μg/ml packed cells. After washing the lysed cells the monomer pool is not re-established, indicating that only a small proportion of the actin subunits are free to dissociate. The actin monomer concentration in the cytosol remains unchanged after equilibration of the cells with cytochalasin E. The ability of actin-containing complexes in the membrane to nucleate the polymerization of added G-actin was measured fluorimetrically; it was found that membranes incubated with cytochalasin E were completely inert with respect to nucleating activity under conditions that favor appreciable growth at the slowly-growing (“pointed”) ends of free actin filaments. This suggests that these ends of the actin “protofilaments” in the red cell are blocked or sterically obstructed. After treatment of the membranes with guanidine hydrochloride under conditions that dissociate F-actin, the measured concentration of actin monomer rises to approximately 180 μg/ml of packed cells, which is nearly 70 percent of the total actin content. On treatment with trypsin in the presence of DNAase, the spectrin and 4.1 are extensively degraded, but the actin remains undamaged. This treatment, followed by exposure to guanidine hydrochloride, causes a further rise in the concentration of actin responsive to the DNAase assay to 250 μg/ml of cells, compared with 270 μg/ml estimated by densitometry of stained gels. The oligomeric complex, consisting of actin, spectrin, and 4.1, that is extracted from the membrane at low ionic strength, generates no detectable actin monomer after the same treatment. From literature data on the number of cytochalasin binding sites per cell and our value for the total actin content, we obtain a number-average degree of polymerization for actin in the membrane of 12-17. The results lead to a model for the structure of the cytoskeletal network and suggest some consequences of metabolic depletion.     

马铁菊头蝠不同状态下回声定位声波分析

在自建网室(9 m×4 m×4 m)内驯养马铁菊头蝠(Rhinolophus ferrumequinum),利用超声波探测仪录制蝙蝠不同状态下回声定位声波,声波录制与红外摄像保持同步。结果表明,马铁菊头蝠回声定位声波为调频(FM)/恒频(CF)/调频(FM)型;在蝙蝠接近猎物过程中,声脉冲持续时间和间隔时间显著变短,下调FM(即tFM)组分变得愈为显著,捕捉猎物瞬间,产生捕食蜂鸣;飞行与悬挂状态相比,声脉冲重复率、主频率、声脉冲时间、声脉冲间隔和能率环的差异均达到显著水平。     

Activation of heavy meromyosin adenosine triphosphatase by various states of actin.

F-actin monomer (F-monomer) is formed upon the addition of neutral salt to G-actin. Since F-monomer has a digestibility similar to that of F-actin and much lower than that of G-actin, it has been proposed that F-monomer has a conformation different from that of G-actin and similar to the conformation of the subunits in F-actin. To examine whether F-monomer will enhance the magnesium-activated myosin adenosine triphosphatase (Mg2+-ATPase) as much as F-actin, the ability of partially polymerized actin populations at equilibrium to activate the Mg2+-ATPase of heavy meromyosin was investigated. Correlations were made between ATPase activities and the polymerization state of actin as determined by measurements of viscosity and digestibility. No significant activation of the heavy meromyosin ATPase was observed under conditions where G-actin or mixtures of G-actin and F-monomer were present. As polymer formation occurred at higher actin concentrations, or with increased KCl concentrations, substantial activation characteristic of F-actin was observed. The data suggest that F-monomer may undergo a further conformational change as it forms nuclei or joins onto polymers. Alternatively, the site of actin which activates the myosin ATPase may involve the crevice between two adjacent actin subunits.