The activation of urease

1. It has been shown that the activity of solutions of twice recrystallized urease is reversibly increased by moderate heating and reversibly decreased by storage in the cold, even in the frozen state. 2. Crude extracts of jack bean meal containing potent urease undergo this same type of reversible activation by heating and inactivation by cooling. Dilution has the same potentiating effect on the activity as moderate heating. As much as a fivefold increase in activity can be obtained when a sample previously inactivated by storage for 24 hours at -10 degrees C. is heated for 5 minutes at 60 degrees C. 3. Solutions of crystalline urease protected by serum albumin and preserved in the cold give a constant "potential" activity over a period of more than 30 days if heated 5 minutes at 60 degrees C. before assay. 4. The data presented have been interpreted to mean that an association between urease molecules (or between urease and other proteins) might occur, resulting in inactivation of the enzyme which would be reversed on dissociation. 5. It has been postulated that the same forces are responsible for the reversible inactivation brought about by standing at temperatures above or below the freezing point.     

Biological function and site II Ca2+-induced opening of the regulatory domain of skeletal troponin C are impaired by invariant site I or II Glu mutations

To investigate the roles of site I and II invariant Glu residues 41 and 77 in the functional properties and calcium-induced structural opening of skeletal muscle troponin C (TnC) regulatory domain, we have replaced them by Ala in intact F29W TnC and in wild-type and F29W N domains (TnC residues 1-90). Reconstitution of intact E41A/F29W and E77A/F29W mutants into TnC-depleted muscle skinned fibers showed that Ca(2+)-induced tension is greatly reduced compared with the F29W control. Circular dichroism measurements of wild-type N domain as a function of pCa (= -log[Ca(2+)]) demonstrated that approximately 90% of the total change in molar ellipticity at 222 nm ([theta](222 nm)) could be assigned to site II Ca(2+) binding. With E41A, E77A, and cardiac TnC N domains this [theta](222 nm) change attributable to site II was reduced to < or =40% of that seen with wild type, consistent with their structures remaining closed in +Ca(2+). Furthermore, the Ca(2+)-induced changes in fluorescence, near UV CD, and UV difference spectra observed with intact F29W are largely abolished with E41A/F29W and E77A/F29W TnCs. Taken together, the data indicate that the major structural change in N domain, including the closed to open transition, is triggered by site II Ca(2+) binding, an interpretation relevant to the energetics of the skeletal muscle TnC and cardiac TnC systems.     

Effects of cooling, cryopreservation and heating on sperm proteins, nuclear DNA, and fertilization capability in mouse

In the present study, we used confocal microscopy and electrophoresis to study the effects of heating to 5 or 100 degrees C or cooling to 4 degrees C or -- 196 degrees C on the stability of sperm proteins and DNA. We used intracytoplasmic sperm injection (ICSI) to determine the fertilizing capability of treated spermatoza. It was shown that sperm cryopreservation at - 196 degrees C or cooling at 4 degrees C altered neither protein and DNA profiles nor the sperm fertilization capability, while the protein and DNA profiles of sperm heated at 100 degrees C were irreversibly degraded and inactivated. The proteins of sperm were severely damaged while the nuclear DNA still maintained its integrity when heated to 58 degrees C. Observation by laser confocal microscopy showed that after being heated to 58 degrees C and 100 degrees C, the nuclear of mouse sperm lost their ability to activate oocytes and they could not transform to male pronuclei though the membrane of some sperm could degrade and induce the formation of sperm asters in ICSI oocytes. The results indicate that the use of 58 degrees C heating only causes the degradation of sperm proteins, while the 100 degrees C heating elicits the irreversible degradation of both sperm proteins and nuclear DNA, and the damage of sperm proteins is primarily responsible for the observed decrease in sperm fertilizing capability.     

Physical properties and subunit structure of l-asparaginase isolated from Erwinia carotovora

1. l-Asparaginases from Erwinia carotovora and Escherichia coli (EC2 enzyme) are both capable of inhibiting and eliminating certain types of tumour cells. The Er. carotovora enzyme is a more basic protein, however, and in contrast with the EC2 enzyme it contains neither tryptophan nor cystine, and disulphide bonds are therefore absent. The molecule is very stable in solution from pH3.0 to about pH12.0, and is somewhat more stable at alkaline pH than is the Esch. coli enzyme. Calculations based on a s(0) (20,w) 7.43S and a sedimentation-equilibrium molecular weight of 135000+/-10000 give a frictional ratio (f/f(0)) of 1.08. The molecular conformation is therefore very compact in solution, and the electron microscope shows the negatively stained molecules as almost spherical particles with a diameter of 7.2+/-0.7nm. 2. Sedimentation-velocity and equilibrium ultracentrifugation, in 5-8m solutions of urea and guanidinium chloride, and also electrophoresis in sodium dodecyl sulphate-polyacrylamide gel, reveal a dissociation of the native protein molecule into four subunits of similar molecular weight in the range 32500-38000. The enzymically inactive subunits can be physically reassembled into an active tetramer when urea is removed by dialysis. Although the subunit structures of the Er. carotovora enzyme and the Esch. coli enzyme molecules are similar, the secondary bonding forces holding the subunits together in the tetramer are somewhat stronger in the Er. carotovora enzyme. 3. The optical-rotatory-dispersion (o.r.d.) parameters that characterize the Cotton effects arising from ordered structure in the molecule are [m'](233)=-3522+/-74 degrees and [m'](200)=9096+/-1700 degrees . These show very marked changes as the secondary structure is disrupted and the molecule dissociates into subunits. A correlation pathway was traced on the basis of o.r.d. parameters and enzyme activity as the polypeptide chains were denatured and renatured (and reconstituted) into active molecules after the dilution of solutions in urea. Subunits resulting from treatment with sodium dodecyl sulphate do not show the typically disordered o.r.d. profile, but nevertheless they are inactive.     

A unique molten globule state occurs during unfolding of cytochrome c by LiClO4 near physiological pH and temperature: structural and thermodynamic characterization

We have carried out denaturation studies of bovine cytochrome c (cyt c) by LiClO4 at pH 6.0 and 25 degrees C by observing changes in difference molar absorbance at 400 nm (Deltaepsilon400), mean residue ellipticities at 222 nm ([theta]222) and difference mean residue ellipticity at 409 nm (Delta[theta]409). The denaturation is a three-step process when measured by Deltaepsilon400 and Delta[theta]409, and it is a two-step process when monitored by [theta]222. The stable folding intermediate state has been characterized by near- and far-UV circular dichroism, tryptophan fluorescence, 8-anilino-1-naphthalene sulfonic acid (ANS) binding, and intrinsic viscosity measurements. A comparison of the conformational and thermodynamic properties of the LiClO4-induced molten globule (MG) state with those induced by other solvent conditions (e.g., low pH, LiCl, and CaCl2) suggests that LiClO4 induces a unique MG state, i.e., (i) the core in the LiClO4-induced state retains less secondary and tertiary structure than that in the MG states obtained in other solvent conditions, and (ii) the thermodynamic stability associated with the LiClO4-induced process, native state <--> MG state, is the same as that observed for each transition between native and MG states induced by other solvent conditions.     

Conformational and thermodynamic characterization of the molten globule state occurring during unfolding of cytochromes-c by weak salt denaturants

The denaturation of bovine and horse cytochromes-c by weak salt denaturants (LiCl and CaCl(2)) was measured at 25 degrees C by observing changes in molar absorbance at 400 nm (Delta epsilon(400)) and circular dichroism (CD) at 222 and 409 nm. Measurements of Delta epsilon(400) and mean residue ellipticity at 409 nm ([theta](409)) gave a biphasic transition for both modes of denaturation of cytochromes-c. It has been observed that the first denaturation phase, N (native) conformation <--> X (intermediate) conformation and the second denaturation phase, X conformation <--> D (denatured) conformation are reversible. Conformational characterization of the X state by the far-UV CD, 8-anilino-1-naphthalene sulfonic acid (ANS) binding, and intrinsic viscosity measurements led us to conclude that the X state is a molten globule state. Analysis of denaturation transition curves for the stability of different states in terms of Gibbs energy change at pH 6.0 and 25 degrees C led us to conclude that the N state is more stable than the X state by 9.55 +/- 0.32 kcal mol(-1), whereas the X state is more stable than the D state by only 1.40 +/- 0.25 kcal mol(-1). We have also studied the effect of temperature on the equilibria, N conformation <--> X conformation and X conformation <--> D conformation in the presence of different denaturant concentrations using two different optical probes, namely, [theta](222) and Delta epsilon(400). These measurements yielded T(m), (midpoint of denaturation) and Delta H(m) (enthalpy change) at T(m) as a function of denaturant concentration. A plot of Delta H(m) versus corresponding T(m) was used to determine the constant-pressure heat capacity change, Delta C(p) (= ( partial differential Delta H(m)/ partial differential T(m))(p)). Values of Delta C(p) for N conformation <--> X conformation and X conformation <--> D conformation is 0.92 +/- 0.02 kcal mol(-1) K(-1) and 0.41 +/- 0.01 kcal mol(-1) K(-1), respectively. These measurements suggested that about 30% of the hydrophobic groups in the molten globule state are not accessible to the water.     

Distinctive influence of two hexafluoro solvents on the structural stabilization of Bombyx mori silk fibroin protein and its derived peptides: 13C NMR and CD studies

Employing high-resolution (13)C solution NMR and circular dichroism (CD) spectroscopic techniques, the distinctive influence of two intimately related hexafluoro solvents, 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and hexafluoroacetone trihydrate (HFA), on the structural characteristics of Bombyx mori (B. mori) silk fibroin, the chymotrypsin precipitate (C(p)) fraction, and two synthetic peptides, (AGSGAG)(5) and (AG)(15), is described. The observed (13)C solution NMR and CD spectra of these polypeptides in HFIP and HFA revealed a distinctive influence on their conformational characteristics. The (13)C NMR spectra, as analyzed from the unique chemical shifts of C(alpha) and C(beta) resonances of constituent residues revealed that fibroin largely assumes helical conformation(s) in both solvents. However, the peak shifts were greater for the samples in HFIP, indicating that the types of helical structure(s) may be different from the one populated in HFA. Similar structural tendencies of these polypeptides were reflected in CD spectra. The observed CD patterns, i.e., a strong positive band at approximately 190 nm and negative bands at approximately 206 and 222 nm, have been attributed to the preponderance of helical structures. Of the two prevalent helical structures, alpha-helix and 3(10)-helix, the evidence emerged for the fibroin protein in favor of 3(10)-helical structure stabilization in HFIP and its significant disruption in HFA, as deduced from the characteristic R1 (=[theta](190)/[theta](202)) and R2 (=[theta](222)/[theta](206)) ratios, determined from the CD data. Conversely, the native polypeptides and synthetic peptide fragments derived from highly crystalline regions of the silk fibroin protein sustained predominantly an unordered structure in HFA solvent.     

Thermal stable and oxidation-resistant variant of subtilisin E

A remarkable thermal stable and oxidation-resistant mutant was obtained using the random mutagenesis PCR technique on the mutant M222A gene of subtilisin E. Sequencing analysis revealed an A was replaced by G at nucleotide 671 of the subtilisin E gene, converting the asparagine codon (AAT) to serine codon (AGT) at position 118. The half-life of M222A/N118S enzyme activity, when heated at 65 degrees C, was approximately 80 min while the half-life of M222A and wild-type subtilisin E were 13 min and 15 min, respectively. This suggested the stability of the M222A/N118S mutant was five times greater than that of the wild-type enzyme. The mutant was also as oxidation resistant as the mutant M222A of subtilisin E. These results indicated the M222A/N118S mutant is both an oxidation-resistant and a heat-stable variant of subtilisin E.     

Effects of Na2SO4 on hydrophobic and electrostatic interactions between amphipathic alpha-helices.

The effects of 2 molal Na2SO4 at neutral pH on hydrophobic and electrostatic interactions between amphipathic alpha-helices were investigated by circular dichroism spectroscopy. The amphipathic peptides that were studied included LEK (acetyl-LEELKKKLEELKKKLEEL-NH2) and LEE (acetyl-LEELEEELEELEEELEEL-NH2). In phosphate buffer at neutral pH, only LEK adopted a predominantly alpha-helical conformation, attributable to glu-lys+ interactions where a major contribution is evidently a hydrogen bond (Biochemistry 32: 9668-9676). Despite the presence of lys+ in the e and g' positions of the abcdefg heptad repeat, LEK exhibited mean-residue ellipticities at 222 nm ([theta]222) which were dependent on peptide concentration, indicating the presence of a coiled coil. In the presence of 2 molal Na2SO4 at 25-75 degrees C, the helical content of LEK increased, with the greatest increase observed at 75 degrees C. The value of the ellipticity ratio R ([theta]222/[theta]208) of LEK in 2 molal Na2SO4 also increased, indicating a stronger interhelical association. At 50 degrees C and 75 degrees C, LEK remained predominantly alpha-helical. In phosphate buffer at neutral pH, LEE was mainly random coil. In the presence of 2 molal Na2SO4, however, the peptide formed alpha-helices that associated to form a coiled coil. At 50 degrees C and 75 degrees C, LEE became predominantly random coil but the remaining alpha-helices were still associating. These results are consistent with the strengthening of interhelical hydrophobic interactions and the absence of screening of helix-stabilizing and helix-destabilizing electrostatic interactions in amphipathic alpha-helices by Na2SO4.     

Structure and metastability of N-lignocerylgalactosylsphingosine (cerebroside) bilayers

Differential scanning calorimetry (DSC) and X-ray diffraction have been used to study hydrated N-lignocerylgalactosylsphingosine (NLGS) bilayers. DSC of fully hydrated NLGS shows an endothermic transition at 69-70 degrees C, immediately followed by an exothermic transition at 72-73 degrees C; further heating shows a high-temperature (Tc = 82 degrees C), high-enthalpy (delta H = 15.3 kcal/mol NLGS) transition. Heating to 75 degrees C, cooling to 20 degrees C and subsequent reheating shows no transitions at 69-73 degrees C; only the high-temperature (82 degrees C), high-enthalpy (15.3 kcal/mol) transition. Two exothermic transitions are observed on cooling; for the upper transition its temperature (about 65 degrees C) and enthalpy (about 6 kcal/mol NLGS) are essentially independent of cooling rate, whereas the lower transition exhibits marked changes in both temperature (30----60 degrees C) and enthalpy (2.2----9.5 kcal/mol NLGS) as the cooling rate decreases from 40 to 0.625 Cdeg/min. On reheating, the enthalpy of the 69-70 degrees C transition is dependent on the previous cooling rate. The DSC data provide clear evidence of conversions between metastable and stable forms. X-ray diffraction data recorded at 26, 75 and 93 degrees C show clearly that NLGS bilayer phases are present at all temperatures. The X-ray diffraction pattern at 75 degrees C shows a bilayer periodicity d = 65.4 A, and a number of sharp reflections in the wide-angle region indicative of a crystalline chain packing mode. This stable bilayer form converts to a liquid-crystal bilayer phase; at 93 degrees C, the bilayer periodicity d = 59.1 A, and a diffuse reflection at 1/4.6 A-1 is observed. The diffraction pattern at 22 degrees C represents a combination of the stable and metastable low-temperature bilayer forms. NLGS exhibits a complex pattern of thermotropic changes related to conversions between metastable (gel), stable (crystalline) and liquid-crystalline bilayer phases. The structure and thermotropic properties of NLGS are compared with those of hydrated N-palmitoylgalactosylsphingosine reported previously (Ruocco, M.J., Atkinson, D., Small, D.M., Skarjune, R.P., Oldfield, E. and Shipley, G.G. (1981) Biochemistry 20, 5957-5966).     

PCR amplification using electrolytic resistance for heating and temperature monitoring

An alternative method of rapid-cycle PCR for DNA amplification is demonstrated using electrolyte resistance for heating and temperature monitoring. The PCR amplification solution is electrically conductive and can be heated by passing an alternating current through the sample. The temperature of the solution is evaluated by monitoring its electrical resistance. Cooling is accomplished by forced air convection at ambient temperature. Heating and cooling rates of up to 20 degrees C/s were achieved. The 35 cycles of PCR were completed in less than 12 min with product yields equivalent to conventional temperature cycling. Electrolyte resistance provides a method for both direct heating and monitoring the temperature of PCR samples.     

Effect of heat treatment on oxidase activity and proton-pumping capability of proteoliposome-incorporated beef heart cytochrome aa3

By incubating beef heart cytochrome c oxidase at 43-45 degrees C, selective inactivation of the H+-pumping function is possible without affecting cytochrome c oxidase activity; proteoliposomes reconstituted with heated enzyme (43.5 degrees C for 60 min at pH 7.0) showed an apparent H+/e- ratio of only 0.3 and a turnover with cytochrome c plus ferrocyanide as substrate of 20 s-1, while those with the intact enzyme showed an apparent H+/e- ratio somewhat greater than 1.0 and a turnover of 19 s-1. This decrease in the H+/e- ratio could not be attributed to a stimulation of H+ permeability upon heating, since the respiratory control ratio and the magnitude of membrane potential formation remained almost the same in the two cases. A pH-dependent Em (midpoint redox potential) change of cytochrome a in the presence of cyanide was still observed after the heat treatment. Heating induced a small spectral shift in the Soret region of the oxidized (resting) enzyme; the peak of the heated enzyme was at 421 nm, while that of the intact enzyme was at 419 nm. The spectral shift obtained by pulsing the enzyme with oxygen under turnover conditions is also altered.     

Effects of cold and heat on behavior and cerebellar function in goldfish

Summary A similar sequence of behavioral effects was observed for either cooling or heating; most effects occurred on changing temperature of entire fish or of only the cerebellum. On moderate heating or cooling, fish are hyperexcitable, spontaneously hyperactive; on further heating or cooling swimming is uncoordinated; when the subcerebellar structures are heated or cooled, equilibrium is disturbed; on further heating or cooling coma and respiratory failure ensue. Critical temperatures are modifiable by acclimation. The behavioral effects of cerebellectomy are additive with temperature effects on motor centers.Electrical activity of Purkinje neurons changes in the same thermal ranges as behavior. Inhibition via cerebellar interneurons is most sensitive and can be modified by acclimation. Ongoing activity increases with warming up to a blocking temperature; interspike interval histograms show pattern changes during warming. Activation via mossy fibers-granule cells is more sensitive than that via climbing fibers, and antidromic impulses are most resistant.A neuronal model based on inhibitory actions of Purkinje neurons on motor centers and parallel feedback excitatory pathways can explain both behavioral and electrical observations.     

Complex of human apolipoprotein C-1 with phospholipid: thermodynamic or kinetic stability?

Thermal unfolding of discoidal complexes of apolipoprotein (apo) C-1 with dimyristoyl phosphatidylcholine (DMPC) reveals a novel mechanism of lipoprotein stabilization that is based on kinetics rather than thermodynamics. Far-UV CD melting curves recorded at several heating/cooling rates from 0.047 to 1.34 K/min show hysteresis and scan rate dependence characteristic of slow nonequilibrium transitions. At slow heating rates, the apoC-1 unfolding in the complexes starts just above 25 degrees C and has an apparent melting temperature T(m) approximately 48 +/- 1.5 degrees C, close to T(m) = 51 +/- 1.5 degrees C of free protein. Thus, DMPC binding may not substantially increase the low apparent thermodynamic stability of apoC-1, DeltaG(25 degrees C) < 2 kcal/mol. The scan rate dependence of T(m) and Arrhenius analysis of the kinetic data suggest an activation enthalpy E(a) = 25 +/- 5 kcal/mol that provides the major contribution to the free energy barrier for the protein unfolding on the disk, DeltaG > or = 17 kcal/mol. Consequently, apoC-1/DMPC disks are kinetically but not thermodynamically stable. To explore the origins of this kinetic stability, we utilized dynode voltage measured in CD experiments that shows temperature-dependent contribution from UV light scattering of apoC-1/DMPC complexes (d approximately 20 nm). Correlation of CD and dynode voltage melting curves recorded at 222 nm indicates close coupling between protein unfolding and an increase in the complex size and/or lamellar structure, suggesting that the enthalpic barrier arises from transient disruption of lipid packing interactions upon disk-to-vesicle fusion. We hypothesize that a kinetic mechanism may provide a general strategy for lipoprotein stabilization that facilitates complex stability and compositional variability in the absence of high packing specificity.     

Overheating is detrimental to meiotic spindles within in vitro matured human oocytes

The present study was designed to examine the effects of overheating on meiotic spindle morphology within in vitro matured human oocytes using a polarized light microscope (Polscope). Immature human oocytes at either germinal vesicle or metaphase I stage were cultured in vitro for 24-36 h until they reached metaphase II (M-II) stage. After maturation, oocytes at M-II stage were imaged in the living state with the Polscope at 37, 38, 39 and 40 degrees C for up to 20 min. After heating, oocytes were returned to 37 degrees C and then imaged for another 20 min at 37 degrees C. The microtubules in the spindles were quantified by their maximum retardance, which represents the amount of microtubules. Spindles were intact at 37 degrees C during 40 min of examination and their maximum retardance (1.72-1.79) did not change significantly during imaging. More microtubules were formed in the spindles heated to 38 degrees C and the maximum retardance was increased from 1.77 before heating to 1.95 at 20 min after heating. By contrast, spindles started to disassemble when the temperature was increased to 39 degrees C for 10 min (maximum retardance was reduced from 1.76 to 1.65) or 40 degrees C for 1 min (maximum retardance was reduced from 1.75 to 1.5). At the end of heating (20 min), fewer microtubules were present in the spindles and the maximum retardance was reduced to 0.8 and 0.78 in the oocytes heated to 39 degrees C and 40 degrees C, respectively. Heating to 40 degrees C also induced spindles to relocate in the cytoplasm in some oocytes. After the temperature was returned to 37 degrees C, microtubules were repolymerized to form spindles, but the spindles were not reconstituted completely compared with the spindles imaged before heating. These results indicate that spindles in human eggs are sensitive to high temperature. Moreover, maintenance of an in vitro manipulation temperature of 37 degrees C is crucial for normal spindle morphology.     

Thermal denaturation of Micrococcus lysodeikticus adenosine triphosphatase. Influence of temperature on the circular dichroism, fluroescence and enzymic activity of the protein.

The soluble ATPase (adenosine triphosphatase) from Micrococcus lysodeikticus underwent a major unfolding transition when solutions of the enzyme at pH 7.5 were heated. The midpoint occurred at 46 degrees C when monitored by changes in enzymic activity and intrinsic fluorescence, and at 49 degrees C when monitored by circular dichroism. The products of thermal denaturation retained much secondary structure, and no evidence of subunit dissociation was detected after cooling at 20 degrees C. The thermal transition was irreversible, and thiol groups were not involved in the irreversibility. The presence of ATP, adenylyl imidodiphosphate, CaCl2 or higher concentrations of ATPase conferred stability against thermal denaturation, but did not prevent the irreversibility one denaturation had taken place. In the presence of guanidinium chloride, thermal denaturation occurred at lower temperatures. The midpoints of the transition were 45 degrees C in 0.25 M-, 38 degrees C in 0.5 M-and 30 degrees C in 0.75 M-denaturant. In the highest concentration of guanidinium chloride a similar unfolding transition induced by cooling was observed. Its midpoint was 9 degrees C, and the temperature of maximum stability of the protein was 20 degrees C. The discontinuities occurring the the Arrhenius plots of the activity of this enzyme had no counterpart in variations in the far-u.v. circular dichroism or intrinsic fluorescence of the protein at the same temperature.     

Specific sequences determine the stability and cooperativity of folding of the C-terminal half of tropomyosin

Tropomyosin is a flexible 410 A coiled-coil protein in which the relative stabilities of specific regions may be important for its proper function in the control of muscle contraction. In addition, tropomyosin can be used as a simple model of natural occurrence to understand the inter- and intramolecular interactions that govern the stability of coiled-coils. We have produced eight recombinant tropomyosin fragments (Tm(143-284(5OHW),) Tm(189-284(5OHW)), Tm(189-284), Tm(220-284(5OHW)), Tm(220-284), Tm(143-235), Tm(167-260), and Tm(143-260)) and one synthetic peptide (Ac-Tm(215-235)) to investigate the relative conformational stability of different regions derived from the C-terminal region of the protein, which is known to interact with the troponin complex. Analytical ultracentrifugation experiments show that the fragments that include the last 24 residues of the molecule (Tm(143-284(5OHW)), Tm(189-284(5OHW)), Tm(220-284(5OHW)), Tm(220-284)) are completely dimerized at 10 microm dimer (50 mm phosphate, 100 mm NaCl, 1.0 mm dithiothreitol, and 0.5 mm EDTA, 10 degrees C), whereas fragments that lack the native C terminus (Tm(143-235),Tm(167-260), and Tm(143-260)) are in a monomer-dimer equilibrium under these conditions. The presence of trifluoroethanol resulted in a reduction in the [theta](222)/[theta](208) circular dichroism ratio in all of the fragments and induced stable trimer formation only in those containing residues 261-284. Urea denaturation monitored by circular dichroism and fluorescence revealed that residues 261-284 of tropomyosin are very important for the stability of the C-terminal half of the molecule as a whole. Furthermore, the absence of this region greatly increases the cooperativity of urea-induced unfolding. Temperature and urea denaturation experiments show that Tm(143-235) is less stable than other fragments of the same size. We have identified a number of factors that may contribute to this particular instability, including an interhelix repulsion between g and e' positions of the heptad repeat, a charged residue at the hydrophobic coiled-coil interface, and a greater fraction of beta-branched residues located at d positions.     

Geometrical and conformational preferences of the 9-fluorenylmethoxycarbonyl-amino moiety.

Structural parameters, originating from x-ray crystallographic data, have been compiled for 13 derivatives of amino acids, peptides and related compounds, which contain a total of 14 Fmoc-NH- moieties. For these moieties, molecular geometries and conformations--described by the omegao, theta1, theta2 and theta3' torsion angles--were analysed and compared with the corresponding parameters for the Z-NH- and Boc-NH-moieties (290 and 553, respectively). To gain a deeper insight into the conformational features of the Fmoc-NH- moiety, ab initio free molecule calculations were performed for fully relaxed minima. Also the potential energy surface as a function of the torsion angles (theta3', theta2) was generated. The conformational features of the Fmoc-NH- moiety: (i) two possible values for the angle omegao (approximately 180 degrees or, rarely, approximately theta degrees) and (ii) the angle theta1 = 180 degrees +/- 15 degrees, are common to the Z-NH- and Boc-NH- systems. By contrast, the theta2 and theta3 angles in the Fmoc, Z and Boc groups differ essentially. In the Fmoc groups theta2 mostly has values of 180 degrees +/- 30 degrees and values up [115 degrees] seem to be forbidden, whereas fewer than half of the Z groups adopt theta2 approximately 180 degrees and the remainder have theta2 in the range of [90 degrees +/- 20 degrees]. On the other hand, the Boc methyl groups are staggered. The theta3 values observed for Fmoc are limited to the regions of 180 degrees +/- 20 degrees and 160 degrees +/- 20 degrees], while for the Z group a variety of theta3 occurs. The orientation of the fluorenyl vs the urethane function is mostly trans. Our results suggest a lower conformational flexibility for the Fmoc group compared with that of the Z group. Our calculations confirm that the observed conformational features for the Fmoc-NH- moiety are inherent properties. The Fmoc-NH-moiety in crystals involves the participation of its O=C-NH functionality in hydrogen bonds.     

Effect of temperature and pH on the ribulose-1,5-diphosphate carboxylase activity of the thermophilic hydrogen bacterium, Pseudomonas thermophila

The activity of ribulose 1,5-diphosphate (RDP) carboxylase was found in the soluble fraction of the cytoplasm from sonicated Pseudomonas thermophila K-2 cells. The enzyme is relatively thermolabile and completely loses its activity at 80 degrees C. The activity of RDP carboxylase at 60 degrees C increases by 40% during the first 10 min of heating in the presence of Mg2+ ions, bicarbonate and dithiothreitol, and again decreases if the enzyme is heated over 20 min. The optimum temperature of the enzyme is 50--55 degrees C. The specific activity of the enzyme in fresh preparations under these conditions reaches 0.22 unit per 1 mg of protein in the extract. The calculated value of the activation energy for RDP carboxylase is 6.4 kcal-mole-1, but 11.6 kcal-mole-1 in frozen preparations. The optimal pH is 7.0--7.3 depending on the buffer. The temperature optimum for the enzyme action does not depend on pH within the range of 7.3 to 8.8. Therefore, RDP carboxylase of Ps. thermophila K-2 differs from RDP carboxylases of mesophilic cultures studied earlier by a higher susceptibility to a decrease in temperature (the enzyme activity is negligible at 30 degrees C), by a lower value of the activation energy at suboptimal temperatures, and by a lower pH optimum of the enzyme action.     

Differences in the processes of beta-lactoglobulin cold and heat denaturations.

The changes in beta-lactoglobulin upon cold and heat denaturation were studied by scanning calorimetry, CD, and NMR spectroscopy. It is shown that, in the presence of urea, these processes of beta-lactoglobulin denaturation below and above 308 K are accompanied by different structural and thermodynamic changes. Analysis of the NOE spectra of beta-lactoglobulin shows that changes in the spin diffusion of beta-lactoglobulin after disruption of the unique tertiary structure upon cold denaturation are much more substantial than those upon heat denaturation. In cold denatured beta-lactoglobulin, the network of residual interactions in hydrophobic and hydrophilic regions of the molecules is more extensive than after heat denaturation. This suggests that upon cold- and heat-induced unfolding, the molecule undergoes different structural rearrangements, passing through different denaturation intermediates. From this point of view, cold denaturation can be considered to be a two stage process with a stable intermediate. A similar equilibrium intermediate can be obtained at 35 degrees C in 6.0 M urea solution, where the molecule has no tertiary structure. Cooling or heating of the solution from this temperature leads to unfolding of the intermediate. However, these processes differ in cooperativity, showing noncommensurate sigmoidal-like changes in efficiency of spin diffusion, ellipticity at 222 nm, and partial heat capacity. The disruption with cooling is accompanied by cooperative changes in heat capacity, whereas with heating the heat capacity changes only gradually. Considering the sigmoidal shape of the heat capacity change an extended heat absorption peak, we propose that the intermediate state is stabilized by enthalpic interactions.