Effects of the temperature range and the lack of beta-ketoacyl acyl-carrier protein synthase II on fatty acid synthesis in Escherichia coli K12 after shifts in temperature

Escherichia coli K12 cells grown at higher temperatures and then subjected to lower temperatures produce fatty acids with higher unsaturated/saturated ratios than cells completely adapted to the lower temperatures (Okuyama et al. (1982) J. Biol. Chem. 257, 4812-4817). This hyper-response was not an artefact of chloramphenicol treatment and was observed when the shift-down was more than 20 degrees C in the cells grown at either 40 degrees C or 35 degrees C. In contrast, cells grown at either 25 degrees C or 30 degrees C showed no appreciable hyper-response in terms of unsaturated/saturated ratio on temperature shifts to as low as 10 degrees C. By combining shift-down and shift-up experiments, we could show the presence of different types of temperature dependency in the fatty acid-synthesizing systems of cells grown at various temperatures. Contrary to wild-type cells which synthesized mainly cis-vaccenate on down-shift to 10 degrees C, a mutant strain lacking beta-ketoacyl acyl-carrier protein synthase II synthesized more palmitoleate (16:1) and less palmitate at 10 degrees C than at 40 degrees C. The average chain lengths of saturated and unsaturated fatty acids also changed, but differently, between the mutant and wild-type cells on shifts of temperature. Thus, the mutant strain has a temperature-dependent fatty acid-synthesizing system qualitatively different from that seen in a wild-type strain.     

Isolation and in vitro characterization of temperature-sensitive mutants of the bacteriophage f1 gene V protein

In vivo selections were used to isolate 43 temperature-sensitive gene V mutants of the bacteriophage f1 from a collection of mutants constructed by saturation mutagenesis of the gene. The sites of temperature-sensitive substitutions are found in both the beta-sheets and the turns of the protein, and some sites are exposed to the solvent while others are not. Thirteen of the variant proteins were purified and characterized to evaluate their free energy changes upon unfolding and their affinities for single-stranded DNA, and eight were tested for their tendencies to aggregate at 42 degrees C. Each of the three temperature-sensitive mutants at buried sites and six of ten at surface sites had free energy changes of unfolding substantially lower (less stabilizing) than the wild-type at 25 degrees C. A seventh mutant at a surface site had a substantially altered unfolding transition and its free energy of unfolding was not estimated. The affinities of the mutant proteins for single-stranded DNA varied considerably, but two mutants at a surface site, Lys69, had much weaker binding to single-stranded DNA than any of the other mutants, while two mutants at another surface site, Glu30, had the highest DNA-binding affinities. The wild-type gene V protein is stable at 42 degrees C, but six of the eight mutants tested aggregated within a few minutes and the remaining two aggregated within 30 minutes at this temperature. Overall, each of the temperature-sensitive proteins tested had a tendency to aggregate at 42 degrees C, and most also had either a low free energy of unfolding (at 25 degrees C), or weak DNA binding. We suggest that any of these properties can lead to a temperature-sensitive gene V phenotype.     

Accurate computer-based design of a new backbone conformation in the second turn of protein L.

The rational design of loops and turns is a key step towards creating proteins with new functions. We used a computational design procedure to create new backbone conformations in the second turn of protein L. The Protein Data Bank was searched for alternative turn conformations, and sequences optimal for these turns in the context of protein L were identified using a Monte Carlo search procedure and an energy function that favors close packing. Two variants containing 12 and 14 mutations were found to be as stable as wild-type protein L. The crystal structure of one of the variants has been solved at a resolution of 1.9 A, and the backbone conformation in the second turn is remarkably close to that of the in silico model (1.1 A RMSD) while it differs significantly from that of wild-type protein L (the turn residues are displaced by an average of 7.2 A). The folding rates of the redesigned proteins are greater than that of the wild-type protein and in contrast to wild-type protein L the second beta-turn appears to be formed at the rate limiting step in folding.     

Stability of interleukin 6, soluble interleukin 6 receptor,interleukin 10 and CC16 in human serum

The stability of interleukin 6 (IL-6), its soluble receptor (sIL-6R), IL-10 and CC16 or uteroglobin (an endogenous cytokine inhibitor) in human serum was examined using an accelerated stability testing protocol according to the Arrhenius equation. Further, the effect of time delay between blood sampling and sample processing, clotting temperature and repeated freeze-thaw cycles on serum levels of these proteins were determined. Paired serum samples were stored at 4 degrees C, 20 degrees C, 30 degrees C and 40 degrees C for 1 to 21 days. We found that IL-6 and CC16 concentrations did not change at 4 degrees C, 20 degrees C and 30 degrees C. Interleukin-6 concentrations significantly declined after 11 days at 40 degrees C. The concentrations of sIL-6R and IL-10 did not change at 4 degrees C but significantly decreased at 20 degrees C (after 21 and 14 days respectively), 30 degrees C and 40 degrees C (after 1 day at both temperatures for both cytokines). Arrhenius-plots indicated that sIL-6R and IL-10 are stable for at least several years at -20 degrees C and -70 degrees C, respectively. Since their relative stability, no Arrhenius-plot could be calculated for IL-6 and CC16. The concentrations of the proteins examined were not significantly altered by repeated freeze-thaw cycles, nor by extended clotting times at 4 degrees C or 20 degrees C. We conclude that serum samples for the determination of IL-6, sIL-6R and CC16 can be stored at -20 degrees C for several years, but for IL-10 determinations, storage at -70 degrees C is recommended.     

Purification, characterization, and rapid inactivation of thermolabile ubiquitin-activating enzyme from the mammalian cell cycle mutant ts85

Conjugation of ubiquitin to certain proteins can trigger their degradation in the in vitro reticulocyte system. In order to determine whether ubiquitin conjugation serves as an intermediate step in the turnover of cellular proteins in vivo, it is necessary to isolate proteolytic intermediates, i.e. ubiquitin-protein adducts of specific cellular proteins. While the steady-state level of conjugates of rapidly turning over proteins is relatively high, that of long-lived proteins is presumably extremely low, and therefore undetectable. Therefore, mutant cell lines with conditionally altered function(s) of the ubiquitin system can serve as powerful tools in studying the degradation of stable cellular proteins. We have characterized a temperature sensitive cell cycle arrest mutant cell (ts85) with a thermolabile ubiquitin-activating enzyme (E1; Finley, D., Ciechanover, A., and Varshavsky, A. (1984) Cell 37, 43-55). Following incubation at the restrictive temperature (39.5 degrees C), these cells fail to degrade short-lived proteins (Ciechanover, A., Finley, D., and Varshavsky, A. (1984) Cell 37, 57-66). However, involvement of the ubiquitin system in the turnover of long-lived proteins has not been addressed in these cells. A slow rate of inactivation of E1 in vivo, and significant rate of cell death following long incubation periods at the restrictive temperature, make this question difficult to address experimentally. In the present study we show that incubation of the cells for 1 h at 43 degrees C leads to rapid inactivation of ubiquitin conjugation in the intact mutant cell. Following heat treatment, the cells can be incubated at 39.5 degrees C for at least 6 h in order to study the possible involvement of the system in the turnover of long-lived cellular proteins. The viability of the cells is excellent at the end of the incubation. Following extraction, we have shown that inactivation occurs much more rapidly in the cell lysate in vitro than in the intact cell (t1/2 of 10 min compared to 4 h at 39.5 degrees C). The enzyme from both the mutant cell and the wild-type cell was purified to homogeneity. The molecular mass of the native enzyme from both cells is approximately 220 kDa with a subunit molecular mass of about 108 kDa. The structure of the enzyme is therefore very similar to that purified from rabbit reticulocytes. At the permissive temperature, the enzymes from both cells catalyze ATP-PPi and ATP-AMP exchange in similar kinetics. However, at the high temperature, the mutated enzyme is at least 7-fold less stable than the wild-type enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

Crystal structure of the disulfide bond-deficient azurin mutant C3A/C26A: how important is the S-S bond for folding and stability?

Azurin has a beta-barrel fold comprising eight beta-strands and one alpha helix. A disulfide bond between residues 3 and 26 connects the N-termini of beta strands beta1 and beta3. Three mutant proteins lacking the disulfide bond were constructed, C3A/C26A, C3A/C26I and a putative salt bridge (SB) in the C3A/S25R/C26A/K27R mutant. All three mutants exhibit spectroscopic properties similar to the wild-type protein. Furthermore, the crystal structure of the C3A/C26A mutant was determined at 2.0 A resolution and, in comparison to the wild-type protein, the only differences are found in the immediate proximity of the mutation. The mutants lose the 628 nm charge-transfer band at a temperature 10-22 degrees C lower than the wild-type protein. The folding of the zinc loaded C3A/C26A mutant was studied by guanidine hydrochloride (GdnHCl) induced denaturation monitored both by fluorescence and CD spectroscopy. The midpoint in the folding equilibrium, at 1.3 M GdnHCl, was observed using both CD and fluorescence spectroscopy. The free energy of folding determined from CD is -24.9 kJ.mol-1, a destabilization of approximately 20 kJ.mol-1 compared to the wild-type Zn2+-protein carrying an intact disulfide bond, indicating that the disulfide bond is important for giving azurin its stable structure. The C3A/C26I mutant is more stable and the SB mutant is less stable than C3A/C26A, both in terms of folding energy and thermal denaturation. The folding intermediate of the wild-type Zn2+-azurin is not observed for the disulfide-deficient C3A/C26A mutant. The rate of unfolding for the C3A/C26A mutant is similar to that of the wild-type protein, suggesting that the site of the mutation is not involved in an early unfolding reaction.     

Thermal and Alkaline Denaturation of Bovine β-Casein

The secondary structure of bovine beta-casein was characterized using circular dichroism (CD) and FTIR spectroscopies under physiologically relevant conditions. Analytical ultracentrifugation technique was used to follow the highly temperature, pH and concentration dependent self-association behavior. CD measurements provide convincing evidence for short segments of polyproline II-like structures in beta-casein in addition to a wide range of secondary structure elements, such as 10-20% alpha-helix, approximately 30% turns, 32-35% extended sheet. Results obtained at extreme pH (10.5) revealed structural destabilization in the monomeric form of the protein. At least four distinct structural transitions at 10, 33, 40 and 78 degrees C were observed at pH 6.75 by CD analysis, compared to only two transitions, 26 and 40 degrees C, at pH 10.5. Calculations from analytical ultracentrifugation suggest that the transitions at lower temperature (< or = 30 degrees C) occur primarily in the monomer. It is hypothesized that the transition at 10 degrees C and neutral pH may represent a general conformational change or cold denaturation. Those middle ranged transitions, i.e. 33 and 40 degrees C are more likely the reflection of hydrophobic changes in the core of beta-casein. As beta-casein undergoes self-association and increases in size, the transition at higher temperature (78 degrees C) is perhaps caused by the apparent conformational change within the micelle-like polymers. It has been shown that beta-casein binds the hydrophobic fluorescent probe ANS with high affinity in much similar fashion to molten globular proteins. The effect of urea denaturation on the bound complex effectively supports this observation.     

Conversion of type I 4:6 to 3:5 beta-turn types in human acidic fibroblast growth factor: effects upon structure, stability, folding, and mitogenic function

Human acidic fibroblast growth factor (FGF-1) is a member of the beta-trefoil superfold, a protein architecture that exhibits a characteristic threefold axis of structural symmetry. FGF-1 contains 11 beta-turns, the majority being type I 3:5; however, a type I 4:6 turn is also found at three symmetry-related locations. The relative uniqueness of the type I 4:6 turn in the FGF-1 structure suggests it may play a key role in the stability, folding, or function of the protein. To test this hypothesis a series of deletion mutations were constructed, the aim of which was to convert existing type I 4:6 turns at two locations into type I 3:5 turns. The results show it is possible to successfully substitute the type I 4:6 turn by a type I 3:5 turn with minimal impact upon protein stability or folding. Thus, these different turn structures, even though they differ in length, exhibit similar energetic properties. Additional sequence swapping mutations within the introduced type I 3:5 turns suggests that the turn sequence primarily affects stability but not turn structure (which appears dictated primarily by the local environment). Although the results suggest that a stable, foldable beta-trefoil protein may be designed utilizing a single turn type (type I 3:5), a type I 4:6 turn at turn 1 of FGF-1 appears essential for efficient mitogenic function.     

A point mutation at the C-terminal half of the repressor of temperate mycobacteriophage L1 affects its binding to the operator DNA

The wild-type repressor CI of temperate mycobacteriophage L1 and the temperature-sensitive (ts) repressor CIts391 of a mutant L1 phage, L1cIts391, have been separately overexpressed in E. coli. Both these repressors were observed to specifically bind with the same cognate operator DNA. The operator-binding activity of CIts391 was shown to differ significantly than that of the CI at 32 to 42 degrees C. While 40-95% operator-binding activity was shown to be retained at 35 to 42 degrees C in CI, more than 75% operator-binding activity was lost in CIts391 at 35 to 38 degrees C, although the latter showed only 10% less binding compared to that of the former at 32 degrees C. The CIts391 showed almost no binding at 42 degrees C. An in vivo study showed that the CI repressor inhibited the growth of a clear plaque former mutant of the L1 phage more strongly than that of the CIts391 repressor at both 32 and 42 degrees C. The half-life of the CIts391-operator complex was found to be about 8 times less than that of the CI-operator complex at 32 degrees C. Interestingly, the repressor-operator complexes preformed at 0 degrees C have shown varying degrees of resistance to dissociation at the temperatures which inhibit the formation of these complexes are inhibited. The CI repressor, but not that of CIts391, regains most of the DNA-binding activity on cooling to 32 degrees C after preincubation at 42 to 52 degrees C. All these data suggest that the 131(st) proline residue at the C-terminal half of CI, which changed to leucine in the CIts391, plays a crucial role in binding the L1 repressor to the cognate operator DNA, although the helix-turn-helix DNA-binding motif of the L1 repressor is located at its N-terminal end.     

A folding pattern that is stable to thermal cycling is achieved by long term storage of recombinant human beta-casein with four extra N-terminals amino-acid residues at -20 degrees C

Studies have followed the turbidity (OD400 nm) of beta-casein (CN) as temperature (T) increased from 4 to 37 degrees C. Native non-phosphorylated beta-CN showed a turbidity increase above 25 degrees C and precipitated at about 22 degrees C in 5mM Ca+2. These patterns were reproducible upon T-cycling while those of recombinant beta-CN proteins are not. Here, a wild-type recombinant that was thermally stable after being frozen in solution and stored at -20 degrees C for a prolonged period of time was denatured with guanidine HCl and refolded by dialysis against buffer. This protein was again not stable to T-cycling. A recombinant mutant with four extra N-terminal amino acids was very stable to T-cycling, both with and without 5mM Ca+2. However, it was still much different than the native protein. These results indicate that there are probably many energy minima for this protein and emphasize the possibility of "chaperon-like" conditions for proper folding of human beta-CN.     

Structural changes in plasma membranes prepared from irradiated Chinese hamster V79 cells as revealed by Raman spectroscopy

The effect of gamma irradiation on the integrity of plasma membranes isolated from Chinese hamster V79 cells was investigated by Raman spectroscopy. Plasma membranes of control V79 cells show transitions between (-) 10 and 5 degrees C (low-temperature transition), 10 and 22 degrees C (middle-temperature transition), and 32 and 40 degrees C (high-temperature transition). Irradiation (5 Gy) alters these transitions markedly. First, the low-temperature transition shifts to higher temperature (onset and completion temperatures 4 and 14 degrees C). Second, the middle-temperature transition shifts up to the range of about 20-32 degrees C, but the width remains unchanged. Third, the higher temperature transition broadens markedly and shifts to the range of about 15-40 degrees C. Protein secondary structure as determined by least-squares analysis of the amide I bands shows 36% total helix, 55% total beta-strand, and 9% turn plus undefined for control plasma membrane proteins. Plasma membrane proteins of irradiated V79 cells show an increase in total helix (40 and 45% at 5 and 10 Gy, respectively) and a decrease in the total beta-strand (48 and 44% at 5 and 10 Gy, respectively) structures. The qualitative analysis of the Raman features of plasma membranes and model compounds in the 1600 cm-1 region, assigned to tyrosine groups, revealed that irradiation alters the microenvironment of these groups. We conclude that the radiation dose used in the survival range of Chinese hamster V79 cells can cause damage to plasma membrane proteins without detectable lipid peroxidation, and that the altered proteins react differently with lipids, yielding a shift in the thermal transition properties.     

Serial increase in the thermal stability of 3-isopropylmalate dehydrogenase from Bacillus subtilis by experimental evolution.

We improved the thermal stability of 3-isopropylmalate dehydrogenase from Bacillus subtilis by an in vivo evolutionary technique using an extreme thermophile, Thermus thermophilus, as a host cell. The leuB gene encoding B. subtilis 3-isopropylmalate dehydrogenase was integrated into the chromosome of a leuB-deficient strain of T. thermophilus. The resulting transformant showed a leucine-autotrophy at 56 degrees C but not at 61 degrees C and above. Phenotypically thermostabilized strains that can grow at 61 degrees C without leucine were isolated from spontaneous mutants. Screening temperature was stepwise increased from 61 to 66 and then to 70 degrees C and mutants that showed a leucine-autotrophic growth at 70 degrees C were obtained. DNA sequence analyses of the leuB genes from the mutant strains revealed three stepwise amino acid replacements, threonine-308 to isoleucine, isoleucine-95 to leucine, and methionine-292 to isoleucine. The mutant enzymes with these amino acid replacements were more stable against heat treatment than the wild-type enzyme. Furthermore, the triple-mutant enzyme showed significantly higher specific activity than that of the wild-type enzyme.     

Dimer formation by a "monomeric" protein

Dimeric proteins can arise by the swapping of structural domains between monomers. The prevalence of this occurrence is unknown. Ribonuclease A (RNase A) is assumed to be a monomer near physiological conditions. Here, this hypothesis is tested and found to be imprecise. The two histidine residues (His12 and His119) in the active site of RNase A arise from two domains (S-peptide and S-protein) of the protein. The H12A and H119A variants have 10(5)-fold less ribonucleolytic activity than does the wild-type enzyme. Incubating a 1:1 mixture of the H12A and H119A variants at pH 6.5 and 65 degrees C results in a 10(3)-fold increase in ribonucleolytic activity. A large quantity of active dimer can be produced by lyophilizing a 1:1 mixture of the H12A and H119A variants from acetic acid. At pH 6.5 and 65 degrees C, the ribonucleolytic activity of this dimer converges to that of the dimer formed by simply incubating the monomers, as expected for a monomer-dimer equilibrium. The equilibrium dissociation constant for the dimer is near 2 mM at both 65 and 37 degrees C. This value of Kd is only 20-fold greater than the concentration of RNase A in the cow pancreas, suggesting that RNase A dimers exist in vivo. The intrinsic ability of RNase A to form dimers under physiological conditions is consistent with a detailed model for the evolution of homodimeric proteins. Dimers of "monomeric" proteins could be more prevalent than is usually appreciated.     

Novel thermophilic and thermostable lipolytic enzymes from a Thailand hot spring metagenomic library

Functional screening for lipolytic enzymes from a metagenomic library (origin: Jae Sawn hot spring, Thailand) resulted in isolation of a novel patatin-like phospholipase (PLP) and an esterase (Est1). PLP contained four conserved domains similar to other patatin-like proteins with lipid acyl hydrolase activity. Likewise, sequence alignment analysis revealed that Est1 can be classified as a family V bacterial lipolytic enzyme. Both PLP and Est1 were expressed heterologously as soluble proteins in E. coli and exhibited more than 50% of their maximal activities at alkaline pH, of 7-9 and 8-10, respectively. In addition, both enzymes retained more than 50% of maximal activity in the temperature range of 50-75 degrees C, with optimal activity at 70 degrees C and were stable at 70 degrees C for at least 120 min. Both PLP and Est1 exhibited high V(max) toward p-nitrophenyl butyrate. The enzymes had activity toward both short-chain (C(4) and C(5)) and long chain (C(14) and C(16)) fatty acid esters. The isolated enzymes, are therefore, different from other known patatin-like phospholipases and esterases, which usually show no activity for substrates longer than C(10). We suggest that PLP and EstA enzymes are novel and have a; b potential use in industrial applications.     

Regulation of hydrogenase formation is temperature sensitive and plasmid coded in Alcaligenes eutrophus

Alcaligenes eutrophus grew well autotrophically with molecular hydrogen at 30 degrees C, but failed to grow at 37 degrees C (Hox Ts). At this temperature the strain grew well heterotrophically with a variety of organic compounds and with formate as an autotrophic substrate, restricting the thermolabile character to hydrogen metabolism. The soluble hydrogenase activity was stable at 37 degrees C. The catalytic properties of the wild-type enzyme were identical to those of a mutant able to grow lithoautotrophically at 37 degrees C (Hox Tr). Soluble hydrogenase was not rapidly degraded at elevated temperatures since the preformed enzyme remained stable for at least 5 h in resting cells or was diluted by growth, as shown in temperature shift experiments. Immunochemical studies revealed that the formation of the hydrogenase proteins was temperature sensitive. No cross-reactivity was detected above temperatures of 34 degrees C. The genetic information of Hox resides on a self-transmissible plasmid in A. eutrophus. Using Hox Tr mutants as donors of hydrogen-oxidizing ability resulted in Hox+ transconjugants which not only had recovered plasmid pHG1 and both hydrogenase activities but also were temperature resistant. This is evidence that the Hox Tr phenotype is coded by plasmid pHG1.     

Regulation of Sindbis virus RNA replication: uncleaved P123 and nsP4 function in minus-strand RNA synthesis, whereas cleaved products from P123 are required for efficient plus-strand RNA synthesis.

Nonstructural proteins of Sindbis virus, nsP1, nsP2, nsP3, and nsP4, as well as intermediate polyproteins, are produced from two precursor polyproteins, P123 and P1234, by a proteolytic enzyme encoded in the C-terminal half of nsP2. We studied the requirements for and the functions of the intermediate and mature processing products for Sindbis virus RNA synthesis by using site-directed mutants which have a defect(s) in processing the 1/2, 2/3, or 3/4 cleavage sites either singly or in various combinations. A mutant defective in cleaving both the 1/2 and 2/3 sites, which makes only uncleavable P123 and mature nsP4 as final products, produced 10(-3) as much virus as did the wild-type virus after 10 h at 30 degrees C and was nonviable at 40 degrees C. A mutant defective in processing the 2/3 site, which makes nsP1, nsP4, and P23 as well as precursor P123, grew 10(-1) as efficiently as wild-type virus at 30 degrees C and 10(-3) as efficiently at 40 degrees C. Early minus-strand RNA synthesis by these mutants was as efficient as that by wild-type virus, whereas plus-strand RNA synthesis was substantially decreased compared with that by wild-type virus. A mutant defective in processing the 3/4 site was nonviable at either 30 or 40 degrees C. The 3/4 site mutant could be complemented by the mutant unable to cleave either the 1/2 or 2/3 site, which can provide mature nsP4. We interpret these results to signify that (i) mature nsP4 is required for RNA replication, (ii) nsP4 and uncleaved P123 function in minus-strand RNA synthesis, and (iii) cleavage of P123 is required for efficient plus-strand RNA synthesis. We propose that Sindbis virus RNA replication is regulated by differential proteolysis of P123. Early in infection, nsP4 and uncleaved P123 form transient minus-strand RNA replication complexes which vanish upon cleavage of P123. Later in infection, an elevated level of viral proteinase activity eliminates de novo synthesis of P123, and no further synthesis of minus-strand RNA is possible. In contrast, nsP4 and cleavage products from P123 form plus-strand RNA replication complexes which are stable and remain active throughout the infection cycle.     

Molecular dynamics simulations of human cystatin C and its L68Q varient to investigate the domain swapping mechanism

Human cystatin C variant (L68Q), one of the amyloidgenic proteins, has been shown to form dimeric structure spontaneously via domain swapping and easily cause amyloid deposits in the brains of patients suffering from Alzheimer's disease or hereditary cystatin C amyloid angiopathy. The monomeric L68Q and wild-type (wt) HCCs share similar structural feature consisting of a core with a five-stranded anti-parallel beta-sheet (beta-region) wrapped around a central helix. In this study, various molecular dynamics simulations were conducted to investigate the conformational fluctuations of the monomeric L68Q and wt HCCs at various combinations of temperature (300 and 500K) and pH (2 and 7) to gain insights into the domain swapping mechanism. The results show that elevated temperature accelerates the disruption of the hydrophobic core and acidic condition promotes the destruction of three salt bridges between beta2 and beta3 in both HCCs. The results also indicate that the interior hydrophobic core of the L68Q variant is relatively unstable, leading to domain swapping more readily comparing to wt HCC under conditions favoring this process. However, these two monomeric HCCs adopt the same mechanism of domain swapping as follows: (i) first, the interior hydrophobic core is disrupted; (ii) subsequently, the central helix departs from the beta-region; (iii) then, the beta2-L1-beta3 hairpin structure unfolds following the so-called "zip-up" mechanism; and (iv) finally, the open form HCC is generated.     

Yeast mutants temperature-sensitive for growth after random mutagenesis of the chromosomal RAS2 gene and deletion of the RAS1 gene.

Saccharomyces cerevisiae strains with a disrupted RAS1 gene and with an intact RAS2 gene (ras1- RAS2 strains) grew well on both fermentable and nonfermentable carbon sources. By constructing isogenic mutants having a disrupted RAS1 locus and a randomly mutagenized chromosomal RAS2 gene, we obtained yeast strains with specific growth defects. The strain TS1 was unable to grow on nonfermentable carbon sources and galactose at 37 degrees C, while it could grow on glucose at the same temperature. The mutated RAS2 gene in TS1 cells encoded a protein with the glycines at positions 82 and 84 replaced by serine and arginine respectively. Both mutations were necessary for temperature sensitivity. We also isolated a mutant yeast that was unable to grow on nonfermentable carbon sources both at 30 and 37 degrees C, while growing on glucose at both temperatures. This phenotype was caused by a single chromosomal mutation, leading to the replacement of aspartic acid 40 of the RAS2 protein by asparagine. A ras1- yeast strain with a chromosomal RAS2 gene harbouring the three mutations together did not grow at any temperature using non-fermentable carbon sources, but it was able to grow on glucose at 30 degrees C, and not at 37 degrees C. The mutated proteins were much less effective than the wild-type RAS2 protein in the stimulation of adenylate cyclase, but were efficiently expressed in vivo. The possible roles of residues 40, 82 and 84 of the RAS2 protein in the regulation of adenylate cyclase are discussed.     

Thermodynamic effects of disulfide bond on thermal unfolding of the starch-binding domain of Aspergillus niger glucoamylase

The thermodynamic effects of the disulfide bond of the fragment protein of the starch-binding domain of Aspergillus niger glucoamylase was investigated by measuring the thermal unfolding of the wild-type protein and its two mutant forms, Cys3Gly/Cys98Gly and Cys3Ser/Cys98Ser. The circular dichroism spectra and the thermodynamic parameters of binding with beta-cyclodextrin at 25 degrees C suggested that the native structures of the three proteins are essentially the same. Differential scanning calorimetry of the thermal unfolding of the proteins showed that the unfolding temperature t1/2 of the two mutant proteins decreased by about 10 degrees C as compared to the wild-type protein at pH 7.0. At t1/2 of the wild-type protein (52.7 degrees C), the mutant proteins destabilized by about 10 kJ mol(-1) in terms of the Gibbs energy change. It was found that the mutant proteins were quite stabilized in terms of enthalpy, but that a higher entropy change overwhelmed the enthalpic effect, resulting in destabilization.     

Thermal dependence of isotonic contractile properties of skeletal muscle and sprint performance of the lizardDipsosaurus dorsalis

Contractile properties of the fast-twitch glycolytic (FG) portion of the iliofibularis muscle and sprint running performance were studied at approximately 5 degrees C intervals from 15-44 degrees C in the lizard Dipsosaurus dorsalis. Maximal running velocity (VR) and stride frequency (f) were both greatest when body temperature (Tb) was 40 degrees C, the field-active Tb in Dipsosaurus. At 40 degrees C VR was 4.3 +/- 0.2 m/s and f was 13.5 +/- 0.5 s-1. Between 25 and 40 degrees C, the thermal dependencies of VR and f were approximately constant (Q10's of 1.31 and 1.36 got VR and f, respectively). Below 25 degrees C performance declined more markedly with decreasing temperature. At 20 degrees C strides were qualitatively normal, but VR was only half of the value at 25 degrees C. At 15 degrees C the lizards were substantially incapacitated, and VR was 10% of the value at 20 degrees C. Stride length was approximately 0.33 m and changed very little with Tb from 20-44 degrees C. The time dependent contractile properties of FG muscle were affected more by temperature than was sprint performance. The maximal velocity of shortening at zero load (VO) was 18.7 0/s at 40 degrees C and had a Q10 of 1.7 from 25-40 degrees C. Maximal power output (Wmax) determined from the force-velocity curve was 464 W/kg at 40 degrees C. Below 40 degrees C max varied with temperature with a Q10 of 2-3. The shape of the force-velocity curve changed little with temperature (Wmax/POVO = 0.11). Between 25 and 40 degrees C a relatively temperature-independent process must modulate the effects of temperature on the contractile properties of the muscles that supply the power for burst locomotion. Storage and recovery of elastic energy appears to be a likely candidate for such a process. Below 25 degrees C, however, the contraction time is prolonged to such an extent that the f attainable is limited by the minimum time taken to contract and relax the muscles.