Infrared spectroscopic discrimination between the loop and alpha-helices and determination of the loop diffusion kinetics by temperature-jump time-resolved infrared spectroscopy for cytochrome c

The infrared (IR) absorption of the amide I band for the loop structure may overlap with that of the alpha-helices, which can lead to the misassignment of the protein secondary structures. A resolution-enhanced Fourier transform infrared (FTIR) spectroscopic method and temperature-jump (T-jump) time-resolved IR absorbance difference spectra were used to identify one specific loop absorption from the helical IR absorption bands of horse heart cytochrome c in D2O at a pD around 7.0. This small loop consists of residues 70-85 with Met-80 binding to the heme Fe(III). The FTIR spectra in amide I' region indicate that the loop and the helical absorption bands overlap at 1653 cm(-1) at room temperature. Thermal titration of the amide I' intensity at 1653 cm(-1) reveals that a transition in loop structural change occurs at lower temperature (Tm=45 degrees C), well before the global unfolding of the secondary structure (Tm approximately 82 degrees C). This loop structural change is assigned as being triggered by the Met-80 deligation from the heme Fe(III). T-jump time-resolved IR absorbance difference spectra reveal that a T-jump from 25 degrees C to 35 degrees C breaks the Fe-S bond between the Met-80 and the iron reversibly, which leads to a loop (1653 cm(-1), overlap with the helical absorption) to random coil (1645 cm(-1)) transition. The observed unfolding rate constant interpreted as the intrachain diffusion rate for this 16 residue loop was approximately 3.6x10(6) s(-1).     

Enhancement of the thermostability of thermophilic bacterium PS-3 PPase on substitution of Ser-89 with carboxylic amino acids

Serine 89 of the inorganic pyrophosphatase (PPase) subunit from thermophilic bacterium PS-3 (PS-3) was replaced with glycine, alanine, threonine, glutamic acid, or aspartic acid by the PCR-mutagenesis method with Mut-1 in order to determine the contribution of this serine residue to the thermostability and structural integrity of the enzyme molecule. S89G, S89A, and S89T showed reduced catalytic activity, whereas S89D and S89E showed increased enzyme activity. S89G, S89A, and S89T as well as the wild-type PPase were stable in the presence of 5 mM MgCl(2) at 70 degrees C for 1 h, but were inactivated rapidly with time at 80 degrees C. On the contrary, S89D and S89E were stable at 80 degrees C, showing more than 95% of the original activity after 1 h incubation. The wild-type PPase, S89D and S89E were each a hexamer before and after incubation at 80 degrees C for 1 h, while S89G and S89A comprised a mixture of a hexamer and a trimer both before and after incubation at 80 degrees C for 1 h. On the other hand, S89T was a mixture of a hexamer, a trimer and a monomer, and it was partially precipitated during heat treatment at 80 degrees C. The CD spectra of the recombinant enzymes in the far-ultraviolet region were the same as that of the wild-type PPase, whereas those of S89G, S89A, and S89T as well as the wild-type PPase were markedly different after heat treatment, although those of S89D and S89E did not change. The present study suggested that local small change(s) in the network of interactions among amino acid residues on replacement at position 89 led to the PS-3 PPase molecule being unable to form a hexamer from trimers or to dissociate into monomers in some cases without a significant change in the backbone conformation. It was also suggested that the partial disordering of the conformation of PS-3 PPase caused by heat depended on the degree of hydrophilicity in the vicinity of position 89.     

海栖热袍菌内切葡聚糖酶Cel12B与木聚糖酶XynA CBD结构域融合基因的构建、表达及融合酶性质分析

海栖热袍菌内切葡聚糖酶Cel12B是极耐热胞外酶,氨基酸序列分析表明不含有纤维素结合结构域(CBD),对结晶纤维素无活性,但同样菌种来源的木聚糖酶XynA有催化结构域和纤维素结合结构城。用同样极耐热酶CBD区域和Cel12B融合构建重组质粒pET-20b-Cel12B-CBD,经诱导表达后,对结晶纤维素有活性,酶学特性研究表明:最适反应温度为100℃、最适pH为5.8、在pH4.5~7.0时酶活力稳定,90℃保温2h仍有87%的酶活。     

Biochemical and structural characterization of mammalian-like purine nucleoside phosphorylase from the Archaeon Pyrococcus furiosus

We report here the characterization of the first mammalian-like purine nucleoside phosphorylase from the hyperthermophilic archaeon Pyrococcus furiosus (PfPNP). The gene PF0853 encoding PfPNP was cloned and expressed in Escherichia coli and the recombinant protein was purified to homogeneity. PfPNP is a homohexamer of 180 kDa which shows a much higher similarity with 5'-deoxy-5'-methylthioadenosine phosphorylase (MTAP) than with purine nucleoside phosphorylase (PNP) family members. Like human PNP, PfPNP shows an absolute specificity for inosine and guanosine. PfPNP shares 50% identity with MTAP from P. furiosus (PfMTAP). The alignment of the protein sequences of PfPNP and PfMTAP indicates that only four residue changes are able to switch the specificity of PfPNP from a 6-oxo to a 6-amino purine nucleoside phosphorylase still maintaining the same overall active site organization. PfPNP is highly thermophilic with an optimum temperature of 120 degrees C and is characterized by extreme thermodynamic stability (T(m), 110 degrees C that increases to 120 degrees C in the presence of 100 mm phosphate), kinetic stability (100% residual activity after 4 h incubation at 100 degrees C), and remarkable SDS-resistance. Limited proteolysis indicated that the only proteolytic cleavage site is localized in the C-terminal region and that the C-terminal peptide is not necessary for the integrity of the active site. By integrating biochemical methodologies with mass spectrometry we assigned three pairs of intrasubunit disulfide bridges that play a role in the stability of the enzyme against thermal inactivation. The characterization of the thermal properties of the C254S/C256S mutant suggests that the CXC motif in the C-terminal region may also account for the extreme enzyme thermostability.     

Structural basis for the thermostability of ferredoxin from the cyanobacterium Mastigocladus laminosus

Plant-type ferredoxins (Fds) carry a single [2Fe-2S] cluster and serve as electron acceptors of photosystem I (PSI). The ferredoxin from the thermophilic cyanobacterium Mastigocladus laminosus displays optimal activity at 65 degrees C. In order to reveal the molecular factors that confer thermostability, the crystal structure of M.laminosus Fd (mFd) was determined to 1.25 A resolution and subsequently analyzed in comparison with four similar plant-type mesophilic ferredoxins. The topologies of the plant-type ferredoxins are similar, yet two structural determinants were identified that may account for differences in thermostability, a salt bridge network in the C-terminal region, and the flexible L1,2 loop that increases hydrophobic accessible surface area. These conclusions were verified by three mutations, i.e. substitution of L1,2 into a rigid beta-turn ((Delta)L1,2) and two point mutations (E90S and E96S) that disrupt the salt bridge network at the C-terminal region. All three mutants have shown reduced electron transfer (ET) capabilities and [2Fe-2S] stability at high temperatures in comparison to the wild-type mFd. The results have also provided new insights into the involvement of the L1,2 loop in the Fd interactions with its electron donor, the PSI complex.     

Temperature-induced conformational change at the catalytic site of Sulfolobus solfataricus alcohol dehydrogenase highlighted by Asn249Tyr substitution. A hydrogen/deuterium exchange, kinetic, and fluorescence quenching study

A combination of hydrogen/deuterium exchange, fluorescence quenching, and kinetic studies was used to acquire experimental evidence for the crystallographically hypothesized increase in local flexibility which occurs in thermophilic NAD(+)-dependent Sulfolobus solfataricus alcohol dehydrogenase (SsADH) upon substitution Asn249Tyr. The substitution, located at the adenine-binding site, proved to decrease the affinity for both coenzyme and substrate, rendering the mutant enzyme 6-fold more active when compared to the wild-type enzyme [Esposito et al. (2003) FEBS Lett. 539, 14-18]. The amide H/D exchange data show that the wild-type and mutant enzymes have similar global flexibility at 22 and 60 degrees C. However, the temperature dependence of the Stern-Volmer constant determined by acrylamide quenching shows that the increase in temperature affects the local flexibility differently, since the K(SV) increment is significantly higher for the wild-type than for the mutant enzyme over the range 18-45 degrees C. Interestingly, the corresponding van't Hoff plot (log K(SV) vs 1/T) proves nonlinear for the apo and holo wild-type and apo mutant enzymes, with a break at approximately 45 degrees C in all three cases due to a conformational change affecting the tryptophan microenvironment experienced by the quencher molecules. The Arrhenius and van't Hoff plots derived from the k(cat) and K(M) thermodependence measured with cyclohexanol and NAD(+) at different temperatures display an abrupt change of slope at 45-50 degrees C. This proves more pronounced in the case of the mutant enzyme compared to the wild-type enzyme due to a conformational change in the structure rather than to an overlapping of two or more rate-limiting reaction steps with different temperature dependencies of their rate constants. Three-dimensional analysis indicates that the observed conformational change induced by temperature is associated with the flexible loops directly involved in the substrate and coenzyme binding.     

Heterologous expression of 5'-methylthioadenosine phosphorylase from the archaeon Sulfolobus solfataricus: characterization of the recombinant protein and involvement of disulfide bonds in thermophilicity and thermostability.

The gene for the extremely thermophilic and thermostable 5'-methylthioadenosine phosphorylase from the archaeon Sulfolobus solfataricus was expressed at a high level in Escherichia coli thus providing a basis for detailed structural and functional studies of the enzyme. The recombinant enzyme was purified to homogeneity by means of a heat treatment (10 min at 100 degrees C) and by a single affinity chromatography step. The appropriate expression vector and host strain were selected and the culture conditions were determined that would ensure a consistent yield of 6 mg of pure enzyme per liter of culture. The heterologously expressed enzyme is identical to the original S. solfataricus 5'-methylthioadenosine phosphorylase regarding molecular weight, substrate specificity, and the presence of intersubunit disulfide bonds. On the other hand, the recombinant 5'-methylthioadenosine phosphorylase is less thermophilic and thermostable than the S. solfataricus enzyme, since an incorrect positioning of disulfide bonds within the molecule generates structures less stable to thermal unfolding.     

Lack of communication between LC2 light chain and the SH1 region of myosin S-1 studied by 19F-NMR

Myosin subfragment-1 (S-1) which contains the LC2 light chain has been labelled with fluorine to allow an 19F-NMR study of the coupling and energetics of structural changes in the myosin head. Two fluorine-containing reagents, N-4-(trifluoromethyl)phenyl iodoacetamide and N-3,5-di(trifluoromethyl)phenyl iodoacetamide, have been used to label the myosin heavy chain at the unusually reactive sulfhydryl-1 (SH1) position. The chemical shift of both reagents on S-1 is sensitive to a structural transition in the region of SH1 which occurs upon increasing the temperature from 0 degrees C to 35 degrees C. The midpoint of the transition in both papain and chymotryptic S-1 is at approximately 11 degrees C at pH 7 (0.1 M CKl). The temperature dependence of the chemical shift may be fit assuming a two-state equilibrium where delta G degree' (T) = 101-110T +0.386 T2 (where T is the temperature in Kelvin). Both delta H degree' (T) and delta S degree' (T) have a small temperature dependence from 0 to 35 degrees C: at 20 degrees C, delta H degree' (T) = -33 kcal/mol. delta S degree' (T) = -116 e.u. and delta Cp = -226 cal/mol per deg (pH 7.0, 0.1 M KCl). The NMR data indicate that the presence of the LC2 light chain in papain S-1 does not modify the structure of S-1 in the vicinity of SH1, nor does it modify the energetics of the structural transition from that seen in its absence with chymotryptic S-1. The presence of calcium which is bound by the LC2 of papain S-1 also does not alter the energetics of the transition. Thus it would appear that the LC2 light chain (on myosin S-1) does not participate in the two-state transition, nor does it interact strongly with regions of the heavy chain which participate in the transition.     

Apurinic and apyrimidinic DNA endonuclease of extremely thermophilic Thermothrix thiopara.

An endonuclease specific for apurinic, apyrimidinic (AP) sites in DNA was purified nearly to homogeneity from the extremely thermophilic bacterium Thermothrix thiopara. The enzyme has a molecular weight of approximately 26,000. It cleaves neither native nor UV- or gamma-irradiated DNAs and has no contaminating exonuclease or uracil-DNA glycosylase activities. The enzyme has no cofactor requirement and is not inhibited by EDTA or N'-ethylmaleimide. It shows maximal activity at 70 degrees C and a pH between 7.5 and 9.0. The Arrhenius activation energy of the reaction is 17 kJ/mol, and the apparent Km for AP sites is 38 nM. The rate of heat inactivation of the enzyme followed first-order kinetics, with a half-life of 10 min at 70 degrees C but about 150 min in the presence of 0.5 M ammonium sulfate or 0.5 mg of bovine serum albumin per ml at the same temperature. One cell of T. thiopara contains sufficient AP endonuclease activity for hydrolysis of about 10(6) phosphodiester bonds per h at 70 degrees C. An extract of these bacteria does not contain detectable Mg-dependent AP endonuclease activity, and the above-mentioned enzyme appears to be the main AP endonuclease of T. thiopara.     

A recombinant exonuclease III homologue of the thermophilic archaeon Methanothermobacter thermautotrophicus

AP endonucleases catalyse an important step in the base excision repair (BER) pathway by incising the phosphodiester backbone of damaged DNA immediately 5' to an abasic site. Here, we report the cloning and expression of the 774 bp Mth0212 gene from the thermophilic archaeon Methanothermobacter thermautotrophicus, which codes for a putative AP endonuclease. The 30.3 kDa protein shares 30% sequence identity with exonuclease III (ExoIII) of Escherichia coli and 40% sequence identity with the human AP endonuclease Ape1. The gene was amplified from a culture sample and cloned into an expression vector. Using an E. coli host, the thermophilic protein could be produced and purified. Characterization of the enzymatic activity revealed strong binding and Mg2+-dependent nicking activity on undamaged double-stranded (ds) DNA at low ionic strength, even at temperatures below the optimum growth temperature of M. thermautotrophicus (65 degrees C). Additionally, a much faster nicking activity on AP site containing DNA was demonstrated. Unspecific incision of undamaged ds DNA was nearly inhibited at KCl concentration of approximately 0.5 M, whereas incision at AP sites was still complete at such salt concentrations. Nicked DNA was further degraded at temperatures above 50 degrees C, probably by an exonucleolytic activity of the enzyme, which was also found on recessed 3' ends of linearized ds DNA. The enzyme was active at temperatures up to 70 degrees C and, using circular dichroism spectroscopy, shown to denature at temperatures approaching 80 degrees C. Considering the high intracellular potassium ion concentration in M. thermautotrophicus, our results suggest that the characterized thermophilic enzyme acts as an AP endonuclease in vivo with similar activities as Ape1.     

A chimeric elongation factor containing the putative guanine nucleotide binding domain of archaeal EF-1 alpha and the M and C domains of eubacterial EF-Tu.

A recombinant chimeric elongation factor containing the region of EF-1 alpha from Sulfolobus solfataricus harboring the site for GDP and GTP binding and GTP hydrolysis (SsG) and domains M and C of Escherichia coli EF-Tu (EcMC) was studied. SsG-EcMC did not sustain poly(Phe) synthesis in either S. solfataricus or E. coli assay system. This was probably due to the inability of the chimera to interact with aa-tRNA. The three-dimensional modeling of SsG-EcMC indicated only small structural differences compared to the Thermus aquaticus EF-Tu in the ternary complex with aa-tRNA and GppNHp, which did not account for the observed inability to interact with aa-tRNA. The addition of the nucleotide exchange factor SsEF-1 beta was not required for poly(Phe) synthesis since the chimera was already able to exchange [(3)H]GDP for GTP at very high rate even at 0 degrees C. Compared to that of SsEF-1 alpha, the affinity of the chimera for guanine nucleotides was increased and the k(cat) of the intrinsic GTPase was 2-fold higher. The heat stability of SsG-EcMC was 3 and 13 degrees C lower than that displayed by SsG and SsEF-1alpha, respectively, but 30 degrees C higher than that of EcEF-Tu. This pattern remained almost the same if the melting curves of the proteins being investigated were considered instead. The chimeric elongation factor was more thermophilic than SsG and SsEF-1 alpha up to 70 degrees C; at higher temperatures, inactivation occurred.     

Engineering out motion: introduction of a de novo disulfide bond and a salt bridge designed to close a dynamic cleft on the surface of cytochrome b5

A previous molecular dynamics (MD) simulation of cytochrome b5 (cyt b5) at 25 degrees C displayed localized dynamics on the surface of the protein giving rise to the periodic formation of a cleft that provides access to the heme through a protected hydrophobic channel [Storch and Daggett (1995) Biochemistry 34, 9682]. Here we describe the production and testing of mutants designed to prevent the cleft from opening using a combination of experimental and theoretical techniques. Two mutants have been designed to close the surface cleft: S18D to introduce a salt bridge and S18C:R47C to incorporate a disulfide bond. The putative cleft forms between two separate cores of the protein: one is structural in nature and can be monitored through the fluorescence of Trp 22, and the other binds the heme prosthetic group and can be tracked via heme absorbance. An increase in motion localized to the cleft region was observed for each protein, except for the disulfide-containing variant, in MD simulations at 50 degrees C compared to simulations at 25 degrees C. For the disulfide-containing variant, the cleft remained closed. Both urea and temperature denaturation curves were nearly identical for wild-type and mutant proteins when heme absorbance was monitored. In contrast, fluorescence studies revealed oxidized S18C:R47C to be considerably more stable based on the midpoints of the denaturation transitions, Tm and U1/2. Moreover, the fluorescence changes for each protein were complete at approximately 50 degrees C and a urea concentration of approximately 3.9 M, significantly below the temperature and urea concentration (62 degrees C, 5 M urea) required to observe heme release. In addition, solvent accessibility based on acrylamide quenching of Trp 22 was lower in the S18C:R47C mutant, particularly at 50 degrees C, before heme release [presented in the accompanying paper (58)]. The results suggest that a constraining disulfide bond can be designed to inhibit dynamic cleft formation on the surface of cyt b5. Located near the heme, the native dynamics of the cleft may be functionally important for protein-protein recognition and/or complex stabilization.     

Residues at the active site of the esterase 2 from Alicyclobacillus acidocaldarius involved in substrate specificity and catalytic activity at high temperature.

The recently solved three-dimensional structure of the thermophilic esterase 2 from Alicyclobacillus acidocaldarius allowed us to have a snapshot of an enzyme-sulfonate complex, which mimics the second stage of the catalytic reaction, namely the covalent acyl-enzyme intermediate. The aim of this work was to design, by structure-aided analysis and to generate by site-directed and saturation mutagenesis, EST2 variants with changed substrate specificity in the direction of preference for monoacylesters whose acyl-chain length is greater than eight carbon atoms. Positions 211 and 215 of the polypeptide chain were chosen to introduce mutations. Among five variants with single and double amino acid substitutions, three were obtained, M211S, R215L, and M211S/R215L, that changed the catalytic efficiency profile in the desired direction. Kinetic characterization of mutants and wild type showed that this change was achieved by an increase in k(cat) and a decrease in K(m) values with respect to the parental enzyme. The M211S/R215L specificity constant for p-nitrophenyl decanoate substrate was 6-fold higher than the wild type. However, variants M211T, M211S, and M211V showed strikingly increased activity as well as maximal activity with monoacylesters with four carbon atoms in the acyl chain, compared with the wild type. In the case of mutant M211T, the k(cat) for p-nitrophenyl butanoate was 2.4-fold higher. Overall, depending on the variant and on the substrate, we observed improved catalytic activity at 70 degrees C with respect to the wild type, which was a somewhat unexpected result for an enzyme with already high k(cat) values at high temperature. In addition, variants with altered specificity toward the acyl-chain length were obtained. The results were interpreted in the context of the EST2 three-dimensional structure and a proposed catalytic mechanism in which k(cat), e.g. the limiting step of the reaction, was dependent on the acyl chain length of the ester substrate.     

The crystal structure of an EST2 mutant unveils structural insights on the H group of the carboxylesterase/lipase family

Esterase 2 (EST2) from the thermophilic eubacterium Alicyclobacillus acidocaldarius is a thermostable serine hydrolase belonging to the H group of the esterase/lipase family. This enzyme hydrolyzes monoacylesters of different acyl-chain length and various compounds with industrial interest. EST2 displays an optimal temperature at 70 degrees C and maximal activity with pNP-esters having acyl-chain bearing from six to eight carbon atoms. EST2 mutants with different substrate specificity were also designed, generated by site-directed mutagenesis, and biochemically characterized. To better define at structural level the enzyme reaction mechanism, a crystallographic analysis of one of these mutants, namely M211S/R215L, was undertaken. Here we report its three-dimensional structure at 2.10A resolution. Structural analysis of the enzyme revealed an unexpected dimer formation as a consequence of a domain-swapping event involving its N-terminal region. This phenomenon was absent in the case of the enzyme bound to an irreversible inhibitor having optimal substrate structural features. A detailed comparison of the enzyme structures before and following binding to this molecule showed a movement of the N-terminal helices resulting from a trans-cis isomerization of the F37-P38 peptide bond. These findings suggest that this carboxylesterase presents two distinct structural arrangements reminiscent of the open and closed forms already reported for lipases. Potential biological implications associated with the observed quaternary reorganization are here discussed in light of the biochemical properties of other lipolytic members of the H group.     

High thermal stability of 3-isopropylmalate dehydrogenase from Thermus thermophilus resulting from low DeltaC(p) of unfolding.

To characterize the thermal stability of 3-isopropylmalate dehydrogenase (IPMDH) from an extreme thermophile, Thermus thermophilus, urea-induced unfolding of the enzyme and of its mesophilic counterpart from Escherichia coli was investigated at various temperatures. The unfolding curves were analyzed with a three-state model for E.coli IPMDH and with a two-state model for T.thermophilus IPMDH, to obtain the free energy change DeltaG degrees of each unfolding process. Other thermodynamic parameters, enthalpy change DeltaH, entropy change DeltaS and heat capacity change DeltaC(p), were derived from the temperature dependence of DeltaG degrees. The main feature of the thermophilic enzyme was its lower dependence of DeltaG degrees on temperature resulting from a low DeltaC(p). The thermophilic IPMDH had a significantly lower DeltaC(p), 1.73 kcal/mol.K, than that of E.coli IPMDH (20.7 kcal/mol.K). The low DeltaC(p) of T.thermophilus IPMDH could not be predicted from its change in solvent-accessible surface area DeltaASA. The results suggested that there is a large structural difference between the unfolded state of T.thermophilus and that of E.coli IPMDH. Another responsible factor for the higher thermal stability of T.thermophilus IPMDH was the increase in the most stable temperature T(s). The DeltaG degrees maximum of T.thermophilus IPMDH was much smaller than that of E.coli IPMDH. The present results clearly demonstrated that a higher melting temperature T(m) is not necessarily accompanied by a higher DeltaG degrees maximum.     

Change in Microbial Numbers during Thermophilic Composting of Sewage Sludge with Reference to CO(2) Evolution Rate

Dewatered sewage sludge was composted in a laboratory-scale autothermal reactor in which a constant temperature of 60 degrees C was kept as long as possible by regulating the air feed rate. The change in CO(2) evolution rate was measured continuously from the start up through the cessation of compositing. The succession of mesophilic bacteria, thermophilic bacteria, and thermophilic actinomycetes was also observed during the composting. Specific CO(2) evolution rates of thermophilic bacteria and actinomycetes in the constant-temperature region of 60 degrees C were assessed quantitatively. It was found that the CO(2) evolution rate was attributed to thermophilic bacteria at the initial stage of 60 degrees C and to thermophilic actinomycetes at the later stage of 60 degrees C.     

Purification and characterization of a novel recombinant highly enantioselective short-chain NAD(H)-dependent alcohol dehydrogenase from Thermus thermophilus

The gene encoding a novel alcohol dehydrogenase (ADH) that belongs to the short-chain dehydrogenase/reductase (SDR) superfamily was identified in the extremely thermophilic, halotolerant gram-negative eubacterium Thermus thermophilus HB27. The T. thermophilus ADH gene (adh(Tt)) was heterologously overexpressed in Escherichia coli, and the protein (ADH(Tt)) was purified to homogeneity and characterized. ADH(Tt) is a tetrameric enzyme consisting of identical 26,961-Da subunits composed of 256 amino acids. The enzyme has remarkable thermophilicity and thermal stability, displaying activity at temperatures up to approximately 73 degrees C and a 30-min half-inactivation temperature of approximately 90 degrees C, as well as good tolerance to common organic solvents. ADH(Tt) has a strict requirement for NAD(H) as the coenzyme, a preference for reduction of aromatic ketones and alpha-keto esters, and poor activity on aromatic alcohols and aldehydes. This thermophilic enzyme catalyzes the following reactions with Prelog specificity: the reduction of acetophenone, 2,2,2-trifluoroacetophenone, alpha-tetralone, and alpha-methyl and alpha-ethyl benzoylformates to (S)-(-)-1-phenylethanol (>99% enantiomeric excess [ee]), (R)-alpha-(trifluoromethyl)benzyl alcohol (93% ee), (S)-alpha-tetralol (>99% ee), methyl (R)-(-)-mandelate (92% ee), and ethyl (R)-(-)-mandelate (95% ee), respectively, by way of an efficient in situ NADH-recycling system involving 2-propanol and a second thermophilic ADH. This study further supports the critical role of the D37 residue in discriminating NAD(H) from NADP(H) in members of the SDR superfamily.     

Solution structure and thermal stability of ribosomal protein L30e from hyperthermophilic archaeon Thermococcus celer

To understand the structural basis of thermostability, we have determined the solution structure of a thermophilic ribosomal protein L30e from Thermococcus celer by NMR spectroscopy. The conformational stability of T. celer L30e was measured by guanidine and thermal-induced denaturation, and compared with that obtained for yeast L30e, a mesophilic homolog. The melting temperature of T. celer L30e was 94 degrees C, whereas the yeast protein denatured irreversibly at temperatures >45 degrees C. The two homologous proteins also differ greatly in their stability at 25 degrees C: the free energy of unfolding was 45 kJ/mole for T. celer L30e and 14 kJ/mole for the yeast homolog. The solution structure of T. celer L30e was compared with that of the yeast homolog. Although the two homologous proteins do not differ significantly in their number of hydrogen bonds and the amount of solvent accessible surface area buried with folding, the thermophilic T. celer L30e was found to have more long-range ion pairs, more proline residues in loops, and better helix capping residues in helix-1 and helix-4. A K9A variant of T. celer L30e was created by site-directed mutagenesis to examine the role of electrostatic interactions on protein stability. Although the melting temperatures of the K9A variant is approximately 8 degrees C lower than that of the wild-type L30e, their difference in T(m) is narrowed to approximately 4.2 degrees C at 0.5 M NaCl. This salt-dependency of melting temperatures strongly suggests that electrostatic interactions contribute to the thermostability of T. celer L30e.     

Endothermic force generation in fast and slow mammalian (rabbit) muscle fibers.

Isometric tension responses to rapid temperature jumps (T-jumps) of 3-7 degrees C were examined in single skinned fibers isolated from rabbit psoas (fast) and soleus (slow) muscles. T-jumps were induced by an infrared laser pulse (wavelength 1.32 microns, pulse duration 0.2 ms) obtained from a Nd-YAG laser, which heated the fiber and bathing buffer solution in a 50-microliter trough. After a T-jump, the temperature near the fiber remained constant for approximately 0.5 s, and the temperature could be clamped for longer periods by means of Peltier units assembled on the back trough wall. A T-jump produced a step decrease in tension in both fast and slow muscle fibers in rigor, indicating thermal expansion. In maximally Ca-activated (pCa approximately 4) fibers, the increase of steady tension with heating (3-35 degrees C) was approximately sigmoidal, and a T-jump at any temperature induced a more complex tension transient than in rigor fibers. An initial (small amplitude) step decrease in tension followed by a rapid recovery (tau(1); see Davis and Harrington, 1993) was seen in some records from both fiber types, which presumably was an indirect consequence of thermal expansion. The net rise in tension after a T-jump was biexponential, and its time course was characteristically different in the two fibers. At approximately 12 degrees C the reciprocal time constants for the two exponential components (tau(2) and tau(3), respectively, were approximately 70.s(-1) and approximately 15.s(-1) in fast fibers and approximately 20.s(-1) and approximately 3.s(-1) in slow fibers. In both fibers, tau(2) ("endothermic force regeneration") became faster with an increase in temperature. Furthermore, tau(3) was temperature sensitive in slow fibers but not in fast fibers. The results are compared and contrasted with previous findings from T-jump experiments on fast fibers. It is observed that the fast/slow fiber difference in the rate of endothermic force generation (three- to fourfold) is considerably smaller than the reported differences in the "phosphate release steps" (> 30-fold).