Spectroscopic Investigations on the Interaction between Carbon Nanotubes and Catalase on Molecular Level

The interactions between well‐dispersed multiwalled carbon nanotubes (MWCNTs) and catalase (CAT) were investigated. The activity of CAT was inhibited with the addition of MWCNTs. After deducting the inner filter effect, the fluorescence spectra revealed that the tryptophan (Trp) residues were exposed and the fluorescence intensities of CAT increased with the increase in the MWCNTs concentration. At the same time, the environment of the Trp residues became more hydrophobic. The results of UV–vis absorption spectroscopy and CD spectra indicated that the secondary structure of CAT had been changed, and the amino acid residues were located in a more hydrophobic environment. Meanwhile, the UV–vis spectra indicated that the conformation of the heme porphyrin rings was changed. The microenvironment of CAT activity sites may be interfered by MWCNTs. This research showed that MWCNTs could not only contribute to the conformational changes of protein but also change the enzyme function.     

Lipid and signal peptide-induced conformational changes within the C-domain of Escherichia coli SecA protein

SecA ATPase is an essential component of the Sec-dependent protein translocation machinery. Upon interaction with the plasma membrane containing SecYE, preprotein, and ATP, SecA undergoes cycles of membrane insertion and retraction resulting in the translocation of segments of the preprotein to the trans side of the membrane. To study the structural basis of SecA function, we employed fluorescence spectroscopy along with collisional quenchers with a set of SecA proteins containing single tryptophan substitutions. Our data show that among the seven naturally occurring tryptophan residues of Escherichia coli SecA, only the three tryptophan residues contained within the C-domain contributed significantly to the fluorescence signal, and they occupied distinct local environments in solution: Trp723 and Trp775 were found to be relatively solvent accessible and inaccessible, respectively, while Trp701 displayed an intermediate level of solvent exposure. Exposure to increased temperature or interaction with model membranes or signal peptide elicited a similar conformational response from SecA based upon the fluorescence signals of the SecA-W775F and SecA-W723F mutant proteins. Specifically, Trp775 became more solvent exposed, while Trp723 became less solvent accessible under these conditions, indicating similarities in the overall conformational change of the C-domain promoted by temperature or translocation ligands. Only Trp701 did not respond in parallel to the different conditions, since its solvent accessibility changed only in the presence of signal peptide. These results provide the first detailed structural information about the C-domain of SecA and its response to translocation ligands, and they provide insight into the conformational changes within SecA that drive protein translocation.     

Nanoparticulate glutathione peroxidase mimics based on selenocystine-pullulan conjugates

We synthesized nanoparticulate glutathione peroxidase (GPx) mimics in which selenocystine (SeCyst) was conjugated to a hydrophilic linear polysaccharide, pullulan (Pul). The SeCyst ester-conjugated Pul derivatives (SeCyst-Pul) in phosphate buffer (pH 7) were treated with a sonicator to spontaneously form particulate materials. Dynamic light scattering measurements revealed that the SeCyst-Pul conjugates could form particulate materials with diameters between 100 and 300 nm. Distinctive endothermic peaks were observed for the SeCyst-Pul aggregate solutions based on a differential scanning calorimetric analysis. The tryptophan (Trp) fluorescence intensity of SeCyst benzyl ester-tryptophanyl-Pul (SeCyst-Bz-Trp-Pul) mostly decreased in comparison to those of the Trp-Pul (its precursor) and free Trp, which indicates that the Trp residues come close to each other during the aggregation of the conjugates. Formation of SeCyst-Pul aggregates could be induced by the hydrophobic interactions between the SeCyst esters and the amino acid residues on Pul. The GPx-like activity of SeCyst-Bz-Trp-Pul aggregates for the reduction of H2O2 was enhanced nearly 20-fold higher than that of free SeCyst. The double-reciprocal plots of the SeCyst-Bz-Trp-Pul aggregate-catalyzed reduction yielded parallel lines by varying the substrate concentrations, indicating a "ping-pong" mechanism that is similar to those of the natural GPxs. The enhanced GPx activity of the SeCyst-Bz-Trp-Pul aggregate was also supported by higher kinetic parameters, k(cat)/K(m) (GSH) and k(cat)/K(m) H2O2. Overall, the enhanced activity of the SeCyst-Bz-Trp-Pul aggregate would be attributed to a hydrophobic environment that was formed at the vicinity of the SeCyst.     

Probing local environments of tryptophan residues in proteins: comparison of 19F nuclear magnetic resonance results with the intrinsic fluorescence of soluble human tissue factor

19F nuclear magnetic resonance (19F NMR) of 5-fluorotryptophan (5F-Trp) and tryptophan (Trp) fluorescence both provide information about local environment and solvent exposure of Trp residues. To compare the information provided by these spectroscopies, the four Trp residues in recombinant soluble human tissue factor (sTF) were replaced with 5F-Trp. 19F NMR assignments for the 5F-Trp residues (14, 25, 45, and 158) were based on comparison of the wild-type protein spectrum with the spectra of three single Trp-to-Phe replacement mutants. Previously we showed from fluorescence and absorption difference spectra of mutant versus wild-type sTF that the side chains of Trpl4 and Trp25 are buried, whereas those of Trp45 and Trp158 are partially exposed to bulk solvent (Hasselbacher et al., Biophys J 1995;69:20-29). 19F NMR paramagnetic broadening and solvent-induced isotope-shift experiments show that position 5 of the indole ring of 5F-Trp158 is exposed, whereas that of 5F-Trp45 is essentially inaccessible. Although 5F-Trp incorporation had no discernable effect on the procoagulant cofactor activity of either the wild-type or mutant proteins, 19F NMR chemical shifts showed that the single-Trp mutations are accompanied by subtle changes in the local environments of 5F-Trp residues residing in the same structural domain.     

Resolution of fluorescence intensity decays of the two tryptophan residues in glutamine-binding protein from Escherichia coli using single tryptophan mutants.

Time correlated single photon counting measurements of tryptophan (Trp) fluorescence intensity decay and other spectroscopic studies were performed on glutamine-binding protein (GlnBP) from Escherichia coli. Using site-specifically mutated forms of the protein in which tyrosine (Tyr) and phenylalanine (Phe) substitute for the Trp residues at positions 32 and 220, we have examined whether wild-type (Wtyp) intensity decay components may be assigned to specific Trp residues. Results indicate that: (a) two exponential intensity decay components are recovered from the Wtyp protein (6.16 ns, 0.46 ns); (b) the long decay component arises from Trp-220 and comprises greater than 90% of the total fluorescence emission; (c) the short component arises from Trp-32 and is highly quenched; (d) all four single-Trp mutants exhibit multiexponential intensity decays, yet equimolar mixtures of two single-Trp mutants yield only two decay components which are virtually indistinguishable from the Wtyp protein; (e) the recovery of additional components in protein mixtures is obscured by statistical noise inherent in the technique of photon counting; (f) various spectroscopic measurements suggest that Trp-Trp interactions occur in the Wtyp protein, but the Wtyp intensity decay may be closely approximated by a linear combination of intensity decays from single-Trp mutants; and (g) inferences derived independently from fluorescence and NMR spectroscopy which pertain to the presence of Trp-Trp interactions and the relative solvent exposure of the two Trp residues are in agreement.     

Fluorescence and chemical modification of tryptophan as probes of structure of GTP:AMP phosphotransferase from beef heart mitochondria

Selective modification of the two Trp residues of GTP:AMP phosphotransferase from beef heart mitochondria (Mr 26 000; MgGTP + AMP in equilibrium MgGDP + ADP) has been attained by treatment of the enzyme with N-bromosuccinimide at pH 4.0. Almost complete loss of activity is observed when one Trp is oxidized. Fluorescence emission spectra (lambda exc 295 nm) were recorded over the pH range 1.9-12.2. Quenching constants, K, with acrylamide were 4.9, 3.4, 3.1, 2.4, 9.2 and 9.4 M-1 at respective pH values of 11.1, 7.5, 5.5, 4.0, 1.9 and 7.5 with 6 M guanidine/HCl. Over the pH range 8.0-5.5 the fluorescence peak has a constant height with maximum at 333-334 nm, which can be segregated by acrylamide quenching into a peak with maximum at 338 nm and another with maximum at 330 nm. Dropping the pH from 5.5 to 4.0 results in the fluorescence at 338 nm decreasing to 335 nm (indicative of less exposure of the Trp) while that at 330 nm remains constant. Thus the limitation of reactivity to N-bromosuccinimide to pH 4.0 or lower cannot be accounted for by increased exposure of the Trp residues but rather must be explained by a change in the microenvironment of each Trp. As shown by K values above, at pH 2.0 Trp residues are exposed to the solvent, as in the case of treatment with 6 M guanidine hydrochloride. In raising the pH from 8.0 to 12.0 a number of changes occur: (a) the lambda max of emission shifts from 333-334 nm to 343 nm; (b) residue(s) become(s) more available to acrylamide quenching; (c) fluorescence decreases and enzymatic activity increases, both with a midpoint at about 10.6; (d) absorption difference spectra show a maximum at 295 nm typical of Tyr ionization. These data are consistent with conformational change as the pH becomes more alkaline making the Trp residue(s) more exposed to the solvent and/or to non-radiative energy transfer to tyrosinate.     

Substrate-induced conformational changes of the mitochondrial oxoglutarate carrier: a spectroscopic and molecular modelling study

The structural and dynamic properties of the oxoglutarate carrier were investigated by introducing a single tryptophan in the Trp-devoid carrier in position 184, 190 or 199 and by monitoring the fluorescence spectra in the presence and absence of the substrate oxoglutarate. In the absence of substrate, the emission maxima of Arg190Trp, Cys184Trp and Leu199Trp are centered at 342, 345 and 348 nm, respectively, indicating that these residues have an increasing degree of solvent exposure. The emission intensity of the Arg190Trp and Cys184Trp mutants is higher than that of Leu199Trp. Addition of substrate increases the emission intensity of Leu199Trp, but not that of Cys184Trp and Arg190Trp. A 3D model of the oxoglutarate carrier was built using the structure of the ADP/ATP carrier as a template and was validated with the experimental results available in the literature. The model identifies Lys122 as the most likely candidate for the quenching of Trp199. Consistently, the double mutant Lys122Ala-Leu199Trp exhibits a higher emission intensity than Leu199Trp and does not display further fluorescence enhancement in response to substrate addition. Substitution of Lys122 with Cys and evaluation of its reactivity with a sulphydryl reagent in the presence and absence of substrate confirms that residue 122 is masked by the substrate, likely through a substrate-induced conformational change.     

Lysyl-tRNA synthetase from Bacillus stearothermophilus: the Trp314 residue is shielded in a non-polar environment and is responsible for the fluorescence changes observed in the amino acid activation reaction

Three Trp variants of lysyl-tRNA synthetase from Bacillus stearothermophilus, in which either one or both of the two Trp residues within the enzyme (Trp314 and Trp332) were substituted by a Phe residue, were produced by site-directed mutagenesis without appreciable loss of catalytic activity. The following two phenomena were observed with W332F and with the wild-type enzyme, but not with W314F: (1) the addition of L-lysine alone decreased the protein fluorescence of the enzyme, but the addition of ATP alone did not; (2) the subsequent addition of ATP after the addition of excess L-lysine restored the fluorescence to its original level. Fluorometry under various conditions and UV-absorption spectroscopy revealed that Trp314, which was about 20A away from the lysine binding site and was shielded in a non-polar environment, was solely responsible for the fluorescence changes of the enzyme in the L-lysine activation reaction. Furthermore, the microenvironmental conditions around the residue were made more polar upon the binding of L-lysine, though its contact with the solvent was still restricted. It was suggested that Trp314 was located in a less polar environment than was Trp332, after comparison of the wavelengths at the peaks of fluorescence emission and of the relative fluorescence quantum yields. Trp332 was thought, based on the fluorescence quenching by some perturbants and the chemical modification with N-bromosuccinimide, to be on the surface of the enzyme, whereas Trp314 was buried inside. The UV absorption difference spectra induced by the L-lysine binding indicated that the state of Trp314, including its electrostatic environment, changed during the process, but Trp332 did not change. The increased fluorescence from Trp314 at acidic pH compared with that at neutral pH suggests that carboxylate(s) are in close proximity to the Trp314 residue.     

Substrate-induced conformational changes of the mitochondrial oxoglutarate carrier: a spectroscopic and molecular modelling study

The structural and dynamic properties of the oxoglutarate carrier were investigated by introducing a single tryptophan in the Trp-devoid carrier in position 184, 190 or 199 and by monitoring the fluorescence spectra in the presence and absence of the substrate oxoglutarate. In the absence of substrate, the emission maxima of Arg190Trp, Cys184Trp and Leu199Trp are centered at 342, 345 and 348 nm, respectively, indicating that these residues have an increasing degree of solvent exposure. The emission intensity of the Arg190Trp and Cys184Trp mutants is higher than that of Leu199Trp. Addition of substrate increases the emission intensity of Leu199Trp, but not that of Cys184Trp and Arg190Trp. A 3D model of the oxoglutarate carrier was built using the structure of the ADP/ATP carrier as a template and was validated with the experimental results available in the literature. The model identifies Lys122 as the most likely candidate for the quenching of Trp199. Consistently, the double mutant Lys122Ala-Leu199Trp exhibits a higher emission intensity than Leu199Trp and does not display further fluorescence enhancement in response to substrate addition. Substitution of Lys122 with Cys and evaluation of its reactivity with a sulphydryl reagent in the presence and absence of substrate confirms that residue 122 is masked by the substrate, likely through a substrate-induced conformational change.     

Site-directed mutagenesis of conserved C-terminal tyrosine and tryptophan residues of PsbO, the photosystem II manganese-stabilizing protein, alters its activity and fluorescence properties

The extrinsic photosystem II PsbO subunit (manganese-stabilizing protein) contains near-UV CD signals from its complement of aromatic amino acid residues (one Trp, eight Tyr, and 13 Phe residues). Acidification, N-bromosuccinimide modification of Trp, reduction or elimination of a disulfide bond, or deletion of C-terminal amino acids abolishes these signals. Site-directed mutations that substitute Phe for Trp241 and Tyr242, near the C-terminus of PsbO, were used to examine the contribution of these residues to the activity and spectral properties of the protein. Although this substitution is, in theory, conservative, neither mutant binds efficiently to PSII, even though these proteins appear to retain wild-type solution structures. Removal of six residues from the N-terminus of the W241F mutant restores activity to near-wild-type levels. The near-UV CD spectra of the mutants are modified; well-defined Tyr and Trp peaks are lost. Characterizations of the fluorescence spectra of the full-length WF and YF mutants indicate that Y242 contributes significantly to PsbO's Tyr fluorescence emission and that an excited-state tyrosinate could be present in PsbO. Deletion of W241 shows that this residue is a major contributor to PsbO's fluorescence emission. Loss of function is consistent with the proposal that a native C-terminal domain is required for PsbO binding and activity, and restoration of activity by deletion of N-terminal amino acids may provide some insights into the evolution of this important photosynthetic protein.     

A predominant role for hydrogen bonding in the stability of the homodimer of bothropstoxin-I, A lysine 49-phospholipase A2

Bothropstoxin-I (BthTx-I) is a homodimeric Lys49-phospholipase A(2) isolated from Bothrops jararacussu venom which damages liposome membranes via a Ca(2+)-independent mechanism. The Glu12/Trp77/Lys80 triad at the dimer interface forms extensive intermolecular hydrogen bonds and hydrophobic contacts, and equilibrium chemical denaturation was used to evaluate the effect on homodimer stability of site-directed mutagenesis of these residues. Changes in the intrinsic fluorescence anisotropy and farUV circular dichroism signals were analyzed using a two-step unfolding model of the BthTx-I dimer to estimate the Gibbs free energy changes of transitions between the dimer and native monomer and between the native and denatured monomers. Whereas the Trp77His, Trp77Gln and Glu12Gln mutants showed native-like dimer stabilities, the Trp77Phe, Lys80Met and Lys80Gly mutants showed significantly reduced K(d) values. A reduced dimer stability is correlated with a decrease in the Ca(2+)-independent membrane damaging activity as monitored by the release of a liposome entrapped fluorescent marker. Although the membrane damaging activity of the monomer is fivefold less than the dimer, the myotoxic activity was unaffected, indicating that these two effects are not correlated. These data suggest that the BthTx-I dimer is predominantly stabilized by hydrogen bonding interactions, and highlight the importance of the homodimeric form for efficient Ca(2+)-independent membrane damage.     

Environments of the four tryptophans in the extracellular domain of human tissue factor: comparison of results from absorption and fluorescence difference spectra of tryptophan replacement mutants with the crystal structure of the wild-type protein.

The local environments of the four tryptophan residues of the extracellular domain of human tissue factor (sTF) were assessed from difference absorption and fluorescence spectra. The difference spectra were derived by subtracting spectra from single Trp-to-Phe or Trp-to-Tyr replacement mutants from the corresponding spectrum of the wild-type protein. Each of the mutants was capable of enhancing the proteolytic activity of factor VIIa showing that the mutations did not introduce major structural changes, although the mutants were more susceptible to denaturation by guanidinium chloride. The difference spectra indicate that the Trp residues are buried to different extents within the protein matrix. This evaluation was compared with the x-ray crystal structure of sTF. There is excellent agreement between predictions from the difference spectra and the environments of the Trp residues observed in the x-ray crystal structure, demonstrating that difference absorption and particularly fluorescence spectra derived from functional single-Trp replacement mutants can be used to obtain information about the local environments of individual Trp residues in multi-tryptophan proteins.     

酶切菌紫质C端引起的紫膜蛋白的发光性质变化

用木瓜蛋白酶切去bRC端尾巴约20—25个氨基酸之后发现:在KCl盐浓度作用下,其蛋白荧光光谱中的色氨酸荧光成份有明显的增强;用Cs~ 对bR蛋白荧光的猝灭实验表明此色氨酸荧光成份的增加是由于在KCl作用下bR中某些色氨酸残基暴露的结果;磷光光谱实验表明在KCl作用下色氨酸磷光的增加也是由于色氨酸残基暴露的结果。本文讨论了这种构象变化可能影响正常bR分子中菌蛋白光循环的进行,从而使质子泵效率降低;并且此构象变化可能与其C端尾巴的构象有关。     

Effects of five-tryptophan mutations on structure, stability and function of Escherichia coli dihydrofolate reductase

To elucidate the roles of tryptophan residues in the structure, stability, and function of Escherichia coli dihydrofolate reductase (DHFR), its five tryptophan residues were replaced by site-directed mutagenesis with leucine, phenylalanine or valine (W22F, W22L, W30L, W47L, W74F, W74L, W133F, and W133V). Far-ultraviolet circular dichroism (CD) spectra of these mutants reveal that exciton coupling between Trp47 and Trp74 strongly affects the peptide CD of wild-type DHFR, and that Trp133 also contributes appreciably. No additivity was observed in the contributions of individual tryptophan residues to the fluorescence spectrum of wild-type DHFR, Trp74 having a dominant effect. These single-tryptophan mutations induce large changes in the free energy of urea unfolding, which showed values of 1.79-7.14 kcal/mol, compared with the value for wild-type DHFR of 6.08 kcal/mol. Analysis of CD and fluorescence spectra suggests that thermal unfolding involves an intermediate with the native-like secondary structure, the disrupted Trp47-Trp74 exciton coupling, and the solvent-exposed Trp30 and Trp47 side chains. All the mutants except W22L (13%) retain more than 50% of the enzyme activity of wild-type DHFR. These results demonstrate that the five tryptophan residues of DHFR play important roles in its structure and stability but do not crucially affect its enzymatic function.     

Cofactor and tryptophan accessibility and unfolding of brain glutamate decarboxylase

Cofactor and tryptophan accessibility of the 65-kDa form of rat brain glutamate decarboxylase (GAD) was investigated by fluorescence quenching measurements using acrylamide, I-, and Cs+ as the quenchers. Trp residues were partially exposed to solvent. I- was less able and Cs+ was more able to quench the fluorescence of Trp residues in the holoenzyme of GAD (holoGAD) than the apoenzyme (apoGAD). The fraction of exposed Trp residues were in the range of 30-49%. In contrast, pyridoxal-P bound to the active site of GAD was exposed to solvent. I- was more able and Cs+ was less able to quench the fluorescence of pyridoxal-P in holoGAD. The cofactor was present in a positively charged microenvironment, making it accessible for interactions with anions. A difference in the exposure of Trp residues and pyridoxal-P to these charged quenchers suggested that the exposed Trp residues were essentially located outside of the active site. Changes in the accessibility of Trp residues upon pyridoxal-P binding strongly supported a significant conformational change in GAD. Fluorescence intensity measurements were also carried out to investigate the unfolding of GAD using guanidine hydrochloride (GdnHCl) as the denaturant. At 0.8-1.5 M GdnHCl, an intermediate step was observed during the unfolding of GAD from the native to the denatured state, and was not found during the refolding of GAD from the denatured to native state, indicating that this intermediate step was not a reversible process. However, at >1.5 M GdnHCl for holoGAD and >2.0 M GdnHCl for apoGAD, the transition leading to the denatured state was reversible. It was suggested that the intermediate step involved the dissociation of native dimer of GAD into monomers and the change in the secondary structure of the protein. Circular dichroism revealed a decrease in the alpha-helix content of GAD from 36 to 28%. The unfolding pattern suggested that GAD may consist of at least two unfolding domains. Unfolding of the lower GdnHCl-resisting domain occurred at a similar concentration of denaturant for apoGAD and holoGAD, while unfolding of the higher GdnHCl-resisting domain occurred at a higher concentration of GdnHCl for apoGAD than holoGAD.     

Functional evaluation of tryptophans in glycolipid binding and membrane interaction by HET-C2, a fungal glycolipid transfer protein

HET-C2 is a fungal glycolipid transfer protein (GLTP) that uses an evolutionarily-modified GLTP-fold to achieve more focused transfer specificity for simple neutral glycosphingolipids than mammalian GLTPs. Only one of HET-C2's two Trp residues is topologically identical to the three Trp residues of mammalian GLTP. Here, we provide the first assessment of the functional roles of HET-C2 Trp residues in glycolipid binding and membrane interaction. Point mutants HET-C2^W208F, HET-C2^W208A and HET-C2^F149Y all retained > 90% activity and 80–90% intrinsic Trp fluorescence intensity; whereas HET-C2^F149A transfer activity decreased to ~ 55% but displayed ~ 120% intrinsic Trp emission intensity. Thus, neither W208 nor F149 is absolutely essential for activity and most Trp emission intensity (~ 85–90%) originates from Trp109. This conclusion was supported by HET-C2^W109Y/F149Y which displayed ~ 8% intrinsic Trp intensity and was nearly inactive. Incubation of the HET-C2 mutants with 1-palmitoyl-2-oleoyl-phosphatidylcholine vesicles containing different monoglycosylceramides or presented by lipid ethanol-injection decreased Trp fluorescence intensity and blue-shifted the Trp λ_max by differing amounts compared to wtHET-C2. With HET-C2 mutants for Trp208, the emission intensity decreases (~ 30–40%) and λ_max blue-shifts (~ 12 nm) were more dramatic than for wtHET-C2 or F149 mutants and closely resembled human GLTP. When Trp109 was mutated, the glycolipid induced changes in HET-C2 emission intensity and λ_max blue-shift were nearly nonexistent. Our findings indicate that the HET-C2 Trp λ_max blue-shift is diagnostic for glycolipid binding; whereas the emission intensity decrease reflects higher environmental polarity encountered upon nonspecific interaction with phosphocholine headgroups comprising the membrane interface and specific interaction with the hydrated glycolipid sugar.     

白茯苓凝集素的荧光光谱研究

白茯苓凝集素（ＳＬＬ）分子中含有４个色氨酸（Ｔｒｐ）残基,ＮＢＳ修饰测得这４个Ｔｒｐ残基位于分子表面。ＳＬＬ在天然状态下荧光发射峰位于３３５ｎｍ处,离子强度和温度对其荧光光谱均无明显的影响。ＮＢＳ修饰后的ＳＬＬ失去凝血活性,相应荧光光谱的强度减弱,荧光发射峰发生蓝移,提示ＳＬＬ的构象发生改变。用ＫＩ·ＣｓＣｌ和丙烯酰胺淬灭剂研究ＳＬＬ分子中Ｔｒｐ残基的微环境,发现丙烯酰胺和ＣｓＣｌ能淬灭分子中１００％和５０％的Ｔｒｐ残基的荧光,而ＫＩ完全不能淬灭ＳＬＬ分子中Ｔｒｐ残基的荧光,因此Ｔｒｐ残基周围存在阴离子区,或者Ｔｒｐ残基处于分子表面的疏水环境中。     

Effects of in situ cobalt ion addition on the activity of a gfp-oph fusion protein: the fermentation kinetics

The effects of cobalt ion addition and inducer concentration were studied in the fermentation of E. coli BL21 expressing a GFP (green fluorescent protein)-OPH (organophosphorus hydrolase) fusion protein. It was found that cobalt ion addition improved the OPH activity significantly. When 2 mM of CoCl(2) was supplied during the IPTG-induction phase, OPH activity was enhanced approximately 10-fold compared to the case without cobalt or by the addition of cobalt to the cell extracts. Results indicate, therefore, that incorporation of the cobalt during synthesis is needed for enhanced activity. Also, the maximum OPH activity was not linearly related to inducer concentration. A mathematical model was then constructed to simulate these phenomena. Model parameters were determined by constrained least-squares and optimal IPTG and cobalt addition concentrations were obtained, pinpointing the conditions for the maximum productivity. Finally, the GFP fluorescence intensity was found linear to the OPH activity in each fermentation, demonstrating the function of GFP for monitoring its fusion partner's quantity in the bioreactor.     

Thermal denaturation of a recombinant mouse amelogenin: circular dichroism and differential scanning calorimetric studies

Conformational analyses of a recombinant mouse tooth enamel amelogenin (rM179) were performed using circular dichroism (CD), fluorescence, differential scanning calorimetry, and sedimentation equilibrium studies. The results show that the far-UV CD spectra of rM179 at acidic pH and 10 degrees C are different from the spectra of random coil in 6 M GdnHCl. A near-UV CD spectrum of rM179 at 10 degrees C is similar to that of rM179 in 6 M GdnHCl, which indicates that aromatic residues of native structure are exposed to solvent and rotate freely. Far-UV CD values of rM179 at 80 degrees C are different from that of random-coil structure in 6 M GdnHCl, which suggests that rM179 at 80 degrees C has specific secondary structures. A gradual thermal transition was observed by far-UV CD, which is interpreted as a weak cooperative transition from specific secondary structures to other specific secondary structures. The fluorescence emission maximum for the spectrum due to Trp residues in rM179 at 10 degrees C shows the same fluorescence emission maximum as rM179 in 6 M GdnHCl and amino acid Trp, which indicates that the three Trp in rM179 are exposed to solvent. Deconvolution of differential scanning calorimetry curve gives the population of three states (A, I, and C states). These results indicate that three states (A, I, and C) have specific secondary structures, in which hydrophobic and Trp residues are exposed to the solvent. The thermodynamic characteristics of rM179 are unique and different from a typical globular protein, proline-rich peptides, and a molten globule state.     

Conformational changes of an exchangeable apolipoprotein, apolipophorin III from Locusta migratoria, at low pH: correlation with lipid binding.

Locusta migratoria apolipophorin III (apoLp-III) is a helix bundle exchangeable apolipoprotein that reversibly binds to lipoprotein surfaces. Structural reorganization of its five amphipathic alpha-helices enables the transition from the lipid-free to lipid-bound state. ApoLp-III-induced transformation of dimyristoylphosphatidylcholine (DMPC) bilayer vesicles into smaller discoidal complexes is enhanced as a function of decreasing pH, with maximal transformation occurring at pH 3.5. Over the entire pH range studied, apoLp-III retains nearly all of its secondary structure content. Whereas no changes in fluorescence emission maximum of the two Trp residues in apoLp-III were observed in the pH range from 7.0 to 4.0, a further decrease in pH resulted in a strong red shift. Near-UV circular dichroism spectra of apoLp-III showed well-defined extrema (at 286 and 292 nm) between pH 7.0 and pH 4.0, which were attributed to Trp115. Below pH 4.0, these extrema collapsed, indicating a less rigid environment for Trp115. Similarly, the fluorescence intensity of 8-anilinonaphthalene-1-sulfonate in the presence of apoLp-III increased 4-fold below pH 4.0, indicating exposure of hydrophobic sites in the protein in this pH range. Taken together, the data suggest two conformational states of the protein. In the first state between pH 7.0 and pH 4.0, apoLp-III retains a nativelike helix bundle structure. The second state, found between pH 3.0 and pH 4.0, is reminiscent of a molten globule, wherein tertiary structure contacts are disrupted without a significant loss of secondary structure content. In both states DMPC vesicle transformation is enhanced by lowering the solution pH, reaching an optimum in the second state. The correlation between tertiary structure and lipid binding activity suggests that helix bundle organization is a determinant of apoLp-III lipid binding activity.