Unfolding-refolding kinetics of the tryptophan synthase alpha subunit by CD and fluorescence measurements.

To elucidate the folding mechanism of the tryptophan synthase alpha subunit from Escherichia coli, the kinetics of the unfolding-refolding were studied by peptidyl circular dichroism (CD) and aromatic fluorescence measurement at pH 7 and 25 degrees C. The reactions were induced by concentration jumps of guanidine hydrochloride (GuHCl). The results can be summarized as follows. (1) The kinetic properties of the unfolding-refolding monitored by CD at 222 nm and aromatic fluorescence coincided with each other, indicating that the changes in the secondary and tertiary structures proceed simultaneously. (2) The unfolding kinetics showed two phases in the range of final GuHCl concentration above 1.8 M. The total amplitudes in the unfolding kinetics accounted for about 100% of the total change. (3) The refolding kinetics also showed two phases in the native condition. The total amplitudes observed in the two phases accounted for only 41% of the total change in maximum, indicating the presence of an undetectable early folding intermediate in the folding process. (4) The fast phases in both the unfolding and refolding were major phases as judged by the magnitudes of the amplitudes. (5) The amplitudes in terms of the CD values at 222 nm for the undetectable early folding intermediate in the refolding kinetics showed little dependence on final GuHCl concentration in the native condition, but depended on final GuHCl concentration in the transition zone, resulting in a similar equilibrium GuHCl unfolding curve. (6) The CD spectrum in the far-UV region for the early folding intermediate was similar to that for the equilibrium unfolding intermediate. (7) It is concluded that the early folding intermediate of the alpha subunit is equivalent to the equilibrium unfolding intermediate, which is assumed to be a molten globule.

Further examination of the intermediate state in the denaturation of the tryptophan synthase alpha subunit. Evidence that the equilibrium denaturation intermediate is a molten globule.

To further characterize the intermediate state in the denaturation of tryptophan synthase alpha subunit from Escherichia coli, we have carried out differential scanning calorimetry in various concentrations of urea near pH 8.5. The heat capacity curve of the intermediate has no excess heat capacity nor any transition. This indicates that the intermediate is a thermodynamically denatured form. Although the intermediate retains significant CD signal in the far-uv region, the tertiary structure of the intermediate is disrupted as judged by the near-uv CD spectra and 1H NMR spectra in the aromatic region. This intermediate might be similar to a molten globule state. These results do not support our earlier proposal that the intermediate of the alpha subunit in the denaturation process retains an intact N-terminal domain, but that the C-terminal domain unfolds.

Absence of the thermal transition in apo-alpha-lactalbumin in the molten globule state. A study by differential scanning microcalorimetry.

To estimate the energy level of the molten globule state, the heat capacity function of apo-alpha-lactalbumin in the molten globule state has been examined using a scanning microcalorimeter at neutral pH. The results showed that the enthalpy difference between the molten globule state and presumed unfolded state by heating was almost zero at neutral pH, demonstrating that the molten globule state does not exhibit any co-operative transition upon heating. This is in agreement with the results already reported at acid pH, but is apparently in conflict with that recently reported with some assumptions at neutral pH.

Crystal structure of methionine aminopeptidase from hyperthermophile, Pyrococcus furiosus.

The structure of methionine aminopeptidase from hyperthermophile Pyrococcus furiosus (PfMAP) with an optimal growth temperature of 100 degreesC was determined by the multiple isomorphous replacement method and refined in three different crystal forms, one monoclinic and two hexagonal, at resolutions of 2.8, 2.9, and 3.5 A. The resolution of the monoclinic crystal form was extended to 1.75 A by water-mediated transformation to a low-humidity form, and the obtained diffraction data used for high-resolution structure refinement. This is the first description of a eukaryotic type methionine aminopeptidase structure. The PfMAP molecule is composed of two domains, a catalytic domain and an insertion domain, connected via two antiparallel beta-strands. The catalytic domain, which possesses an internal 2-fold symmetry and contains two cobalt ions in the active site, resembles the structure of a prokaryotic type MAP from Escherichia coli (EcMAP), while the structure of the insertion domain containing three helices has a novel fold and accounts for a major difference between the eukaryotic and prokaryotic types of methionine aminopeptidase. Analysis of the PfMAP structure in comparison with EcMAP and other mesophile proteins reveals several factors which may contribute to the hyperthermostability of PfMAP: (1) a significantly high number of hydrogen bonds and ion-pairs between side-chains of oppositely charged residues involved in the stabilization of helices; (2) an increased number of hydrogen bonds between the positively charged side-chain and neutral oxygen; (3) a larger number of buried water molecules involved in crosslinking the backbone atoms of sequentially separate segments; (4) stabilization of two antiparallel beta-strands connecting the two domains of the molecule by proline residues; (5) shortening of N and C-terminal tails and stabilization of the loop c3E by deletion of three residues.

Contribution of hydrophobic residues to the stability of human lysozyme: calorimetric studies and X-ray structural analysis of the five isoleucine to valine mutants.

In order to understand the contribution of hydrophobic residues to the conformational stability of human lysozyme, five Ile mutants (Ile --> Val) in the interior of the protein were constructed. The thermodynamic parameters characterizing the denaturation of these mutant proteins were determined by scanning calorimetry, and the three-dimensional structure of each mutant protein was solved at high resolution by X-ray crystallography. The thermodynamic analyses at 64.9 degrees C and at pH 2.7 revealed the following. (1) The stabilities of all the mutant proteins were decreased as compared with that of the wild-type protein. (2) The changes in the calorimetric enthalpies were larger than those in the Gibbs energies, and were compensated by entropy changes. (3) The destabilization mechanism of the mutant proteins differs, depending on the location of the mutation sites. X-ray analyses showed that the overall structures of all the mutant human lysozymes examined were identical to that of the wild-type protein, and only small structural rearrangements were observed locally around some of the mutation sites. The most striking change among the mutant proteins was found in the mutant protein, 159V, which contains a new water molecule in the cavity created by the mutation. The thermodynamic stabilities of the mutant proteins are discussed in light of the high-resolution X-ray structures of the wild-type and five mutant human lysozymes examined.

Characterization of soluble artificial proteins with random sequences.

The structural and catalytic properties of two soluble random proteins, RP3-42 and RP3-45, of 141 amino acid residues were investigated. Although no marked secondary structure was detected by CD spectrum, sedimentation equilibrium and small-angle X-ray scattering studies showed that they form an oligomeric structure and are as compact as the molten globule. The random proteins have low but distinct esterase activity; the values of the second-order rate constant for the hydrolysis of p-nitrophenol were 0.78 and 1.39 M(-1) s(-1) for RP3-42 and RP3-45, respectively. The differences in the properties of the random and the native proteins are discussed from the evolutionary point of view.

Comparison of denaturation by guanidine hydrochloride of the wild type tryptophan synthase alpha-subunit of Escherichia coli and two mutant protein (Glu 49 replaced by Met or Gln).

In order to elucidate the roles of individual amino acid residues in the conformational stability of proteins, the denaturation by guanidine hydrochloride of the wild-type trytophan synthase alpha-subunit of Escherichia coli and two mutant proteins, trpA33 (Glu 49 leads to Met) and trpA11 (Glu 49 leads to Gln), has been compared by means of CD measurements at pH 7.0 and various temperatures. CD spectra of the two mutant proteins were similar to that of the wild-type protein. The trpA33 and the trpA11 proteins were more and less resistant, respectively, to guanidine hydrochloride than the wild-type protein at 9.7 to 49.6 degrees C. The free energy change of unfolding in water delta delta Gnd H2O, was evaluated assuming a three state denaturation, since the denaturation curves of three proteins suggested the presence of one stable intermediate. The values of delta Gnd H2O for the trpA33, the wild-type, and the trpA11 proteins at 25.8 degrees C and pH 7.0 were 13.4,8.8, and 6.3 kcal/mol, respectively. The delta Gnd H2O of the trpA11 protein was almost independent of temperature, though that of the trpA33 protein was remarkably dependent on temperature. The conformation stabilities of the three proteins were correlated with the hydrophobicities of the substituted amino acid residues.

Effect of guanidine hydrochloride on the holo- and apo-enzymes of pig heart lipoamide dehydrogenase.

1. The effect of guanidine hydrochloride (GuHCl) on pig heart lipoamide dehydrogenase [NADH: lipoamide oxidoreductase, EC 1.6.4.3.] was investigated by means of enzymatic activity and optical measurements (CD, absorption, and fluorescence spectra). The activity of the enzyme decreased on increasing the concentration of GuHCl and the enzyme was completely inactivated in 2.0 M GuHCl. 2. The contents of alpha-helix, beta, and unordered forms in lipoamide dehydrogenase were estimated to be 34, 14, and 52%, respectively. On increasing the concentration of GuHCl, the content of alpha-helix in lipoamide dehydrogenase decreased, whereas the content of the beta form hardly changed. 3. The native lipoamide dehydrogenase showed absorption, CD, and fluorescence spectra characteristic of bound FAD in the visible region, suggesting hydrophobic interaction between the protein moiety and FAD chromophore. The absorption, CD, and fluorescence spectra of the enzyme in 2.0 M GuHCl were similar to those of free FAD in the buffer, suggesting the release of FAD from the protein moiety. 4. The protein fluorescence spectrum of lipoamide dehydrogenase had a maximum at 350 nm blue-shifted by 8 nm from that of tryptophan in aqueous solution. The maximum of the enzyme in 2.0 M GuHCl was red-shifted to 357 nm. This suggests exposure of tryptophan residues to a polar environment. The maximum, 352nm, of the apoenzyme shifted to 350 nm on addition of FAD. These results show that the conformation in the microenvironment of some tryptophan residues in lipoamide dehydrogenase is affected by the dissociation-association of FAD. 5. The contents of alpha-helix, beta, and unordered forms in the apoenzyme were estimated to be 35, 8, and 57%, respectively. These values are similar to those of the native holoenzyme. The alpha-helical structure in the apoenzyme molecule was more sensitive to GuHCl than that in the holoenzyme. FAD and two hydrophobic probes, 8-anilinonaphthalene-1-sulfonate (ANS) and 4 benzolamido-4'-aminostilbene-2,2'-disulfonate (MBAS), which can bind to the apoenzyme, stabilized the alpha-helical structure in the apoenzyme molecule.

Interaction of hydrophobic probes with the apoenzyme of pig heart lipoamide dehydrogenase.

The interaction of hydrophobic probes, 8-anilinonaphthalene-1-sulfonate (ANS) and 4-benzoylamido-4'-aminostilbene-2, 2'-disulfonate (MBAS), with pig heart lipoamide dehydrogenase [NADH: lipoamide oxidoreductase, EC 1.6.4.3] was investigated. When ANS or MBAS was mixed with the apoenzyme of lipoamide dehydrogenase, the fluorescence quantum yield, of each dye was enhancedd markedly and the emission maxima concurrently shifted to the blue. The quantum yield, 0.038, of ANS bound to the apoenzyme, calculated from the corrected emission spectrum, was eight times higher than that in buffer solution, and the value, 0.0090, for bound MBAS was eighteen times higher than that in buffer solution. Moreover, the absortion bands of both ANS and MBAS shifted to the red upon binding with the apoenzyme. A general feature of the absorption spectra of these dyes observed on changing the solvent from polar to apolar was a red shift of the absorption bands. These results indicate that ANS or MBAS bound to the apoenzyme of lipoamide dehydrogenase is situated in a hydrophobic region of the apoenzyme molecule. It was found that 2 moles of each dye was bound per mole of the apoenzyme, which contains two polypeptide chains. The dissociation constants for the ANS- and MBAS-apoenzyme complexes were estimated to be 1.03X10(-5) and 1.54X10(-5) M, respectively. The enhanced fluorescence of both dyes bound to the apoenzyme decreased linearly upon adding FAD and disappeared at about 2 moles of FAD per mole of the apoenzyme. This suggests that both ANS and MBAS were displaced from their binding sites on the apoenzyme by FAD. The protein fluorescence spectrum of the apoenzyme had a maximum at 352 nm, which was blue-shifted by 6 nm from that of tryptophan in the buffer. Upon binding ANS or MBAS, the maximum of the protein fluorescence of the apoenzyme returned to 350 nm for the holoenzyme, and the fluorescence intensity decreased. Thus, the conformation around some tryptophan residues was affected by the binding of the dyes. When guanidine hydrochloride (GuHCl) was added to the ANS-apoenzyme complex solution, the enhanced fluorescence due to the bound ANS decreased and the emission maximum concurrently shifted to the red. Further, the maximum of the protein fluorescence of the apoenzyme shifted to the red, indicating the exposure of some tryptophan residues buried in an apolar region of the apoenzyme. Thus the binding of ANS to the apoenzyme was inhibited by protein denaturation due to GuHCL. In contrast, the holoenzyme of lipoamide dehydrogenase did not bind ANS or MBAS at all.

Amino-terminally formylated tryptophan synthase alpha-subunit produced by the trp operon cloned in a plasmid vector.

The trp operon of Escherichia coli cloned in a multicopy plasmid produced an amino-terminally formylated tryptophan synthase alpha-subunit, as well as the normal deformylated one.

Purification properties and subunit composition of pig heart lipoate acetyltransferase.

Lipoate acetyltransferase [acetyl-CoA: dihydrolipoate S-acetyl-transferase, EC 2.3.1.12], the core enzyme of the pyruvate dehydrogenase complex, has been highly purified by gel chromatography on Sepharose 6B and sucrose density gradient centrifugation in the presence of potassium iodide. The native enzyme has a sedimentation coefficient (S020,W) of 26.7S and a diffusion coefficient (D020,W) of 1.25 x 10(-7) cm2.-sec-1. The weight-average molecular weight was estimated to be 1.8 million from the sedimentation equilibrium data. The content of right-handed alpha helix in the enzyme molecule was estimated to be about 25% by optical rotatory dispersion and about 22% from the circular dichroism spectra. The enzyme was found to contain about 23 moles of protein-bound lipoic acid per mole of enzyme; some other properties are also reported. Lipoate acetyltransferase dissociated to yield a single subunit with a molecular weight of 74,000 as estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate and by gel filtration on Bio-Gel in 6 M guanidine-HCl. The molecular weight was also estimated to be 74,000 from sedimentation equilibrium data in 6 M guanidine-HCl] containing 0.1 M 2-mercaptoethanol. Evidence is presented that 1 molecule of lipoate acetyltransferase apparently consists of 24 very similar subunits, each of which contains NH2-terminal alanine. Each subunit contains 1 molecule of covalently bound lipoic acid.

Properties and subunit structure of pig heart pyruvate dehydrogenase.

Pyruvate dehydrogenase [EC 1.2.4.1] was separated from the pyruvate dehydrogenase complex and its molecular weight was estimated to be about 150,000 by sedimentation equilibrium methods. The enzyme was dissociated into two subunits (alpha and beta), with estimated molecular weights of 41,000 (alpha) and 36,000 (beta), respectively, by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The subunits were separated by phosphocellulose column chromatography and their chemical properties were examined. The subunit structure of the pyruvate dehydrogenase was assigned as alpha2beta2. The content of right-handed alpha-helix in the enzyme molecule was estimated to be about 29 and 28% by optical rotatory dispersion and by circular dichroism, respectively. The enzyme contained no thiamine-PP, and its dehydrogenase activity was completely dependent on added thiamine-PP and partially dependent on added Mg2+ and Ca2+. The Km value of pyruvate dehydrogenase for thiamine diphosphate was estimated to be 6.5 X 10(-5) M in the presence of Mg2+ or Ca2+. The enzyme showed highly specific activity for thiamine-PP dependent oxidation of both pyruvate and alpha-ketobutyrate, but it also showed some activity with alpha-ketovalerate, alpha-ketoisocaproate, and alpha-ketoisovalerate. The pyruvate dehydrogenase activity was strongly inhibited by bivalent heavy metal ions and by sulfhydryl inhibitors; and the enzyme molecule contained 27 moles of 5,5'-dithiobis(2-nitrobenzoic acid)-reactive sulfhydryl groups and a total of 36 moles of sulfhydryl groups. The inhibitory effect of p-chloromercuribenzoate was prevented by preincubating the enzyme with thiamine-PP plus pyruvate. The structure of pyruvate dehydrogenase necessary for formation of the complex is also reported.

The structure, stability, and folding process of amyloidogenic mutant human lysozyme.

The physicochemical properties of an amyloidogenic mutant human lysozyme (Ile56Thr) were examined in order to elucidate the mechanism of amyloid formation. The crystal structure of the mutant protein was the same as the wild-type structure, except that the hydroxyl group of the introduced Thr56 formed a hydrogen bond with a water molecule in the interior of the protein. The other physicochemical properties of the mutant protein in the native state were not different from those of the wild-type protein. However, the equilibrium and kinetic stabilities of the mutant protein were remarkably decreased due to the introduction of a polar residue (Thr) in the interior of the molecule. It can be concluded that the amyloid formation of the mutant human lysozyme is due to a tendency to favor (partly or/and completely) denatured structures.

Tyr-341 of the beta subunit is a major Km-determining residue of TF1-ATPase: parallel effect of its mutations on Kd(ATP) of the beta subunit and on Km(ATP) of the alpha 3 beta 3 gamma complex.

Residue Tyr-341 of the F1-ATPase beta subunit from a thermophilic Bacillus strain, PS3, was mutagenized to leucine, cysteine or alanine. Each of the mutated beta subunits was isolated and its affinity for ATP-Mg was examined by means of difference circular dichroism and differential titration calorimetry. The Kd values for ATP-Mg obtained were: beta Y341 (wild type), 0.015 mM; beta Y341L, 0.7 mM; beta Y341C and beta Y341A, > 3 mM. All the mutant beta subunits could be reconstituted into the alpha 3 beta 3 gamma complex with alpha and gamma subunits. The alpha 3 beta (mutant)3 gamma complexes hydrolyzed ATP with apparent Vmax values larger than that of the alpha 3 beta (WILD)3 gamma complex. The apparent Km values of the alpha 3 beta (mutant)3 gamma complexes increased in parallel with the Kd values for ATP-Mg of the isolated mutant beta subunits. These results indicate that residue beta Y341 is directly involved in the catalytic ATP-Mg binding and is a major Km-determining residue of F1-ATPase.

State of Tyr49 in a mutant tryptophan synthase alpha-subunit substituted at position 49.

The states of tyrosine residues in an alpha-subunit of wild-type tryptophan synthase from Escherichia coli and a mutant protein which has tyrosine in place of glutamic acid at position 49, were examined by absorption spectrum, spectrophotometric titration of phenolic hydroxyl ionization, and deuteration kinetics of phenolic hydrogen monitored by fluorescence measurement. The difference absorption spectrum of the mutant protein against the wild-type protein at pH 7.0 and 25 degrees C had peaks at 289, 282, and 276.5 nm. These positions corresponded to those in the absorption spectrum of L-tyrosine derivatives in a non-aqueous solvent at 77 K and these bands were well-resolved even at 25 degrees C as if the tyrosine residue were fixed at lower temperature. The titration curve of the mutant protein at 3 degrees C differed from that of the wild-type protein only above pH 12.7, where the difference molar extinction coefficient at 295 nm reached a plateau, indicating that ionization of Tyr49 took place at an abnormally high pH. These results suggest that Tyr49 is buried in the hydrophobic interior and fixed in a certain orientation. The deuterium exchanges of phenolic hydrogen at pH 7.0 and 13 degrees C in the wild-type and mutant proteins consisted of a single and two first order processes, respectively, all three having smaller rate constants than that of free tyrosine, indicating that these tyrosine residues are buried. It is concluded that Tyr49 in the mutant protein is not on the surface of the molecule.

X-ray crystalline structures of pyrrolidone carboxyl peptidase from a hyperthermophile, Pyrococcus furiosus, and its cys-free mutant.

In order to elucidate the mechanism of the thermostability of proteins from hyperthermophiles, X-ray crystalline structures of pyrrolidone carboxyl peptidase from a hyperthermophile, Pyrococcus furiosus (PfPCP), and its mutant protein with Ser substituted at Cys142 and Cys188 were determined at 2.2 and 2.7 A resolution, respectively. The obtained structures were compared with those previously reported for pyrrolidone carboxyl peptidases from a hyperthermophilie, Thermococcus litoralis (TlPCP), and from a mesophile, Bacillus amyloliquefaciens (BaPCP). The PfPCP structure is a tetramer of four identical subunits similar to that of the TlPCP and BaPCP. The largest structural changes among the three PCPs were detected in the C-terminal protrusion, which interacts with that of another subunit. A comparison of the three structures indicated that the high stability of PfPCP is caused by increases in hydrophobic interactions and hydrogen bonds, the formation of an intersubunit ion-pair network, and improvement to an ideal conformation. On the basis of the structures of the three proteins, it can be concluded that PfPCP does not have any special factors responsible for its extremely high stability and that the conformational structure of PfPCP is superior in its combination of positive and negative stabilizing factors compared with BaPCP.

Hepatosplenic gamma delta T-cell lymphoma associated with hepatitis B virus infection.

Hepatitis B virus (HBV) infection has been implicated in the development of hepatocellular and hematopoietic malignancies. We describe a patient with chronic hepatitis B who developed hepatosplenic gamma delta T-cell lymphoma. A 45-year-old woman presented with marked hepatosplenomegaly and hepatic failure during the course of chronic hepatitis B. Peripheral blood examination revealed 57% abnormal lymphoid cells which expressed the gamma delta T-cell receptor. The cytogenetic analysis of tumor cells showed an abnormal karyotype; 47, XX, -13, +2mar in all 20 metaphases examined. A clonal rearrangement of the T-cell receptor genes was demonstrated by Southern blot analysis, showing monoclonal expansion of tumor cells. A liver biopsy specimen showed fibrosis of the portal areas and sinusoidal infiltration of tumor cells. HBV infection was documented by the presence of IgG anti-HBc and anti-HBs antibodies in serum. Although HBV-DNA was not detected in tumor cells by polymerase chain reaction analysis, there is a possibility that proliferation of gamma delta T cells in response to HBV infection played a role in the pathogenesis of hepatosplenic gamma delta T-cell lymphoma.

Effects of amino acid substitution on the physicochemical properties of artificial proteins with random sequences.

Physicochemical properties of four random proteins, each consisting of about 150 amino acid residues with different sequence identity, were compared to know the correlation between the physicochemical properties and its sequence. The results showed that the extent of the sequence alterations correlated well with the extent of differences in CD spectra, roughly with those in pH-solubility profiles and sedimentation velocity, and not with that in the binding of a hydrophobic fluorescent dye (ANS). Therefore, proteins with similar sequences can have different physicochemical properties, indicating that the extent of mutational effects varies in response to the sequence being altered. This warrants the evolution of a protein in a sequence-specific manner.

[A case of breast cancer with pleural metastasis responding to administration of intrapleural adriamycin and systemic chemotherapy with FEMP].

A 51-year-old woman with breast cancer metastatic to the pleura was treated with intrapleural instillation of adriamycin after thoracentesis, and systemic administration of FEMP, with complete response for more than 11 months. She underwent radical mastectomy and bilateral oophorectomy for advanced breast cancer, and 18 months later presented radiological evidence of pleural fluid contralateral to the affected breast. Initially, thoracentesis for symptomatic relief was done with a positive cytology in the pleural effusion. Intrapleural instillation of adriamycin was followed by partial resolution, and administration of FEMP achieved complete disappearance of both pleural metastasis and pleural effusion.

[A case of portal hypertension with massive gastrointestinal bleeding from ileal varices].

A rare case of massive gastrointestinal bleeding from ileal varices was reported. On December 28, 1982, a 49-year-old male was admitted to Kurashiki Central Hospital because of massive gastrointestinal bleeding. Eleven years ago, the patient underwent an emergency operation for rupture of esophageal varices. The venous phase of selective superior mesenteric and celiac angiography showed mesenteric varices, but no definite bleeding point was noted. Endoscopy revealed esophageal varices, but no area of bleeding was encountered. Because of frequently repeated hemorrhages, laparotomy was performed. A large vein was found on the surface of the ileal wall, through an adhesion to the lateral pelvic wall. Partial resection of ileum was performed. Histological examination of the resected small bowel demonstrated ruptured submucosal varices. The postoperative course was uneventful and he was discharged on the 40th postoperative day. It is stressed that this disease should be considered as a differential diagnosis for a patient with lower gastrointestinal bleeding and portal hypertension.