Resistance exercise combined with essential amino acid supplementation improved walking ability in elderly people.

We investigated the effects of resistance exercise combined with essential amino acid supplementation on psoas major muscle (PMM) hypertrophy and walking ability in elderly individuals. Twenty-nine healthy elderly individuals were assigned to 3 groups: (1) E (exercise), (2) A3 (exercise combined with 3.0 g of essential amino acid supplementation), and (3) A6 (exercise combined with 6.0 g of essential amino acid supplementation). To evaluate walking ability, the participants underwent the following 3 types of tests: the (1) 10-meter walk (10-W), (2) 10-meter walk involving crossing of obstacles (10-W + O), and (3) 6-minute walk (6M-W) tests. The 6-month training program resulted in significant PMM hypertrophy in all groups independent of amino acid supplementation. The extent of hypertrophy in the participants who took amino acids was dose-dependent, although the differences were not significant. Groups A3 and A6 demonstrated improvements in the 10-W and 10-W + O tests, whereas no improvement was observed in group E, regardless of PMM hypertrophy. Furthermore, group A6 showed an improvement in the 6M-W test. These results suggest that our training program causes PMM hypertrophy, whereas the training program combined with essential amino acid supplementation improves walking ability.

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.

Thermal stability of pyrrolidone carboxyl peptidases from the hyperthermophilic Archaeon, Pyrococcus furiosus.

The temperature adaptation of pyrrolidone carboxyl peptidase (PCP) from a hyperthermophile, Pyrococcus furiosus (Pf PCP), was characterized in the context of an assembly form of the protein which is a homotetramer at neutral pH. The Pf PCP exhibited maximal catalytic activity at 90-95 degrees C and its activity was higher in the temperature range 30-100 degrees C than its counterpart from the mesophilic Bacillus amyloliquefaciens (BaPCP). Thermal stability was monitored by differential scanning calorimetry (DSC). Two clearly separated peaks appeared on the DSC curves for Pf PCP at alkaline and acidic pH. Using the oxidized Pf PCP and two mutant proteins (Pf C188S and Pf C142/188S), it was found that the peaks on the high and low temperature sides of the DSC curve of Pf PCP were produced by the forms with an intersubunit disulfide bridge between the two subunits and without the bridge, respectively, indicating the stabilization effect of intersubunit disulfide bridges. The denaturation temperature (Td) of Pf PCP with intersubunit disulfide bridges was higher by 53 degrees C at pH 9.0 than that of BaPCP. An analysis of the equilibrium ultracentrifugation patterns showed that the tetrameric Pf C142/188S dissociated into dimers with decreasing pH in the acidic region and became monomer subunits at pH 2.5. The heat denaturation of Pf PCP and its two Cys mutants was highly reversible in the dimeric forms, but completely irreversible in the tetrameric form. The Td of Pf C142/188S decreased as the enzyme became dissociated, but the monomeric form of the protein was still folded at pH 2.5, although BaPCP was completely denatured at acidic pH. These results indicate that subunit interaction plays an important role in stabilizing PCP from P. furiosus in addition to the intrinsic enhanced stability of its monomer.

Structural evidence for entropic contribution of salt bridge formation to a protein antigen-antibody interaction: the case of hen lysozyme-HyHEL-10 Fv complex.

A structural and thermodynamic study of the entropic contribution of salt bridge formation to the interaction between hen egg white lysozyme (HEL) and the variable domain fragment (Fv) of anti-HEL antibody, HyHEL-10, was carried out. Three Fv mutants (HD32A, HD96A, and HD32AD96A) were prepared, and the interactions between the mutant Fvs and HEL were investigated. Crystallography revealed that the overall structures of these mutant complexes were almost identical to that of wild-type Fv. Little structural changes were observed in the HD32AD96A mutant-HEL complex, and two water molecules were introduced into the mutation site, indicating that the two water molecules structurally compensated for the complete removal of the salt bridges. This result suggests that the entropic contribution of the salt bridge originates from dehydration. In the singly mutated complexes, one water molecule was also introduced into the mutated site, bridging the antigen-antibody interface. However, a local structural difference was observed in the HD32A Fv-HEL complex, and conformational changes occurred due to changes in the relative orientation of the heavy chain to the light chain upon complexation in HD96A Fv-HEL complexes. The reduced affinity of these single mutants for the antigen originates from the increase in entropy loss, indicating that these structural changes also introduced an increase in entropy loss. These results suggest that salt bridge formation makes an entropic contribution to the protein antigen-antibody interaction through reduction of entropy loss due to dehydration and structural changes.

Entropic stabilization of the tryptophan synthase alpha-subunit from a hyperthermophile, Pyrococcus furiosus. X-ray analysis and calorimetry.

The structure of the tryptophan synthase alpha-subunit from Pyrococcus furiosus was determined by x-ray analysis at 2.0-A resolution, and its stability was examined by differential scanning calorimetry. Although the structure of the tryptophan synthase alpha(2)beta(2) complex from Salmonella typhimurium has been already determined, this is the first report of the structure of the alpha-subunit alone. The alpha-subunit from P. furiosus (Pf-alpha-subunit) lacked 12 and 6 residues at the N and C termini, respectively, and one residue each in two loop regions as compared with that from S. typhimurium (St-alpha-subunit), resulting in the absence of an N-terminal helix and the shortening of a C-terminal helix. The structure of the Pf-alpha-subunit was essentially similar to that of the St-alpha-subunit in the alpha(2)beta(2) complex. The differences between both structures were discussed in connection with the higher stability of the Pf-alpha-subunit and the complex formation of the alpha- and beta-subunits. Calorimetric results indicated that the Pf-alpha-subunit has extremely high thermostability and that its higher stability is caused by an entropic effect. On the basis of structural information of both proteins, we analyzed the contributions of each stabilization factor and could conclude that hydrophobic interactions in the protein interior do not contribute to the higher stability of the Pf-alpha-subunit. Rather, the increase in ion pairs, decrease in cavity volume, and entropic effects due to shortening of the polypeptide chain play important roles in extremely high stability in Pf-alpha-subunit.

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.

The process of amyloid-like fibril formation by methionine aminopeptidase from a hyperthermophile, Pyrococcus furiosus.

Amyloid is associated with serious diseases including Alzheimer's disease and senile-systemic amyloidosis due to misfolded proteins. In the course of study of the denaturation process of methionine aminopeptidase (MAP) from the hyperthermophile P. furiosus, we found that MAP forms amyloid-like fibrils, and we then investigated the mechanism of amyloid fibril formation. The kinetic experiments on denaturation monitored by CD at 222 nm indicated that MAP in the presence of 3.37 M GuHCl at pH 3.31 changed to a conformation containing a considerable content of beta-sheet structure after the destruction of the alpha-helical structure. MAP in this beta-rich conformation was highly associated, and its stability was remarkably high: the midpoint of the GuHCl denaturation curve was 4.82 M at pH 3.0, and a thermal transition was not observed up to 125 degrees C by calorimetry. The amyloid-like fibril formation of MAP was confirmed by Congo red staining with a typical peak at 542 nm in the difference spectrum, showing a cross-beta X-ray diffraction pattern with a clear sharp reflection at 4.7 A and a characteristic unbranched fibrillar appearance with a length of about 1000 A and a diameter of about 70 A in the electron micrographs. Present results indicate that the amyloid-like form of MAP appears just after the protein is almost completely denatured, and even highly stable proteins can also form amyloid-like conformation under conditions where the denatured state of the protein is abundantly populated.

Experimental verification of the 'stability profile of mutant protein' (SPMP) data using mutant human lysozymes.

The stability profile of mutant protein (SPMP) (Ota,M., Kanaya,S. and Nishikawa,K., 1995, J. Mol. Biol., 248, 733-738) estimates the changes in conformational stability due to single amino acid substitutions using a pseudo-energy potential developed for evaluating structure-sequence compatibility in the structure prediction method, the 3D-1D compatibility evaluation. Nine mutant human lysozymes expected to significantly increase in stability from SPMP were constructed, in order to experimentally verify the reliability of SPMP. The thermodynamic parameters for denaturation and crystal structures of these mutant proteins were determined. One mutant protein was stabilized as expected, compared with the wild-type protein. However, the others were not stabilized even though the structural changes were subtle, indicating that SPMP overestimates the increase in stability or underestimates negative effects due to substitution. The stability changes in the other mutant human lysozymes previously reported were also analyzed by SPMP. The correlation of the stability changes between the experiment and prediction depended on the types of substitution: there were some correlations for proline mutants and cavity-creating mutants, but no correlation for mutants related to side-chain hydrogen bonds. The present results may indicate some additional factors that should be considered in the calculation of SPMP, suggesting that SPMP can be refined further.

The unusually slow unfolding rate causes the high stability of pyrrolidone carboxyl peptidase from a hyperthermophile, Pyrococcus furiosus: equilibrium and kinetic studies of guanidine hydrochloride-induced unfolding and refolding.

To elucidate the energetic features of the anomalously high-level stabilization of a hyperthermophile pyrrolidone carboxyl peptidase (PfPCP) from a hyperthermophilic archaeon, Pyrococcus furiosus, equilibrium and kinetic studies of the guanidine hydrochloride (GuHCl)-induced unfolding and refolding were carried out with CD measurements at 220 nm in comparison with those from the mesophile homologue (BaPCP) from Bacillus amyloliquefaciens. The mutant protein of PfPCP substituted with Ser at both Cys142 and Cys188 (PfC142/188S) was used. The GuHCl unfolding for PfC142/188S and BaPCP was reversible. It was difficult to obtain the equilibrated unfolding curve of the hyperthermophile proteins at temperatures below 50 degreesC and pH 7, because of the remarkably slow rate of the unfolding. The unfolding for PfC142/188S attained equilibrium after 7 days at 60 degreesC, resulting in the coincidence between the unfolding and refolding curves. The Gibbs energy change of unfolding, DeltaGH2O (56.6 kJ/mol), for PfC142/188S at 60 degreesC and pH 7 was dramatically higher than that (7.6 kJ/mol) for BaPCP at 40 degreesC and pH 7. The unfolding and refolding kinetics for PfC142/188S and BaPCP at both 25 and 60 degreesC at pH 7 were approximated as a single exponential. The rate constant in water (kuH2O) of the unfolding reaction for PfC142/188S (1.6 x 10(-)15 s-1) at 25 degreesC and pH 7 was drastically reduced by 7 orders of magnitude compared to that (1.5 x 10(-)8 s-1) for BaPCP, whereas the refolding rates (krH2O) in water for PfC142/188S (9.3 x 10(-)2 s-1) and BaPCP (3.6 x 10(-)1 s-1) at 25 degreesC and pH 7 were similar. These results indicate that the greater stability of the hyperthermophile PCP was characterized by the drastically slow unfolding rate.

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.

Electrostatic stabilization in methionine aminopeptidase from hyperthermophile Pyrococcus furiosus.

The thermostability of methionine aminopeptidase from a hyperthermophile P. furiosus (PfMAP) was extremely high: the denaturation temperature was 106.2 degreesC at pH 10.2. To explore the contribution of electrostatic interaction to the superior thermostability of PfMAP, the thermostability of PfMAP was examined by differential scanning calorimetry (DSC) in various salt concentrations in the acidic region far from the isoelectric point of PfMAP. (1) In 20 mM glycine buffer, the DSC curve of PfMAP exhibited a single peak. Transition temperatures (Tm) were lowered with decreasing pH from 4 to 3. The heat denaturation of PfMAP was not reversible. (2) Denaturation enthalpy (DeltaH) measured at different pHs linearly correlated with Tm up to 102 degreesC, suggesting that the denaturation heat capacity (DeltaCp) for PfMAP is constant up to 100 degreesC. DeltaCp was estimated to be 0.82 J K-1 g-1. (3) In the presence of 10-100 mM KCl at pH 3.2, two peaks appeared on the DSC curves. The first peak shifted to lower temperatures with increasing concentration of KCl and, oppositely, the second one to higher temperatures. It was found that the first and second peaks originated from the heat denaturation of the native form of PfMAP and the melting of the non-native associated form having molten globule-like structure, respectively, judged from the CD spectra and ultracentrifugation analyses. This indicates the following: first, the attractive electrostatic interaction is an important factor in stabilizing the native form of PfMAP; second, the presence of KCl stimulates the formation of the molten globule-like state of PfMAP and stabilizes it. (4) In a comparison of the sequence and crystal structure of PfMAP, which has been recently determined (1xgs.pdb), with those of MAP from Escherichia coli (EcMAP), it was predicted that the extra four short-range ion pairs less than 3 A involved in PfMAP are crucial candidates as determinants for the superior thermostability of PfMAP.

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.

High-resolution crystals of methionine aminopeptidase from Pyrococcus furiosus obtained by water-mediated transformation.

The monoclinic crystal form of methionine amino-peptidase from Pyrococcus furiosus (MAP-Pfu) has been crystallized from four different conditions. Native crystals belong to space group P2(1) with typical unit-cell dimensions a = 53.4, b = 85.1, c = 72.7 A, beta = 107.7 degrees and diffract to 2.9-4.5 A resolution. However, there is a problem of nonisomorphism among the crystals. Water-mediated transformation to low-humidity form occurs by reduction of the relative humidity of crystal environment to 79%. The unit-cell dimensions of transformed crystals are a = 51.9, b = 83.3, c = 70.3 A, beta = 105.9 degrees, and the calculated solvent content is 3.9% less than in original crystals. Transformation to low-humidity form is accompanied by 1.7 times reduction of overall temperature factors, extension of diffraction resolution up to 1.75 A, without change or reduction of crystal mosaicity, and improvement in stability to X-ray radiation. The water-mediated transformation also appears to relieve the problem of nonisomorphism among the original MAP-Pfu crystals.

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.

Thermal conversion from low- to high-activity forms of catalase I from Bacillus stearothermophilus.

Catalase I from Bacillus stearothermophilus has the interesting property of increasing its enzyme activity on heating. It was confirmed that after heating at 70 degrees C for 10 min or 65 degrees C for 20 min, almost all the enzyme molecules were converted irreversibly to the activated form. The increase in kcat from 1400 to 3930 s-1 and the decrease in Km for H2O2 from 4.4 to 2.7 mM by heat activation indicate changes in the kinetic property of the enzyme molecule. Therefore, it follows that catalase I has two active forms, a high-activity form and a low-activity form. The heat activation process followed the first-order kinetics with an activation enthalpy (DeltaH*) of 191 kJ/mol while the heat denaturation process had a DeltaH* of 545 kJ/mol. The CD spectra of the two enzyme forms had small but marked differences. The conversion of the low-activity form to the high-activity form was an endothermic process with a Tm of 56 degrees C, which is much lower than that of the heat denaturation (Tm = 76 degrees C), and the enthalpy change for the transition was only 5% of that for the denaturation. It has to be noted that the high-activity form of the enzyme was converted back to a low-activity form through the process of denaturation, refolding, and reconstitution with heme. In addition, the newly obtained low-activity form was brought to a high-activity form by heating. These results suggest that the native state of catalase I has two active conformations that are roughly the same but not identical and are separated by a high energy barrier.

Immunohistochemical expression of nm23 gene product, nucleotide diphosphate kinase, in pancreatic neoplasms.

CONCLUSION: Our findings suggest that contrary to the proposed role for the nm23 protein as a tumor metastasis suppressor, in pancreatic tumors, the nm23 protein does not play an important role as a suppressor against tumor metastasis. BACKGROUND: The nm23 gene product, nucleotide diphosphate kinase, is believed to suppress tumor metastasis. Although a number of studies on many kinds of tumors have examined the relationship between nm23 expression and metastatic potential, the antimetastatic activity of nm23 remains controversial. The expression of the nm23 protein has not been examined in pancreatic tumors, except for a few reports on pancreatic duct cell carcinomas. METHODS: We have investigated nm23 expression in pancreatic duct cell carcinomas, islet cell tumors, and ampullary carcinomas by immunohistochemical methods. RESULTS: In 73 cases of pancreatic duct cell carcinomas, the nm23 expression was increased when compared with the adjacent normal pancreatic ducts; diffuse immunostaining was detected in 21 (29%) cases, focally positive immunostaining in 47 (64%) cases, and negative immunostaining in 5 cases (7%). All five negative samples were obtained from distant metastatic regions. However, there was no significant difference in the nm23 expression between primary tumors and regional lymph node metastases. Furthermore, there was no significant correlation between nm23 expression and the prognosis of the 55 resected cases. In the 15 cases of ampullary carcinomas, all 15 tumors were positive for nm23 protein (6 diffuse and 9 focal), and the staining intensity was stronger than in normal pancreatic ducts. There was no significant difference in the nm23 expression in the primary regions between patients with and without lymph node metastasis (2 diffuse and 5 focal out of 7 patients with lymph node metastasis, and 4 diffuse and 4 focal out of 8 patients without lymph node metastasis). All 12 islet cell tumors showed strong and diffuse staining for the nm23 protein.

Equilibrium and kinetic analyses of unfolding and refolding for the conserved proline mutants of tryptophan synthase alpha subunit.

To elucidate the role of conserved proline residues of the tryptophan synthase alpha subunit from Escherichia coli in stability and folding, equilibrium and kinetic studies of the unfolding-refolding induced by guanidine hydrochloride for six mutant alpha subunits (Pro-->Ala) were carried out by peptidyl circular dichroism and aromatic fluorescence measurements at pH 7 and 25 degrees C. These results were analyzed assuming the presence of one intermediate (I) state in the denaturation process. (I) For all mutant and wild-type proteins, the Gibbs energy change (delta Gni(H2O)) in water between the native (N) and I states coincided with the difference (delta G++u(H2O)-delta G++r(H2O)) between the activation Gibbs energy changes in water for the unfolding (delta G++u(H2O) and refolding (delta G++r(H2O) reactions. This means that the early folding intermediate of the alpha subunit corresponds to the equilibrium intermediate. Delta Gni(H2O) values of all mutant proteins decreased compared with that of the wild-type protein. Gibbs energy change (delta Gid(H2O) in water between I and the denatured (D) states was not substantially affected by the substitutions. Delta G++u(H2O) and delta G++r(H2O) decreased and increased, respectively, for all mutant proteins. (2) Six conserved prolines played roles in stability and folding of the alpha subunit in a different manner: prolines 28 and 96 by stabilizing the N state and prolines 28, 96, 132, and 207 by destabilizing the I state. The contributions of prolines 57 and 62 to the stability were marginal. (3) Cis proline 28 was not the origin of the slow phase in the refolding kinetics assumed to arise from the cis-trans isomerization reaction of proline.

Calorimetric observation of a GroEL-protein binding reaction with little contribution of hydrophobic interaction.

Binding of Escherichia coli chaperonin, GroEL, to substrate proteins with non-native structure, reduced alpha-lactalbumin (rLA) and denatured pepsin, were analyzed by isothermal titration calorimetry at various temperatures in the presence of salt (0.2 M KCl). Both proteins bound to GroEL with 1:1 stoichiometry and micromolar affinity at all temperatures tested. However, thermodynamic properties of their binding to GroEL are remarkably different from each other. While heat capacity changes (DeltaCp) of rLA-GroEL binding showed large negative values, -4.19 kJ mol-1 K-1, that of denatured pepsin-GroEL binding was only -0.2 kJ mol-1 K-1. These values strongly indicate that the hydrophobic interaction is a major force of rLA-GroEL binding but not so for denatured pepsin-GroEL binding. When salt was omitted from the solution, the affinity and DeltaCp of the rLA-GroEL binding reaction were not significantly changed whereas denatured pepsin lost affinity to GroEL. Thus, in the non-native protein-GroEL binding reaction, thermodynamic properties, as well as the effect of salt, differ from protein to protein and hydrophobic interaction may not always be a major driving force.

Crystallization and preliminary X-ray analysis of methionine aminopeptidase from the hyperthermophilic bacterium Pyrococcus furiosus.

Methionine aminopeptidase (MAP) from Pyrococcus furiosus (Pfu) has been crystallized in four different forms (A, B, C and D). Form A crystals belong to space group P2(1) with unit-cell dimensions a = 54.18, b = 85.72, c = 72.84 A, beta = 108.34 degrees. Forms B, C and D belong to space group P6(2(4)) with unit-cell dimensions a = 139.1, c = 63.7 A for form B, a = 198.6, c = 243.8 A for form C, and a = 111.0, c = 125.0 A for form D. Forms A and D diffract to 2.9 A, form B diffracts to 3.5 A, and form C crystals diffract to 4.5 A. Form A contains two molecules of MAP-Pfu per asymmetric unit. The binuclear metal center positions and a non-crystallographic twofold symmetry matrix has been determined for the form A crystals.

Novel selection method for engineered antibodies using the mechanism of Fv fragment stabilization in the presence of antigen.

Although the heavy and light chain domains of some antibody variable region fragments (Fvs) readily dissociate under physiological conditions, the Fvs are stable in the presence of antigen. This 'antigen-driven Fv stabilization mechanism' was applied to the selection of clones with specificity toward target antigens. The results can be summarized as follows. (i) Some of the residues in the heavy chain complementarity determining region 2 (HCDR2) of anti-hen egg white lysozyme (HEL) monoclonal antibody HyHEL10 heavy chain variable region (VH) were randomized. (ii) The randomized VH fragments of HyHEL10 were displayed on a filamentous bacteriophage and mixed with the target antigen, before being applied to a light chain variable region (VL) which was immobilized on microtiter plates and subjected to selection by panning. (iii) After four rounds of panning, four clones that showed significant binding to human lysozyme (hL), which HyHEL10 recognized poorly, were selected from the HCDR2 library. (iv) The soluble Fv fragments selected were expressed in Escherichia coli, purified, and subjected to an inhibition assay of lysozyme enzymatic activities and an isothermal titration calorimetry. These Fv fragments had increased affinity toward hL, and thermodynamic analysis suggested that the reduced entropy loss due to binding by the replacement of residues in HCDR2 resulted in the higher hL binding activity.