Effects of carboxyl-terminal truncations on the activity and solubility of human monoamine oxidase B

Monoamine oxidase is an outer mitochondrial membrane protein that catalyzes the deamination of a number of neurotransmitters and dietary amines. To determine the roles of the carboxyl-terminal amino acids on the activity and solubility of human monoamine oxidase (MAO B), 10 sequential mutants were made with stop codons at amino acid positions 511, 504, 498, 492, 486, 481, 476, 467, 417, and 397, respectively. All truncated mutants were expressed in Sf21 insect cells using baculovirus, and the enzyme kinetic parameters were determined. Truncations at amino acid positions 511, 504, and 498 slightly decreased MAO B catalytic activity and had no significant changes on deprenyl inhibition. Further deletions up to amino acid 417 decreased the specific activity 10--100-fold without significant changes of the K(m) for phenylethylamine or dopamine or the IC(50) for deprenyl and clorgyline. The truncation mutant C397, which lacks covalently attached FAD, was inactive. Progressive carboxyl-terminal truncations up to position 481 were correlated with increased solubility of MAO B mutants. 47% of the activity of the truncated C481 was found in the 105,000 x g supernatant in the absence of detergent. However, further truncated mutants, C476, C467, and C417, remained associated with the membrane fraction. In contrast to crude homogenate, the water-soluble C481 mutant was rapidly inactivated at 4 degrees C and 37 degrees C, which indicates that the membrane environment is required for the stability of MAO B. Expression of the green fluorescent protein-MAO B C481 fusion protein revealed that this mutant was located in the cytoplasm, whereas its counterpart in MAO A, truncated mutant C490, was located on the mitochondria. These results suggest that the carboxyl-terminal amino acid residues 417--520 of MAO B are not directly involved in the active site but are required for maintaining the appropriate conformation and interaction with the outer mitochondrial membrane. The different solubilities of the various carboxyl-terminal truncation mutants indicate that the interaction of MAO B with mitochondrial membrane is not simply anchoring through the carboxyl-terminal hydrophobic tail. Further, our results suggest that the carboxyl-terminal of MAO A and B plays different roles in mitochondrial attachment.     

Very fast folding and association of a trimerization domain from bacteriophage T4 fibritin

The foldon domain constitutes the C-terminal 30 amino acid residues of the trimeric protein fibritin from bacteriophage T4. Its function is to promote folding and trimerization of fibritin. We investigated structure, stability and folding mechanism of the isolated foldon domain. The domain folds into the same trimeric beta-propeller structure as in fibritin and undergoes a two-state unfolding transition from folded trimer to unfolded monomers. The folding kinetics involve several consecutive reactions. Structure formation in the region of the single beta-hairpin of each monomer occurs on the submillisecond timescale. This reaction is followed by two consecutive association steps with rate constants of 1.9(+/-0.5)x10(6)M(-1)s(-1) and 5.4(+/-0.3)x10(6)M(-1)s(-1) at 0.58 M GdmCl, respectively. This is similar to the fastest reported bimolecular association reactions for folding of dimeric proteins. At low concentrations of protein, folding shows apparent third-order kinetics. At high concentrations of protein, the reaction becomes almost independent of protein concentrations with a half-time of about 3 ms, indicating that a first-order folding step from a partially folded trimer to the native protein (k=210 +/- 20 s(-1)) becomes rate-limiting. Our results suggest that all steps on the folding/trimerization pathway of the foldon domain are evolutionarily optimized for rapid and specific initiation of trimer formation during fibritin assembly. The results further show that beta-hairpins allow efficient and rapid protein-protein interactions during folding.     

The extended signal peptide of the trimeric autotransporter EmaA of Aggregatibacter actinomycetemcomitans modulates secretion

The extracellular matrix protein adhesin A (EmaA) of the Gram-negative bacterium Aggregatibacter actinomycetemcomitans is a fibrillar collagen adhesin belonging to the family of trimeric autotransporters. The protein forms antenna-like structures on the bacterial surface required for collagen adhesion. The 202-kDa protein monomers are proposed to be targeted and translocated across the inner membrane by a long signal peptide composed of 56 amino acids. The predicted signal peptide was functionally active in Escherichia coli and A. actinomycetemcomitans using truncated PhoA and Aae chimeric proteins, respectively. Mutations in the signal peptide were generated and characterized for PhoA activity in E. coli. A. actinomycetemcomitans strains expressing EmaA with the identical mutant signal peptides were assessed for cellular localization, surface expression, and collagen binding activity. All of the mutants impaired some aspect of EmaA structure or function. A signal peptide mutant that promoted alkaline phosphatase secretion did not allow any cell surface presentation of EmaA. A second mutant allowed for cell surface exposure but abolished protein function. A third mutant allowed for the normal localization and function of EmaA at 37°C but impaired localization at elevated temperatures. Likewise, replacement of the long EmaA signal peptide with a typical signal peptide also impaired localization above 37°C. The data suggest that the residues of the EmaA signal peptide are required for protein folding or assembly of this collagen adhesin.     

Structure function relationships in the ribosomal stalk proteins of archaebacteria.

The ribosomal L12 protein gene of Sulfolobus solfataricus (SsoL12) has been subcloned and overexpressed in Escherichia coli. Five protein L12 mutants were designed: two NH2-terminal and two COOH-terminal truncated mutants and one mutant lacking the highly charged part of the COOH-terminal region. The mutant protein genes were overexpressed in E. coli and the products purified. The amino acid composition was verified and the NH2 terminally truncated mutants were subjected to Edman degradation. The SsoL12 protein was selectively removed from entire S. solfataricus ribosomes by an ethanol wash. The remaining ribosomal core particles showed a substantial decrease in the in vitro translational activity. S. solfataricus L12 protein overexpressed in E. coli (SsoL12e) was incorporated into these ribosomal cores and restored their translational activity. Mutants lacking any part of the COOH-terminal region could be incorporated into these cores, as proven by two-dimensional polyacrylamide gels of the reconstituted particles. Mutant SsoL12 MC2 (residue 1-70) was sufficient for dimerization and incorporation into ribosomes. In contrast to the COOH terminally truncated mutants, L12 proteins lacking the 12 highly conserved NH2-terminal residues or the entire NH2-terminal region (44 amino acids) are unable to bind to ribosomes, suggesting that the SsoL12 protein binds with its NH2-terminal portion to the ribosome. None of the mutants could significantly increase the translational activity of the core particles suggesting that every deleted part of the protein was needed directly or indirectly for translational activity. Our results suggest that the COOH terminally truncated mutants were bound to ribosomes but not functional for translation. Cores preincubated with these COOH terminally truncated mutants regained activity when a second incubation with the entire overexpressed SsoL12e protein followed. This finding suggests that archaebacterial L12 proteins are freely exchanged on the ribosome.     

Purification and characterization of serine acetyltransferase from Escherichia coli partially truncated at the C-terminal region

Incubation of serine acetyltransferase (SAT) from Escherichia coli at 25 degrees C in the absence of protease inhibitors yielded a truncated SAT. The truncated SAT was much less sensitive to feedback inhibition than the wild-type SAT. Analyses of the N- and C-terminal amino acid sequences found that the truncated SAT designated as SAT delta C20 was a resultant form of the wild-type SAT cleaved between Ser 253 and Met 254, deleting 20 amino acid residues from the C-terminus. Based on these findings, we constructed a plasmid containing an altered cysE gene encoding the truncated SAT. SAT delta C20 was produced using the cells of E. coli JM70 transformed with the plasmid and purified to be homogeneous on an SDS-polyacrylamide gel. Properties of the purified SAT delta C20 were investigated in comparison with those of the wild-type SAT and Met-256-Ile mutant SAT, which was isolated by Denk and B?ck but not purified (J. Gen. Microbiol., 133, 515-525 (1987)). SAT delta C20 was composed of four identical subunits like the wild-type SAT and Met-256-Ile mutant SAT. Specific activity, optimum pH for reaction, thermal stability, and stability to reagents for SAT delta C20 were similar those for the wild-type SAT and Met-256-Ile mutant SAT. However, SAT delta C20 did not form a complex with O-acetylserine sulfhydrylase-A (OASS-A), a counterpart of the cysteine synthetase and did not reduce OASS activity in contrast to the wild-type SAT and Met-256-Ile mutant SAT.     

Interaction of the small heat shock protein with molecular mass 25 kDa (hsp25) with actin.

The interaction of heat shock protein with molecular mass 25 kDa (HSP25) and its point mutants S77D + S81D (2D mutant) and S15D + S77D + S81D (3D mutant) with intact and thermally denatured actin was analyzed by means of fluorescence spectroscopy and ultracentrifugation. Wild type HSP25 did not affect the polymerization of intact actin. The HSP25 3D mutant decreased the initial rate without affecting the maximal extent of intact actin polymerization. G-actin heated at 40-45 degrees C was partially denatured, but retained its ability to polymerize. The wild type HSP25 did not affect polymerization of this partially denatured actin. The 3D mutant of HSP25 increased the initial rate of polymerization of partially denatured actin. Heating at more than 55 degrees C induced complete denaturation of G-actin. Completely denatured G-actin cannot polymerize, but it aggregates at increased ionic strength. HSP25 and especially its 2D and 3D mutants effectively prevent salt-induced aggregation of completely denatured actin. It is concluded that the interaction of HSP25 with actin depends on the state of both actin and HSP25. HSP25 predominantly acts as a chaperone and preferentially interacts with thermally unfolded actin, preventing the formation of insoluble aggregates.     

Site-directed mutagenesis of the calcium-binding site of α-amylase of <Emphasis Type="Italic">Bacillus licheniformis</Emphasis>

Amylases that are active under acidic conditions (pH <6), at higher temperatures (>70 degrees C) and have less reliance on Ca(2+) are required for starch hydrolysis. The alpha-amylase gene of Bacillus licheniformis MTCC 6598 was cloned and expressed in Escherichia coli BL21. The calcium-binding site spanning amino acid residues from 104 to 200 in the loop regions of domain B and D430 in domain C of amylase were changed by site-directed mutagenesis and the resultant mutant amylases were analyzed. Calcium-binding residues, N104, D161, D183, D200 and D430, were replaced with D104 and N161, N183, N200 and N430, respectively. Mutant amylase with N104D had a slightly decreased activity at 30 degrees C but a significantly improved specific activity at pH 5 and 70 degrees C, which is desirable character for a food enzyme. The amylase mutants with D183N or D200N lost all activity while the mutant amylase with D161N retained its activity at 30 degrees C but had significantly less activity at 70 degrees C. On the other hand, the activity of the mutant amylase with D430N was not changed at 30 degrees C but had an improved activity at 70 degrees C.     

Mutations in the tether region of the iron-sulfur protein affect the activity and assembly of the cytochrome bc(1) complex of yeast mitochondria

Resolution of the crystal structure of the mitochondrial cytochrome bc(1) complex has indicated that the extra-membranous extrinsic domain of the iron-sulfur protein containing the 2Fe2S cluster is connected by a tether to the transmembrane helix that anchors the iron-sulfur protein to the complex. To investigate the role of this tether in the cytochrome bc(1) complex, we have mutated the conserved amino acid residues Ala-86, Ala-90, Ala-92, Lys-93 and Glu-95 and constructed deletion mutants DeltaVLA(88-90) and DeltaAMA(90-92) and an insertion mutant I87AAA88 in the iron-sulfur protein of the yeast, Saccharomyces cerevisiae. In cells grown at 30 degrees C, enzymatic activities of the bc(1) complex were reduced 22-56% in mutants A86L, A90I, A92C, A92R and E95R, and the deletion mutants, DeltaVLA(88-90) and DeltaAMA(90-92), while activity of the insertion mutant was reduced 90%. No loss of cytochromes b or c-c(1), detected spectrally, or the iron-sulfur protein, determined by quantitative immunoblotting, was observed in these mutants with the exception of the mutants of Ala-92 in which the loss of activity paralleled a loss in the amount of the iron-sulfur protein. EPR spectroscopy revealed no changes in the iron-sulfur cluster of mutants A86L, A90I, A92R or the deletion mutant DeltaVLA(88-90). Greater losses of both protein and activity were observed in all of the mutants of Ala-92 as well as in A90F grown at 37 degrees C. suggesting that these conserved alanine residues may be involved in maintaining the stability of the iron-sulfur protein and its assembly into the bc(1) complex. By contrast, no significant loss of iron-sulfur protein was observed in the mutants of Ala-86 in cells grown at either 30 degrees C or 37 degrees C despite the 50-70% loss of enzymatic activity suggesting that Ala-86 may play a critical role in catalysis in the bc(1) complex.     

Isolation and characterization of temperature-sensitive mutants of adenovirus type 7.

Fifty temperature-sensitive mutants, which replicate at 32 degrees C but not at 39.5 degrees C, were isolated after mutagenesis of the vaccine strain of adenovirus type 7 with hydroxylamine (mutation frequency of 9.0%) or nitrous acid (mutation frequency of 3.8%). Intratypic complementation analyses separated 46 of these mutants into seven groups. Intertypic complementation tests with temperature-sensitive mutants of adenovirus type 5 showed that the mutant in complementation group A failed to complement H5ts125 (a DNA-binding protein mutant), that mutants in group B and C did not complement adenovirus type 5 hexon mutants, and that none of the mutants was defective in fiber production. Further phenotypic characterization showed that at the nonpermissive temperature the mutant in group A failed to make immunologically reactive DNA-binding protein, mutants in groups B and C were defective in transport of trimeric hexons to the nucleus, mutants in groups D, E, and F assembled empty capsids, and mutants in group G assembled DNA-containing capsids as well as empty capsids. The mutants of the complementation groups were physically mapped by marker rescue, and the mutations were localized between the following map coordinates: groups B and C between 50.4 and 60.2 map units (m.u.), groups D and E between 29.6 and 36.7 m.u., and group G between 36.7 and 42.0 m.u. or 44.0 and 47.0 m.u. The mutant in group A proved to be a double mutant.     

Transformation of a Fragment of β-Structural Bacteriophage T4 Adhesin to Stable α-Helical Trimer

Gene product 12 of bacteriophage T4, adhesin, serves to adhere the virus to host cells. Adhesin is a fibrous homotrimer, and a novel tertiary structure element, a -helix, is supposed to be a major structural feature of this protein. We have constructed two truncated gp12 mutants, 12N1 and 12N2, containing 221 and 135 N-terminal residues, respectively. When expressed in E. coli cells, these gp12 fragments formed labile -structural trimers. Another hybrid protein, 12FN, containing 179 N-terminal amino acid residues of gp12 fused to the C-terminal domain (31 amino acids) of T4 fibritin, was shown to have a trimeric proteolytically resistant a-helical structure. This structure is probably similar to that of fibritin, which has a triple -helical coiled-coil structure. Hence, we have demonstrated the possibility of global transformation of fibrous protein structure using fusion with a C-terminal domain that initiates trimerization.     

Domain organization, folding and stability of bacteriophage T4 fibritin, a segmented coiled-coil protein.

Fibritin is a segmented coiled-coil homotrimer of the 486-residue product of phage T4 gene wac. This protein attaches to a phage particle by the N-terminal region and forms fibrous whiskers of 530 A, which perform a chaperone function during virus assembly. The short C-terminal region has a beta-annulus-like structure. We engineered a set of fibritin deletion mutants sequentially truncated from the N-termini, and the mutants were studied by differential scanning calorimetry (DSC) and CD measurements. The analysis of DSC curves indicates that full-length fibritin exhibits three thermal-heat-absorption peaks centred at 321 K (Delta H=1390 kJ x mol trimer(-1)), at 336 K (Delta H=7600 kJ x mol trimer(-1)), and at 345 K (Delta H=515 kJ x mol trimer(-1)). These transitions were assigned to the N-terminal, segmented coiled-coil, and C-terminal functional domains, respectively. The coiled-coil region, containing 13 segments, melts co-operatively as a single domain with a mean enthalpy Delta Hres=21 kJ x mol residue(-1). The ratio of Delta HVH/Delta Hcal for the coiled-coil part of the 120-, 182-, 258- and 281-residue per monomer mutants, truncated from the N-termini, and for full-length fibritin are 0.91, 0.88, 0.42, 0.39, and 0.13, respectively. This gives an indication of the decrease of the 'all-or-none' character of the transition with increasing protein size. The deletion of the 12-residue-long loop in the 120-residue fibritin increases the thermal stability of the coiled-coil region. According to CD data, full-length fibritin and all the mutants truncated from the N-termini refold properly after heat denaturation. In contrast, fibritin XN, which is deleted for the C-terminal domain, forms aggregates inside the cell. The XN protein can be partially refolded by dilution from urea and does not refold after heat denaturation. These results confirm that the C-terminal domain is essential for correct fibritin assembly both in vivo and in vitro and acts as a foldon.     

Zinc and an N-terminal extra stretch of the ferredoxin from a thermoacidophilic archaeon stabilize the molecule at high temperature.

Ferredoxin from the thermoacidophilic archaeon Sulfolobus sp. strain 7 has a 36-residue extra domain at its N-terminus and a 67-residue core domain carrying two iron-sulfur clusters. A zinc ion is held at the interface of the two domains through tetrahedral coordination of three histidine residues (-6, -19 and -34) and one aspartic acid residue (-76) [Fujii, T., Hata, Y., Oozeki, M., Moriyama, H., Wakagi, T., Tanaka, N. & Oshima, T. (1997) Biochemistry 36, 1505-1513]. To elucidate the roles of the novel zinc ion and the extra N-terminal domain, a series of truncated mutants was constructed: G1, V12, S17, G23, L31 and V38, which lack residues 0, 11, 16, 22, 30 and 37 starting from the N-terminus, respectively. A mutant with two histidine residues each replaced by an alanine residue, H16A/H19A, was also constructed. All the mutant ferredoxins had two iron-sulfur clusters, while zinc was retained only in G1 and V12. The thermal stability of the proteins was investigated by monitoring A408; the melting temperature (Tm) was approximately 109 degrees C for the natural ferredoxin, approximately 109 degrees C for G1, 97.6 degrees C for V12, 89.0 degrees C for S17, 89.2 degrees C for G23, 89.3 degrees C for L31, 82.1 degrees C for V38, and 89.4 degrees C for H16A/H19A. Km and Vmax values of 2-oxoglutarate:ferredoxin oxidoreductase for natural ferredoxin, G1, S17 and L31 were similar, suggesting that electron-accepting activities were not affected by the deletion. The combination of CD and fluorescent spectroscopic analyses with truncated mutant S17 indicated that not only the clusters but also the secondary and tertiary structures were simultaneously degraded at a Tm around 89 degrees C. These results unequivocally demonstrate that the zinc ion and certain parts, but not all, of the extra sequence stretch in the N-terminal domain are responsible not for function but for thermal stabilization of the molecule.     

Characterization of viable mutants of polyomavirus cold sensitive for maintenance of cell transformation

We mutagenized a cloned fragment of polyoma DNA encoding portions of the middle size (MT) and large T antigens. We regenerated infectious viral genomes containing the mutagenized DNA and tested their transforming ability at 32 and 39 degrees C. We isolated three nontransforming mutants and two mutants which were cold sensitive for the maintenance of cell transformation. The nontransforming mutants contained amber termination codons in the reading frame for the MT antigen. They synthesized truncated MT antigens which lacked MT-associated protein kinase activity. The cold-sensitive mutants synthesized MT antigens indistinguishable from wild type with regard to size, stability at 32 and 39 degrees C, intracellular location, and associated protein kinase activity. One of the mutants was shown by nucleotide sequence analysis to contain a single amino acid change in the MT antigen, located two residues upstream from the C-terminal hydrophobic region, and no changes in the large T antigen. The other mutant contained two amino acid changes in the MT antigen and two amino acid changes in the large T antigen.     

Control of ribonucleotide reductase in heat- and cold-sensitive mammalian cell-cycle mutants

Two heat-sensitive (reversibly arrested in G1 phase at 39.5 degrees C, multiplying at 33 degrees C) and two cold-sensitive (reversibly arrested in G1 phase at 33 degrees C, multiplying at 39.5 degrees C) cell-cycle mutants of the P-815-X2 murine mastocytoma line were tested for ribonucleotide reductase activity, using cells made permeable to nucleotides. After transfer of the heat-sensitive mutant cells to 39.5 degrees C, ribonucleotide reductase activity, similar to thymidine kinase (Schneider, E., Müller, B. and Schindler, R. (1983) Biochim. Biophys. Acta 741, 77-85), but unlike DNA polymerase alpha (Schneider, E., Müller, B. and Schindler, R. (1985) Biochim. Biophys. Acta 825, 375-383), decreased rapidly and in parallel with numbers of cells in S phase, whereas in the cold-sensitive mutant cells brought to 33 degrees C, ribonucleotide reductase activity decreased approx. 8 h later than numbers of DNA-synthesizing cells. When arrested heat- or cold-sensitive mutant cells were returned to the permissive temperature, ribonucleotide reductase activities, similar to DNA polymerase alpha and to thymidine kinase in heat-sensitive mutants, increased essentially in parallel with reentry of cells into S phase, whereas the increase in thymidine kinase activity in the cold-sensitive mutants was previously shown to occur approx. one cell-cycle time later. This indicates that ribonucleotide reductase and thymidine kinase are coordinately expressed in the heat-sensitive, but independently regulated in the cold-sensitive mutants.     

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

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

Protein synthesis defects in temperature-sensitive mutants of Escherichia coli with altered ribosomal proteins

The ribosomes from four temperature-sensitive mutants of Escherichia coli have been examined for defects in cell-free protein synthesis. The mutants examined had alterations in ribosomal proteins S10, S15, or L22 (two strains). Ribosomes from each mutant showed a reduced activity in the translation of phage MS2 RNA at 44 degrees C and were more rapidly inactivated by heating at this temperature compared to control ribosomes. Ribosomal subunits from three of the mutants demonstrated a partial or complete inability to reassociate at 44 degrees C. 70-S ribosomes from two strains showed a reducton in messenger RNA binding. tRNA binding to the 30 S subunit was reduced in the strains with altered 30-S proteins and binding to the 50 S subunit was affected in the mutants with a change in 50 S protein L22. The relation between ribosomal protein structure and function in protein synthesis in these mutants is discussed.     

Roles of two surface residues near the access channel in the substrate recognition by cytochrome P450cam

Detailed stopped-flow kinetics of binding of 1R-camphor to cytochrome P450cam has been studied at different temperatures for the wild type as well as for two site specific mutants T192E and S190D of the enzyme, where the surface exposed Threonine and Serine residues were mutated by acidic amino acids. The near-UV and visible circular dichroism spectra as well as the intrinsic fluorescence spectra of the WT and mutant enzymes showed that the mutation of the enzyme did not affect the tertiary structure of the enzyme over the temperature range 4-30 degrees C. The S190D mutation did not show any significant change in the rate constants of the substrate association while they were much lower in the T192E mutant compared to the WT enzyme. The activation energies for substrate association and dissociation processes were determined from the analysis of temperature dependence of the rate constants by the Arrhenius equation over the temperature range 4-19 degrees C. The activation energy for the substrate association was found to be significantly higher in the T192E mutant compared to the S190D mutant or the WT enzyme. The results showed that the Threonine 192 that resides on the F-G loop and directed towards the putative substrate access channel of the enzyme, plays an important role in recognition of the substrate at the surface of the enzyme. These results showed that though the active site of the enzyme resides deep inside the protein matrix, the substrate is recognized at the surface of the enzyme and directed towards the active site through the access channel.     

Identification of a discrete intermediate in the assembly/disassembly of physalis mottle tymovirus through mutational analysis.

Assembly intermediates of icosahedral viruses are usually transient and are difficult to identify. In the present investigation, site-specific and deletion mutants of the coat protein gene of physalis mottle tymovirus (PhMV) were used to delineate the role of specific amino acid residues in the assembly of the virus and to identify intermediates in this process. N-terminal 30, 34, 35 and 39 amino acid deletion and single C-terminal (N188) deletion mutant proteins of PhMV were expressed in Escherichia coli. Site-specific mutants H69A, C75A, W96A, D144N, D144N-T151A, K143E and N188A were also constructed and expressed. The mutant protein lacking 30 amino acid residues from the N terminus self-assembled to T=3 particles in vivo while deletions of 34, 35 and 39 amino acid residues resulted in the mutant proteins that were insoluble. Interestingly, the coat protein (pR PhCP) expressed using pRSET B vector with an additional 41 amino acid residues at the N terminus also assembled into T=3 particles that were more compact and had a smaller diameter. These results demonstrate that the amino-terminal segment is flexible and either the deletion or addition of amino acid residues at the N terminus does not affect T=3 capsid assembly. In contrast, the deletion of even a single residue from the C terminus (PhN188Delta1) resulted in capsids that were unstable. These capsids disassembled to a discrete intermediate with a sedimentation coefficent of 19.4 S. However, the replacement of C-terminal asparagine 188 by alanine led to the formation of stable capsids. The C75A and D144N mutant proteins also assembled into capsids that were as stable as the pR PhCP, suggesting that C75 and D144 are not crucial for the T=3 capsid assembly. pR PhW96A and pR PhD144N-T151A mutant proteins failed to form capsids and were present as heterogeneous aggregates. Interestingly, the pR PhK143E mutant protein behaved in a manner similar to the C-terminal deletion protein in forming unstable capsids. The intermediate with an s value of 19.4 S was the major assembly product of pR PhH69A mutant protein and could correspond to a 30mer. It is possible that the assembly or disassembly is arrested at a similar stage in pR PhN188Delta1, pR PhH69A and pR PhK143E mutant proteins.     

Reconstitution of denatured E. coli alkaline phosphatase with E. coli ribosome.

E. coli alkaline phosphatase was denatured by physical/chemical means. In vitro reconstitution of this denatured enzyme was assisted by 70S E. coli ribosome, as shown by the recovery of its catalytic competence. Almost total recovery of activity of the totally inactivated enzyme was obtained in presence of equimolar concentration of 70S ribosome at 50 degrees C.