Functional analysis of three amino acid residues of purR repressor, Trpl47, Gln-218 and Gln-292 inSalmonella typhimurium

The amber mutation sites of 6 purR(am) mutants were determined by cloning and DNA sequencing. The results showed that the mutations were distributed at three different sites in PurR coding region, G⁷²¹(→A), C⁹³³(→T) and C¹¹⁵⁵(→T), which respectively turn Trp-147,Gln-218 and Gln-292 of PurR into TAG terminal codon. To determine the effect of the three amino acid residues on regulatory function of PurR protein 5 different kinds of tRNA suppressor genes, Su3, Su4, Su6, Su7 and Su9 were used for creating the PurR protein variants with single amino acid substitution. The results indicated that Cys, Glu, Gly, His and Arg which substituted Trp-147 respectively all could not recover the regulation function of PurR. It confirmed that Trp-147 is a critical amino acid for the PurR function. Gln-292 substituted respectively by the same amino acids also could not recover the PurR function, demonstrating that Gln-292 is also an important amino acid residue in PurR.     

Functional analysis of three amino acid residues of purR repressor, Trpl47, Gln-218 and Gln-292 in Salmonella typhimurium

The amber mutation sites of 6 purR(am) mutants were determined by cloning and DNA sequencing. The results showed that the mutations were distributed at three different sites in PurR coding region, G⁷²¹(→A), C⁹³³(→T) and C¹¹⁵⁵(→T), which respectively turn Trp-147, Gln-218 and Gln-292 of PurR into TAG terminal codon. To determine the effect of the three amino acid residues on regulatory function of PurR protein 5 different kinds of tRNA suppressor genes, Su3, Su4, Su6, Su7 and Su9 were used for creating the PurR protein variants with single amino acid substitution. The results indicated that Cys, Glu, Gly, His and Arg which substituted Trp-147 respectively all could not recover the regulation function of PurR. It confirmed that Trp-147 is a critical amino acid for the PurR function. Gln-292 substituted respectively by the same amino acids also could not recover the PurR function, demonstrating that Gln-292 is also an important amino acid residue in PurR.     

鼠伤寒沙门氏菌嘌呤生物合成的调控研究X.purR琥珀突变体(am)的分离和氨基酸取代的初步研究

以超阻遏突变体３—１８为出发株,采用以乳糖为唯一碳源的ＮＣＥ平板的方法分离到４３９ 株调节突变体。通过转导引入ｔＲＮＡ抑制基因从中检测到 １１株 ｐｕｒＲ（ａｍ）候选株。共转导分 析证明,这些突变株的琥珀浪突变均发生在ｐｕｒＲ上。用 ｓｕｐＤ． ｓｕｐＥ和 ｓｕｐＦ分别对上述各ａｍｂｅｒ 突变体作了氨基酸取代实验,初步结果表明：同一氨基酸对ｐｕｒＲ不同位点（ａｍ）的氨基酸取 代,对ＰｕｒＲ调节功能有不同程度的影响。不同氨基酸（３种）对ｐｕｒＲ同一位点（ａｍ）的氨基酸取 代,对其调节功能的影响也存在差异。     

Mutagenic analysis of the a subunit of the F1F0 ATP synthase in Escherichia coli: Gln-252 through Tyr-263.

The a subunit of F1F0 ATP synthase contains a highly conserved region near its carboxyl terminus which is thought to be important in proton translocation. Cassette site-directed mutagenesis was used to study the roles of four conserved amino acids Gln-252, Phe-256, Leu-259, and Tyr-263. Substitution of basic amino acids at each of these four sites resulted in marked decreases in enzyme function. Cells carrying a subunit mutations Gln-252-->Lys, Phe-256-->Arg, Leu-259-->Arg, and Tyr-263-->Arg all displayed growth characteristics suggesting substantial loss of ATP synthase function. Studies of both ATP-driven proton pumping and proton permeability of stripped membranes indicated that proton translocation through F0 was affected by the mutations. Other mutations, such as the Phe-256-->Asp mutation, also resulted in reduced enzyme activity. However, more conservative amino acid substitutions generated at these same four positions produced minimal losses of F1F0 ATP synthase. The effects of mutations and, hence, the relative importance of the amino acids for enzyme function appeared to decrease with proximity to the carboxyl terminus of the a subunit. The data are most consistent with the hypothesis that the region between Gln-252 and Tyr-263 of the a subunit has an important structural role in F1F0 ATP synthase.     

Conformational changes in subdomain 2 of G-actin: fluorescence probing by dansyl ethylenediamine attached to Gln-41.

Gln-41 on G-actin was specifically labeled with a fluorescent probe, dansyl ethylenediamine (DED), via transglutaminase reaction to explore the conformational changes in subdomain 2 of actin. Replacement of Ca2+ with Mg2+ and ATP with ADP on G-actin produced large changes in the emission properties of DED. These substitutions resulted in blue shifts in the wavelength of maximum emission and increases in DED fluorescence. Excitation of labeled actin at 295 nm revealed energy transfer from tryptophans to DED. Structure considerations and Cu2+ quenching experiments suggested that Trp-79 and/or Trp-86 serves as energy donors to DED. Energy transfer from these residues to DED on Gln-41 increased with the replacement of Ca2+ with Mg2+ and ATP with ADP. Polymerization of Mg-G-actin with MgCl2 resulted in much smaller changes in DED fluorescence than divalent cation substitution. This suggests that the conformation of loop 38-52 on actin is primed for the polymerization reaction by the substitution of Ca2+ with Mg2+ on G-actin.     

Functional analysis of Gln-237 mutants of HhaI methyltransferase.

When the HhaI (cytosine-5) methyltransferase (M.HhaI) binds DNA it causes the target cytosine to be flipped 180 degrees out of the helix. The space becomes occupied by two amino acids, Ser-87 and Gln-237, which enter the helix from opposite sides and form a hydrogen bond to each other. Gln-237 may be involved in specific sequence recognition since it forms three hydrogen bonds to the orphan guanosine, which is the partner of the target cytosine. We have prepared all 19 mutants of Gln-237 and tested their biochemical properties. We find that mutations of this residue greatly affect the stability of the M.HhaI-DNA complex without affecting the enzyme's specificity for the target sequence. Surprisingly, all mutants retain detectable levels of enzymatic activity.     

Domain wise docking analyses of the modular chitin binding protein CBP50 from Bacillus thuringiensis serovar konkukian S4

This paper presents an in silico characterization of the chitin binding protein CBP50 from B. thuringiensis serovar konkukian S4 through homology modeling and molecular docking. The CBP50 has shown a modular structure containing an N-terminal CBM33 domain, two consecutive fibronectin-III (Fn-III) like domains and a C-terminal CBM5 domain. The protein presented a unique modular structure which could not be modeled using ordinary procedures. So, domain wise modeling using MODELLER and docking analyses using Autodock Vina were performed. The best conformation for each domain was selected using standard procedure. It was revealed that four amino acid residues Glu-71, Ser-74, Glu-76 and Gln-90 from N-terminal domain are involved in protein-substrate interaction. Similarly, amino acid residues Trp-20, Asn-21, Ser-23 and Val-30 of Fn-III like domains and Glu-15, Ala-17, Ser-18 and Leu-35 of C-terminal domain were involved in substrate binding. Site-directed mutagenesis of these proposed amino acid residues in future will elucidate the key amino acids involved in chitin binding activity of CBP50 protein.     

Optical detection of triplet-state magnetic resonance studies on individual tryptophan residues of serum albumin: correlation between phosphorescence and zero-field splittings

Cyanogen bromide cleavage of bovine serum albumin (BSA) yields two fragments, N (1-183) and C (184-582), containing 183 and 399 amino acid residues, respectively. Each in each fragment are characterized in this study by phosphorescence and optically detected magnetic resonance spectroscopy, and the results are compared with those of the intact albumin. Trp-134 in fragment N is located in a hydrophobic environment in the interior of the protein, as reflected by its red-shifted phosphorescence and characteristic zero-field splittings. The spectral properties of Trp-212 in fragment C suggest its location in a partially buried, inhomogeneous environment. They show great similarity to those of human serum albumin, which contains a single Trp at position 214. The Trp phosphorescence 0,0-bands of fragments C and N are fitted with Gaussian functions by computer, and their relative contributions to the phosphoresence 0,0-band of BSA are adjusted to fit the observed BSA 0,0-band. The wavelength dependence of the [D[-[E[ transition frequencies of fragments N and C is then weighted by their 0,0-band intensity, taking into account differences in spin alignment, and summed to predict the peak frequency of the [D[-[E[ band profile as a function of phosphorescence wavelength for the intact BSA. Good agreement between predicted and observed behavior of [D[-[E[ vs. wavelength for the intact protein provides strong evidence for the additivity of the phosphorescence and ODMR spectra of the individual Trp sites in BSA. We find that Trp-134 and Trp-212 have wavelength-independent and wavelength-dependent zero-field splittings, respectively.     

Perturbation of tryptophan residues by point mutations in bacteriophage T4 lysozyme studied by optical detection of triplet-state magnetic resonance spectroscopy

We have investigated perturbations of the triplet-state properties of Trp residues in bacteriophage T4 lysozyme caused by point mutations using low-temperature phosphorescence and optical detection of triplet-state magnetic resonance (ODMR) spectroscopy. Five temperature-sensitive mutants have been studied in detail. These include lysozymes with the point mutations Gln-105----Ala, Gln-105----Gly, Gln-105----Glu, Ala-146----Thr, and Trp-126----Gln. Changes in phosphorescence 0,0 band wavelength, intensity, the triplet-state zero-field splitting (ZFS), and the wavelength dependence of the ZFS were detected only from Trp-138 in each mutant. In the case of the Q105A mutation, the perturbations on Trp-138 have been ascribed to the combination of an increase in the polarizability of the environment and to the loss of hydrogen bonding of the enamine nitrogen of indole. For the Q105G mutation, we believe that Q is replaced by a solvent molecule in H bonding, leading to relatively small changes. In the Q105E mutation, the perturbation results largely from the introduction of a charged residue. In the case of the mutation A146T, the perturbation is associated with a local conformational change in which Trp-138 is shifted to a more solvent-exposed location. On the other hand, no significant spectroscopic changes in Trp-126 and Trp-158 were found in any of the mutants, suggesting that the perturbations are probably localized near Trp-138 for the mutations of positions 105 and 146. However, in the mutation W126Q, which occurs approximately 16 A away from Trp-138, significant changes of Trp-138 are detected, suggesting that the effects of this mutation are propagated over large distances.     

Mapping of the amino acids in membrane-embedded helices that interact with the retinal chromophore in bovine rhodopsin

By using a photoactivatable analog of 11-cis-retinal in rhodopsin, we have previously identified the amino acids Phe-115, Ala-117, Glu-122, Trp-126, Ser-127, and Trp-265 as major sites of cross-linking to the chromophore. To further investigate the amino acids that interact with retinal, we have now used site-directed mutagenesis to replace a variety of amino acids in the membrane-embedded helices in bovine rhodopsin, including those that were indicated by cross-linking studies. The mutant rhodopsin genes were expressed in monkey kidney cells (COS-1) and purified. The mutant proteins were studied for their spectroscopic properties and their ability to activate transducin. Substitution of the two amino acids, Trp-265 and Glu-122 by Tyr, Phe, and Ala and by Gln, Asp and Ala, respectively, resulted in blue-shifted (20-30 nm) chromophore, and substitution of Trp-265 by Ala resulted in marked reduction in the extent of chromophore regeneration. Light-dependent bleaching behavior was significantly altered in Ala-117----Phe, Trp-265----Phe, Ala, and Ala-292----Asp mutants. Transducin activation was reduced in these mutants, in particular Trp-265 mutants, as well as in Glu-122----Gln, Trp-126----Leu (Ala), Pro-267----Ala (Asn, Ser), and Tyr-268----Phe mutants. These findings indicate that Trp-265 is located close to retinal and Glu-122, Trp-126, and probably Tyr-268 are also likely to be near retinal.     

Functional validation of hydrophobic adaptation to physiological temperature in the small heat shock protein αA-crystallin

Small heat shock proteins (sHsps) maintain cellular homeostasis by preventing stress and disease-induced protein aggregation. While it is known that hydrophobicity impacts the ability of sHsps to bind aggregation-prone denaturing proteins, the complex quaternary structure of globular sHsps has made understanding the significance of specific changes in hydrophobicity difficult. Here we used recombinant protein of the lenticular sHsp α A-crystallin from six teleost fishes environmentally adapted to temperatures ranging from -2°C to 40°C to identify correlations between physiological temperature, protein stability and chaperone-like activity. Using sequence and structural modeling analysis we identified specific amino acid differences between the warm adapted zebrafish and cold adapted Antarctic toothfish that could contribute to these correlations and validated the functional consequences of three specific hydrophobicity-altering amino acid substitutions in αA-crystallin. Site directed mutagenesis of three residues in the zebrafish (V62T, C143S, T147V) confirmed that each impacts either protein stability or chaperone-like activity or both, with the V62T substitution having the greatest impact. Our results indicate a role for changing hydrophobicity in the thermal adaptation of α A-crystallin and suggest ways to produce sHsp variants with altered chaperone-like activity. These data also demonstrate that a comparative approach can provide new information about sHsp function and evolution.     

Catalytic Mechanism of S-type Phycobiliprotein Lyase: CHAPERONE-LIKE ACTION AND FUNCTIONAL AMINO ACID RESIDUES

The phycobilin:cysteine 84-phycobiliprotein lyase, CpcS1, catalyzes phycocyanobilin (PCB) and phycoerythrobilin (PEB) attachment at nearly all cysteine 82 binding sites (consensus numbering) of phycoerythrin, phycoerythrocyanin, phycocyanin, and allophycocyanin (Zhao, K. H., Su, P., Tu, J. M., Wang, X., Liu, H., Plöscher, M., Eichacker, L., Yang, B., Zhou, M., and Scheer, H. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 14300–14305). We now show that CpcS1 binds PCB and PEB rapidly with bi-exponential kinetics (38/119 and 12/8300 ms, respectively). Chromophore binding to the lyase is reversible and much faster than the spontaneous, but low fidelity chromophore addition to the apo-protein in the absence of the lyase. This indicates kinetic control by the enzyme, which then transfers the chromophore to the apo-protein in a slow (tens of minutes) but stereo- and regioselectively corrects the reaction. This mode of action is reminiscent of chaperones but does not require ATP. The amino acid residues Arg-18 and Arg-149 of the lyase are essential for chromophore attachment in vitro and in Escherichia coli, mutations of His-21, His-22, Trp-75, Trp-140, and Arg-147 result in reduced activity (<30% of wild type in vitro). Mutants R147Q and W69M were active but had reduced capacity for PCB binding; additionally, with W69M there was loss of fidelity in chromophore attachment. Imidazole is a non-competitive inhibitor, supporting a bilin-binding function of histidine. Evidence was obtained that CpcS1 also catalyzes exchange of C-β84-bound PCB in biliproteins by PEB.     

Mutational Insights into the Roles of Amino Acid Residues in Ligand Binding for Two Closely Related Family 16 Carbohydrate Binding Modules

Carbohydrate binding modules (CBMs) are specialized proteins that bind to polysaccharides and oligosaccharides. Caldanaerobius polysaccharolyticus Man5ACBM16-1/CBM16-2 bind to glucose-, mannose-, and glucose/mannose-configured substrates. The crystal structures of the two proteins represent the only examples in CBM family 16, and studies that evaluate the roles of amino acid residues in ligand binding in this family are lacking. In this study, we probed the roles of amino acids (selected based on CBM16-1/ligand co-crystal structures) on substrate binding. Two tryptophan (Trp-20 and Trp-125) and two glutamine (Gln-81 and Gln-93) residues are shown to be critical in ligand binding. Additionally, several polar residues that flank the critical residues also contribute to ligand binding. The CBM16-1 Q121E mutation increased affinity for all substrates tested, whereas the Q21G and N97R mutants exhibited decreased substrate affinity. We solved CBM/substrate co-crystal structures to elucidate the molecular basis of the increased substrate binding by CBM16-1 Q121E. The Gln-121, Gln-21, and Asn-97 residues can be manipulated to fine-tune ligand binding by the Man5A CBMs. Surprisingly, none of the eight residues investigated was absolutely conserved in CBM family 16. Thus, the critical residues in the Man5A CBMs are either not essential for substrate binding in the other members of this family or the two CBMs are evolutionarily distinct from the members available in the current protein database. Man5A is dependent on its CBMs for robust activity, and insights from this study should serve to enhance our understanding of the interdependence of its catalytic and substrate binding modules.     

Epitope peptides of influenza H3N2 virus neuraminidase gene designed by immunoinformatics

The virus surface protein neuraminidase (NA) is a main subtype-specific antigen in influenza type A viruses. Neuraminidase functions as an enzyme to break the bonds between hemagglutinin (HA) and sialic acid to release newly formed viruses from infected cells. In this study, NA genes from the H3N2 subtype virus were sequenced and NA proteins were screened for B-cell epitopes and assessed based on immunoinformatics. Based on this information, three peptides ES8, RR9, and WK7 (covering amino acid residues 221-228, 292-300, and 383-389, respectively) of the NA protein were selected and synthesized artificially. These peptides were used to immunize New Zealand rabbits subcutaneously to raise antisera. Results showed that these three peptides were capable of eliciting antibodies against H3N2 viruses in a specific and sensitive manner, detected in vitro by enzyme-linked immunosorbent assay. Furthermore, hemadsorption anti-releasing effects occurred in three antisera mixtures at a dilution of 1:40. Alignment using database software showed that amino acid residues in these three epitope peptides were substituted at specific sites in all the NAs sequenced in this study. We suggest that these NA epitope peptides might be used in conjunction with HA proteins as vaccine antigens.     

Fluorescence spectrum of barnase: contributions of three tryptophan residues and a histidine-related pH dependence

Fluorescence spectra of wild-type barnase and mutants in which tryptophan and histidine residues have been substituted have been analyzed to give the individual contributions of the three tryptophan residues. The spectrum is dominated by the contribution of Trp-35. The fluorescence intensity varies with pH according to an ionization of a pKa of 7.75. This pKa is close to that previously determined by NMR titration of the C2-H resonances of His-18 as a function of pH (Sali et al., 1989). This histidine residue is close to Trp-94. The pH dependence of the spectrum is abolished when either His-18 or Trp-94 is mutated, and so appears to be caused by the His-18/Trp-94 interaction. The spectral response of this interaction can serve as a probe of the folding pathway and of electrostatic effects within the protein. Changes in the fluorescence spectra on substitution of Trp-94 and His-18 suggest that there is net energy transfer from Trp-71 to Trp-94.     

Mutational replacements of conserved amino acid residues in the beta subunit resulted in defective assembly of H+-translocating ATPase (F0F1) in Escherichia coli

Mutant genes for the beta subunit of H+-translocating ATPase (F0F1) were cloned from Escherichia coli strains isolated in this laboratory. Determination of their nucleotide sequence revealed four missense mutations (strain KF39, Glu-41----Lys; strain KF16 and KF42, Glu-185----Lys; strain KF48, Gly-223----Asp; KF26 and 4 other strains, Ser-292----Phe). Two nonsense mutants (strain KF40, Gln-361----end; strain KF20, Gln-397----end) were also identified. Glu-41, Glu-185, and Ser-292 are conserved in the amino acid sequences of the beta subunits so far studied, and Gly-223, Gln-361, and Gln-397 are conserved in beta subunits from bacteria and mitochondria, but not in those from chloroplasts. The amounts of F1 subunits in the membranes of these strains were studied by immunochemical assay and two-dimensional gel electrophoresis. In the mutants studied, the amounts of alpha and beta subunits in the membranes were 69-21 and 46-2%, respectively, of the amounts in wild-type membranes, the amount depending on the strain. No delta and epsilon subunits were detected in membranes of a missense mutant KF16, although reduced amounts of alpha and beta subunits could be detected, suggesting that the F1 portion may not be connected to F0 through the delta and epsilon subunits. The altered residues in missense mutants or missing domains in nonsense mutants may be important for the subunit-subunit interactions or assembly of the entire complex. Genetic experiments on introduction of suppressor tRNA into strains KF40 and KF20 suggested that F1 could be active even when residue 361 or 397 was replaced by a Ser, Leu, or Tyr residue.     

Difference between amino acid residues in the metal-ligand environments of iron- and manganese-superoxide dismutases

Alignment of the amino acid sequences of the Pseudomonas ovalis and Photobacterium leiognathi iron-superoxide dismutases (Fe-SODs) with the known sequences of the manganese-superoxide dismutases (Mn-SODs) shows that both types of SOD are highly homologous (33-53% identity) and share residues for the metal coordination. The amino acid residues that form the environment of the metal ions appear to be also conserved between the Fe- and Mn-SODs, except that the Phe-84 and Gln-154 in the Mn-SODs are replaced by Tyr and Ala, respectively, in the Fe-enzymes. Since this latter residue contributes to formation of the hydrophobic metal-ligand environment through hydrogen bonding with Trp-133 and Tyr-34 in the Mn-SODs, its substitution by Ala should cause different micro environments between the metal centers of the Fe- and Mn-SODs. This difference may account for the metal specificity of both types of SODs demonstrated by previous reconstitution experiments.     

Calcium- and magnesium-dependent interactions between the C-terminus of troponin I and the N-terminal, regulatory domain of troponin C

The muscle thin filament protein troponin (Tn) regulates contraction of vertebrate striated muscle by conferring Ca2+ sensitivity to the interaction of actin and myosin. Troponin C (TnC), the Ca2+ binding subunit of Tn contains two homologous domains and four divalent cation binding sites. Two structural sites in the C-terminal domain of TnC bind either Ca2+ or Mg2+, and two regulatory sites in the N-terminal domain are specific for Ca2+. Interactions between TnC and the inhibitory Tn subunit troponin I (TnI) are of central importance to the Ca2+ regulation of muscle contraction and have been intensively studied. Much remains to be learned, however, due mainly to the lack of a three-dimensional structure for TnI. In particular, the role of amino acid residues near the C-terminus of TnI is not well understood. In this report, we prepared a mutant TnC which contains a single Trp-26 residue in the N-terminal, regulatory domain. We used fluorescence lifetime and quenching measurements to monitor Ca2+- and Mg2+-dependent changes in the environment of Trp-26 in isolated TnC, as well as in binary complexes of TnC with a Trp-free mutant of TnI or a truncated form of this mutant, TnI(1-159), which lacked the C-terminal 22 amino acid residues of TnI. We found that full-length TnI and TnI(1-159) affected Trp-26 similarly when all four binding sites of TnC were occupied by Ca2+. When the regulatory Ca2+-binding sites in the N-terminal domain of TnC were vacant and the structural sites in the C-terminal domain of were occupied by Mg2+, we found significant differences between full-length TnI and TnI(1-159) in their effect on Trp-26. Our results provide the first indica- tion that the C-terminus of TnI may play an important role in the regulation of vertebrate striated muscle through Ca2+-dependent interactions with the regula- tory domain of TnC.