Substitution Of Amino Acid Residues Research Articles

Protein Core Adaptability: Crystal Structures of the Cavity-Filling Variants of Escherichia coli RNase HI

It has generally been accepted that an increase in protein stability is proportional to the increase in hydrophobicity. When a cavity is created by large-to-small substitutions of amino acid residues in protein cores, protein stability decreases 5.3 kJ/mol per single methyl(ene) group removal. In contrast, many reported cavity-filling mutations either failed to increase stability or produced marginal increases in stability; even in successful cases, the increase in stability was much lower than expected from the cost of single methyl(ene) group removal in cavity-creating mutations. Previously it was found that some cavity-filling mutant proteins at Ala52 in E. coli RNase HI increased stability, but decreased activity and they did not increase the stability to the degree expected by the hydrophobic effect alone. The present study attempted to structurally analyze these variant proteins, and it was found that substitutions have little effect on the overall fold but cause conformational strains with the neighboring residues. The present results and literature on cavity-creating/-filling variants provide insight into protein architecture, indicating that natural protein cores are able to accommodate larger side-chain residue by substitution; in other words, excess-packing may not be chosen in natural selection.

Molecular characterization and comparative transcriptional analysis of LMW-m-type genes from wheat (Triticum aestivum L.) and Aegilops species

Twelve new LMW-GS genes were characterized from bread wheat (Triticum aestivum L.) cultivar Zhongyou 9507 and five Aegilops species by AS-PCR. These genes belong to the LMW-m type and can be classified into two subclasses designated as 1 and 2, with the latter predominant in both wheat and related wild species. Genes in the two subclasses were significantly different from each other in SNPs and InDels variations. In comparison to subclass 1, the structural features of subclass 2 differs in possessing 21 amino acid residue substitutions, two fragment deletions (each with 7 amino acid residues), and a double-residue deletion and two fragment insertions (12 and 2-5 residues). Phylogenetic analysis revealed that the two subclasses were divergent at about 6.8 MYA, earlier than the divergence of C, M, N, S(s) and U genomes. The S(s) and B genomes displayed a very close relationship, whereas the C, M, N and U genomes appeared to be related to the D genome of bread wheat. The presently characterized genes ZyLMW-m1 and ZyLMW-m2 from Zhongyou 9507 were assigned to the D genome. Moreover, these genes were expressed successfully in Escherichia coli. Their transcriptional levels during grain developmental stages detected by quantitative real-time PCR (qRT-PCR) showed that both genes started to express at 5 days post-anthesis (DPA), reaching the maximum at 14 DPA after which their expressions decreased. Furthermore, the expression level of ZyLMW-m2 genes was much higher than that of ZyLMW-m1 during all grain developmental stages, suggesting that the expression efficiency of LMW-GS genes between the two subclasses was highly discrepant.

Cetacean Toll-like receptor 4 and myeloid differentiation factor 2, and possible cetacean-specific responses against Gram-negative bacteria

Toll-like receptor 4 (TLR4) and myeloid differentiation factor 2 (MD-2) are essential for recognizing the lipopolysaccharides (LPS) of Gram-negative bacteria. We determined the sequences of cDNAs encoding TLR4 and MD-2 from cetaceans and generated three-dimensional (3D) models for a better understanding of their modes of interaction and LPS recognition. The 3D reconstructions showed that cetacean TLR4 and MD-2 formed a horseshoe-like structure comprised of parallel β-strands and a β-cup structure consisting of two anti-parallel β-sheets, respectively. The (TLR4-MD-2)(2) duplex-heterodimer was shown to form a symmetrical structure. Comparison with the interfaces of the complexes in other mammals revealed that cetacean TLR4s have some amino acid residue substitutions involved in duplex-heterodimer formation and in species variation for LPS recognition. These substitutions in the changed amino acid residues may alter the interaction among TLR4, MD-2, and LPS and modify the TLR4/MD-2 immunological responses.

Man‐6‐P‐GlcNAc recognition by domain 5 of the cation‐independent mannose 6‐phosphate receptor

The 300kDa cation‐independent mannose 6‐phosphate receptor (CI‐MPR) targets newly synthesized acid hydrolases displaying mannose 6‐phosphate (Man‐6‐P) modified glycans to lysosomes. The extracellular region of the CI‐MPR is comprised of 15 domains and contains two high and one low affinity Man‐6‐P binding sites (localized to domains 1‐3, 9, and 5 respectively) which bind through four conserved residues (Gln, Glu, Tyr, Arg). Unlike domains 1‐3 and 9, domain 5 exhibits a higher affinity for phosphodiester‐containing lysosomal enzymes than Man‐6‐P. The goal of this study is to determine the residues of domain 5 that are responsible for phosphodiester recognition. Domain 5 containing single amino acid substitutions of residues in the binding pocket were expressed in Pichia pastoris. Surface plasmon resonance analyses revealed that the Y679F mutant exhibited similar affinities to lysosomal enzymes, acid α‐glucosidase (GAA) monoester and GAA diester, as wild‐type domain 5. In contrast, Y679L displayed a 10‐fold decreased binding affinity to GAA diester and no detectable binding to GAA monoester. Furthermore, substitution at position 653 (W653K/T) resulted in no detectable binding to either ligand. Taken together, Y679 and W653 are important for the architecture of the binding pocket of domain 5 and its specificity for diester.

Rapid Identification of Protein Biomarkers of Escherichia coli O157:H7 by Matrix-Assisted Laser Desorption Ionization-Time-of-Flight−Time-of-Flight Mass Spectrometry and Top-Down Proteomics

Six protein biomarkers from two strains of Escherichia coli O157:H7 and one non-O157:H7, nonpathogenic strain of E. coli have been identified by matrix-assisted laser desorption ionization time-of-flight-time-of-flight tandem mass spectrometry (MALDI-TOF-TOF-MS/MS) and top-down proteomics. Proteins were extracted from bacterial cell lysates, ionized by MALDI, and analyzed by MS/MS. Protein biomarker ions were identified from their sequence-specific fragment ions by comparison to a database of in silico fragment ions derived from bacterial protein sequences. Web-based software, developed in-house, was used to rapidly compare the mass-to-charge (m/z) of MS/MS fragment ions to the m/z of in silico fragment ions derived from hundreds of bacterial protein sequences. A peak matching algorithm and a p-value algorithm were used to independently score and rank identifications on the basis of the number of MS/MS-in silico matches. The six proteins identified were the acid stress chaperone-like proteins, HdeA and HdeB; the cold shock protein, CspC; the YbgS (or homeobox protein); the putative stress-response protein YjbJ (or CsbD family protein); and a protein of unknown function, YahO. HdeA, HdeB, YbgS, and YahO proteins were found to be modified post-translationally with removal of an N-terminal signal peptide. Gene sequencing of hdeA, hdeB, cspC, ybgS, yahO, and yjbJ for 11 strains of E. coli O157:H7 and 7 strains of the "near-neighbor" serotype O55:H7 revealed a high degree sequence homology between these two serotypes. Although it was not possible to distinguish O157:H7 from O55:H7 from these six biomarkers, it was possible to distinguish E. coli O157:H7 from a nonpathogenic E. coli by top-down proteomics of the YahO and YbgS. In the case of the YahO protein, a single amino acid residue substitution in its sequence (resulting in a molecular weight difference of only 1 Da) was sufficient to distinguish E. coli O157:H7 from a non-O157:H7, nonpathogenic E. coli by MALDI-TOF-TOF-MS/MS, whereas this would be difficult to distinguish by MALDI-TOF-MS. Finally, a protein biomarker ion at m/z approximately 9060 observed in the MS spectra of non-O157:H7 E. coli strains but absent from MS spectra of E. coli O157:H7 strains was identified by top-down analysis to be the HdeB acid stress chaperone-like protein consistent with previous identifications by gene sequencing and bottom-up proteomics.

An enhancer peptide for membrane-disrupting antimicrobial peptides.

BackgroundNP4P is a synthetic peptide derived from a natural, non-antimicrobial peptide fragment (pro-region of nematode cecropin P4) by substitution of all acidic amino acid residues with amides (i.e., Glu → Gln, and Asp → Asn).ResultsIn the presence of NP4P, some membrane-disrupting antimicrobial peptides (ASABF-α, polymyxin B, and nisin) killed microbes at lower concentration (e.g., 10 times lower minimum bactericidal concentration for ASABF-α against Staphylococcus aureus), whereas NP4P itself was not bactericidal and did not interfere with bacterial growth at ≤ 300 μg/mL. In contrast, the activities of antimicrobial agents with a distinct mode of action (indolicidin, ampicillin, kanamycin, and enrofloxacin) were unaffected. Although the membrane-disrupting activity of NP4P was slight or undetectable, ASABF-α permeabilized S. aureus membranes with enhanced efficacy in the presence of NP4P.ConclusionsNP4P selectively enhanced the bactericidal activities of membrane-disrupting antimicrobial peptides by increasing the efficacy of membrane disruption against the cytoplasmic membrane.

The Biophysical Costs Associated With Tetrodotoxin Resistance in the Garter Snake, Thamnophis Sirtalis

Tetrodotoxin (TTX) is a potent toxin that specifically binds to voltage gated sodium channels (NaV). TTX binding physically blocks the flow of sodium ions through NaV, thereby preventing action potential generation and propagation. Populations of the garter snake, Thamnophis sirtalis, have evolved TTX resistance by substituting amino acid residues in the highly conversed domain IV P-loop of NaV1.4, allowing them to feed on tetrodotoxic newts. Different populations of the garter snake have different degrees of TTX-resistance closely related to the number of amino acid substitutions. Here, we tested the voltage dependence, kinetics, and ion selectivity of NaV1.4 containing sequences from different garter snake populations. Using Xenopus oocytes under cut-open voltage clamp, we observed significant changes in voltage dependent gating properties of the TTX resistant NaV. The most TTX-resistant channel had hyperpolarized activation midpoint (−6 mV) compared to TTX-sensitive channels. Fast inactivation of the TTX-resistant NaV had significant depolarizing shifts of 2.2 mV in mildly TTX-resistant channels, and 4.5 mV in strongly TTX-resistant channels. Depolarizing shifts in slow inactivation were also observed in TTX-resistant channels with 20 mV and 16 mV shifts in mildly resistant and strongly resistant channels, respectively. In addition, ion selectivity and permeability of TTX-resistant channels were significantly different from those of the TTX-sensitive channel. These results suggest TTX resistance comes at a cost to channel performance caused by changes in the gating properties and ion selectivity of TTX-resistant sodium channels.Supported by a NSERC Discovery Grant to PCR.

Characterization of newly emerging Newcastle disease viruses isolated during 2002–2008 in Taiwan

To elucidate the epidemiological relationships between ND outbreaks and genetic lineages, a portion of the F gene (535 bp) and the full-length HN gene (1922 bp) of recent Taiwanese NDVs isolated in 2002–2008 was amplified by reverse transcription (RT)-polymerase chain reaction (PCR) and sequenced. Only a portion of above amplified PCR products of the F and HN genes (374 and 1713 bp) and their deduced amino acid residues were compared with the other 60 NDVs retrieved from GenBank. Most (29/30) of the recent Taiwanese isolates were clustered in subgenotype VIIe while only one isolate was classified as subgenotype VIIc. All the 29 isolates of subgenotype VIIe were further subclassified and termed provisionally as sub-subgenotypes VIIe2 (13 isolates), VIIe3 (5 isolates), and VIIe4 (11 isolates). The sub-subgenotype VIIe2 isolates possessing the motif 112R-R-Q-K-R-F 117 and amino acid residue substitutions at positions 23 (L to F) and 90 (T to A) were collected during 2002–2005. The sub-subgenotype VIIe3 isolates possessing the motif 112R-R-K-K-R-F 117 and amino acid residue substitutions at positions 74 (E to G) and 75 (A to G) within epitopes and 114 (Q to K) within cleavage site of F protein were collected during 2003–2006. The sub-subgenotype VIIe4 isolates possessing the motif 112R-R-Q-K-R-F 117 and amino acid residue substitutions at positions 23 (L to F), 26 (I to T), and 90 (T to A) were collected during 2007–2008. All the NDV isolates in this study exhibited a high intra-cerebral pathogenicity index (ICPI), they were all classified as velogenic type of NDVs. The sub-subgenotype VIIe2 and VIIe4 viruses are now dominant and have been implicatd in most of the recent ND outbreaks in Taiwan. Phylogenetic analysis of these isolates revealed that they may have evolved from previously reported local strains (VIIe1). This finding is essential for improving the disease control strategies and development of vaccines for ND.

New Mutation in ALAS2 as the Cause of X-Linked Sideroblastic Anemia Responsive to Pyridoxine: Comparison of ALAS2-Defective and DMT1-Defective BFU-E Growth.

Abstract 4051Poster Board III-986Congenital sideroblastic anemias (SA) are hypochromic microcytic anemias with secondary iron overload. Local iron accumulation in mitochondria results in the formation of ringed sideroblasts in the bone marrow. Defects in heme synthesis, Fe-S cluster biogenesis and export have been reported in SA patients. X-linked sideroblastic anemia is a rare gonosomal recessive disorder, resulting from mutations in erythroid-specific delta-aminolevulinate synthase (ALAS2), the first enzyme of heme biosynthesis. It affects mostly males. Few cases of affected females, due to skewed X-inactivation pattern favoring the mutant allele have been reported. Inefficiency of ALAS2 leads to decreased heme production and ineffective erythropoiesis. We investigated a group of 4 young males (average age 28) with congenital SA. Two of the patients had family history of SA and iron overload in males corresponding with the X-linked inheritance. All the patients had hemoglobin level lower than 119 g/L, mean corpuscular volume under 71 fL, increased serum ferritin and numerous sideroblasts in their bone marrow. We performed screening of all exons and promoter region of ALAS2. One patient was negative for ALAS2 mutation, and is a candidate for SLC25A38 mutation screening which only recently has been published as the second most frequent lesion in congenital SA. In two patients we found previously published R452H and R452C mutations; a novel K156E substitution was discovered in one patient. All these three patients responded partially to high dose of pyridoxine. K156 is a conserved amino acid residue and K156E substitution is SIFT predicted as affecting the protein function (the functional study is ongoing). This mutation was not found in 40 healthy controls or 10 patients with myelodysplastic syndrome with SA (RARS, RCMD-RS). Based on our previous study with DMT-1-deficient erythroid progenitors (Priwitzerova et al. Blood 2004;103:3991-2), we also evaluated a possible erythropoietic defect of ALAS2-mutant erythroid progenitors using methylcellulose-based colony assays. We observed that the in vitro growth of all aforementioned ALAS2-mutant patient's erythroid progenitors (BFU-Es) is not affected, which is in contrast to the defective growth of DMT1-mutant erythroid progenitors. These results suggest that impaired heme synthesis is better tolerated by erythroid progenitors than general iron deficiency due to the block in erythroid iron uptake by DMT1 and that the defect in heme synthesis does not fully account for the impaired hemoglobinization and poor growth of DMT1-defective BFU-Es. These data support the role of iron in other processes involved in erythroid-colony development apart from heme synthesis. In conclusion, we present a novel K156E ALAS2 mutation leading to pyridoxine-responsive X-linked sideroblastic anemia. Using BFU-E assays we also submit that lesions in iron-dependent proteins apart from defective heme synthesis contribute to impaired erythroid colony formation in previously described DMT1-mutant erythroid progenitors.Grant support: Ministry of Health Czech Republic grants NS9935-3 and NS10281-3 and Ministry of Education, Youth and Sports program MSM 6198959205. Disclosures:No relevant conflicts of interest to declare.

Allosteric control of catalysis by the F loop of RNA polymerase

Bacterial RNA polymerases (RNAPs) undergo coordinated conformational changes during catalysis. In particular, concerted folding of the trigger loop and rearrangements of the bridge helix at the RNAP active center have been implicated in nucleotide addition and RNAP translocation. At moderate temperatures, the rate of catalysis by RNAP from thermophilic Thermus aquaticus is dramatically reduced compared with its closest mesophilic relative, Deinococcus radiodurans. Here, we show that a part of this difference is conferred by a third element, the F loop, which is adjacent to the N terminus of the bridge helix and directly contacts the folded trigger loop. Substitutions of amino acid residues in the F loop and in an adjacent segment of the bridge helix in T. aquaticus RNAP for their D. radiodurans counterparts significantly increased the rate of catalysis (up to 40-fold at 20 degrees C). A deletion in the F loop dramatically impaired the rate of nucleotide addition and pyrophosphorolysis, but it had only a moderate effect on intrinsic RNA cleavage. Streptolydigin, an antibiotic that blocks folding of the trigger loop, did not inhibit nucleotide addition by the mutant enzyme. The resistance to streptolydigin likely results from the loss of its functional target, the folding of the trigger loop, which is already impaired by the F-loop deletion. Our results demonstrate that the F loop is essential for proper folding of the trigger loop during nucleotide addition and governs the temperature adaptivity of RNAPs in different bacteria.

Molecular cloning and functional analysis of Chinese sturgeon (Acipenser sinensis) growth hormone receptor

A full length cDNA encoding the growth hormone receptor (GHR) of Chinese sturgeon was cloned in order to investigate the mechanism of growth hormone in regulating the growth of Chinese sturgeon. The open reading frame of the cloned Chinese sturgeon growth hormone receptor (csGHR) cDNA encodes a trans-membrane protein of 611 amino acids containing all the characteristic motifs of GHR. By sequence alignment, substitutions of amino acid residues highly conserved in other species were identified. Using the CHO cell culture system, the function of csGHR and the biological significance of the amino acid substitution in csGHR were examined. The promoter of serine protease inhibitor 2.1 (Spi2.1) was trans-activated upon stimulation of seabream GH (sbGH) in the csGHR-expressing CHO cells. Furthermore, CHO cells stably expressing csGHR were stimulated to proliferate by sbGH. In agreement with our previous report, Chinese sturgeon growth hormone-binding protein (csGHBP) was detected in the culture medium of CHO cells stably expressing csGHR. Mutation of Asp residue in the ligand binding motif in csGHR to Glu significantly enhanced csGHR's biological function, whereas mutation of Asp residue to Ala decreased its biological function. The results demonstrated that the cloned csGHR was of full biological function and the csGHBP could be generated through proteolysis of csGHR. These findings might provide new insights into thoroughly understanding the regulatory mechanism of Chinese sturgeon growth.

Enhancement of the organic solvent‐stability of the LST‐03 lipase by directed evolution

LST-03 lipase from an organic solvent-tolerant Pseudomonas aeruginosa LST-03 has high stability and activity in the presence of various organic solvents. In this research, enhancement of organic solvent-stability of LST-03 lipase was attempted by directed evolution. The structural gene of the LST-03 lipase was amplified by the error prone-PCR method. Organic solvent-stability of the mutated lipases was assayed by formation of a clear zone of agar which contained dimethyl sulfoxide (DMSO) and tri-n-butyrin and which overlaid a plate medium. And the organic solvent-stability was also confirmed by measuring the half-life of activity in the presence of DMSO. Four mutated enzymes were selected on the basis of their high organic solvent-stability in the presence of DMSO. The organic solvent-stabilities of mutated LST-03 lipase in the presence of various organic solvents were measured and their mutated amino acid residues were identified. The half-lives of the LST-03-R65 lipase in the presence of cyclohexane and n-decane were about 9 to 11-fold longer than those of the wild-type lipase, respectively. Some substituted amino acid residues of mutated LST-03 lipases have been located at the surface of the enzyme molecules, while some other amino acid residues have been changed from neutral to basic residues.

Mapping a diploid wheat abscisic acid 8′-hydroxylase homologue in the seed dormancy QTL region on chromosome 5Am

In Arabidopsis, two genes of abscisic acid (ABA) 8′-hydroxylase (cytochrome P450 (CYP) 707A1 and A2) have been found to play important roles in seed dormancy through the regulation of ABA content in seeds. In order to examine the role of wheat ABA 8′-hydroxylase gene in seed dormancy, a diploid wheat ABA 8′-hydroxylase gene was cloned that showed high similarity to a barley ABA8′-hydroxylase gene (HvABA8′OH-2), and the cloned gene was designated as TmABA8′OH-2. Using recombinant inbred lines derived from a cross between diploid wheat Triticum boeoticum L. (Tb) and Triticum monococcum L. (Tm), TmABA8′OH-2 has been mapped to near the centromeric region of the long arm of chromosome 5Am, where the major seed dormancy QTL has been previously found. Comparison of the deduced amino acid sequences of TmABA8′OH-2 between Tb and Tm revealed five amino acid residue substitutions. These amino acid residues have distinctly different characteristics, and one of the substitutions occurs in the highly conserved amino acid residues in CYP707A family, indicating that these substitutions may have effects on the enzyme activities. Moreover, hexaploid wheat TmABA8′OH-2 homologue revealed that the level of its expression during seed development peaks at mid-maturation stage. This resembles the expression pattern of the Arabidopsis CYP707A1, which was shown to control seed dormancy. These results imply a possibility that TmABA8′OH-2 might be involved in seed dormancy, and associated with the QTL on chromosome 5Am.

Ala Scanning of the Inhibitory Region of Cardiac Troponin I

In skeletal and cardiac muscles, troponin (Tn), which resides on the thin filament, senses a change in intracellular Ca(2+) concentration. Tn is composed of TnC, TnI, and TnT. Ca(2+) binding to the regulatory domain of TnC removes the inhibitory effect by TnI on the contraction. The inhibitory region of cardiac TnI spans from residue 138 to 149. Upon Ca(2+) activation, the inhibitory region is believed to be released from actin, thus triggering actin-activation of myosin ATPase. In this study, we created a series of Ala-substitution mutants of cTnI to delineate the functional contribution of each amino acid in the inhibitory region to myofilament regulation. We found that most of the point mutations in the inhibitory region reduced the ATPase activity in the presence of Ca(2+), which suggests the same region also acts as an activator of the ATPase. The thin filaments can also be activated by strong myosin head (S1)-actin interactions. The binding of N-ethylmaleimide-treated myosin subfragment 1 (NEM-S1) to actin filaments mimics such strong interactions. Interestingly, in the absence of Ca(2+) NEM-S1-induced activation of S1 ATPase was significantly less with the thin filaments containing TnI(T144A) than that with the wild-type TnI. However, in the presence of Ca(2+), there was little difference in the activation of ATPase activity between these preparations.

Site-directed mutagenesis of cytochrome c: Reactions with respiratory chain components and superoxide radical

Three forms of horse heart cytochrome c with specific substitutions of heme cleft surface located amino acid residues involved in specific interactions with ubiquinol:cytochrome c reductase (complex III) and cytochrome c oxidase (complex IV) were constructed, and their reactions with superoxide radical produced by NADH:ubiquinone reductase (complex I) were studied. The proteins with six (K27E/E69K/K72E/K86E/K87E/E90K and K8E/E62K/E69K/K72E/K86E/K87E) and eight (K8E/K27E/E62K/E69K/K72E/K86E/K87E/E90K) substitutions were inactive in the cytochrome c oxidase reaction, and their reduction rates by complex III were significantly lower than that seen with acetylated cytochrome c. The reduction of these modified cytochromes c under conditions where complex I generates superoxide was almost completely (about 90%) inhibited by superoxide dismutase. The genetically modified cytochromes c are useful analytical reagents for studies on superoxide generation by the mitochondrial respiratory chain. Quantitative comparison of superoxide-mediated cytochrome c reduction with hydrogen peroxide-mediated Amplex Red oxidation suggests that complex I within its native environment (submitochondrial particles) produces both superoxide (~50%) and hydrogen peroxide (~50%).

Importin 13 Mediates Nuclear Import of Histone Fold-containing Chromatin Accessibility Complex Heterodimers

The histone fold is a structural element that facilitates heterodimerization, and histone fold heterodimers play crucial roles in gene regulation. Here, we investigated the nuclear import of two human histone fold pairs, which belong to the H2A/H2B family: CHRAC-15/CHRAC-17 and p12/CHRAC-17. Our results from in vitro nuclear import assays with permeabilized cells and in vivo cotransfection experiments reveal that importin 13 facilitates nuclear import of both histone fold heterodimers. Using glutathione S-transferase pulldown experiments, we provide evidence that heterodimers are required for efficient binding of importin 13 because the monomers alone do not significantly interact. Mutational analysis shows that stepwise substitution of basic amino acid residues conserved among the histone fold subunits leads to a progressive loss of importin 13 binding and nuclear accumulation of CHRAC-15/CHRAC-17 and p12/CHRAC-17. The distribution of basic amino acid residues among the histone fold subunits essential for nuclear uptake suggests that heterodimerization of the histone fold motif-containing proteins forms an importin 13-specific binding platform.

RNA helicase module in an acetyltransferase that modifies a specific tRNA anticodon

Post-transcriptional RNA modifications in the anticodon of transfer RNAs frequently contribute to the high fidelity of protein synthesis. In eubacteria, two genome-encoded transfer RNA (tRNA) species bear the same CAU sequence as the anticodons, which are differentiated by modified cytidines at the wobble positions. The elongator tRNA(Met) accepts an acetyl moiety at the wobble base to form N(4)-acetylcytidine (ac(4)C): an inherent modification ensures precise decoding of the AUG codon by strengthening C-G base-pair interaction and concurrently preventing misreading of the near cognate AUA codon. We have determined the crystal structure of tRNA(Met) cytidine acetyltransferase (TmcA) from Escherichia coli complexed with two natural ligands, acetyl-CoA and ADP, at 2.35 A resolution. The structure unexpectedly reveals an idiosyncratic RNA helicase module fused with a GCN5-related N-acetyltransferase (GNAT) fold, which intimately cross-interact. Taken together with the biochemical evidence, we further unravelled the function of acetyl-CoA as an enzyme-activating switch, and propose that an RNA helicase motor driven by ATP hydrolysis is used to deliver the wobble base to the active centre of the GNAT domain.

A Comparison of the Anorexic Effects of Chicken, Porcine, Human and Bovine Insulin on the Central Nervous System of Chicks

The aim of the present study was to determine if some naturally-occurring substitutions of amino acid residues of insulin could act differentially within the central nervous system (CNS) of neonatal chicks to control ingestive behavior. Intracerebroventricular (ICV) administration of chicken insulin (20ng) inhibited food intake in ad libitum chicks over the 120-min experiment period. A lower dose (2ng) also shows a suppressive effect at 60min post-injection, but not at 30 and 120min. Similar to chicken insulin, a higher dose of porcine insulin (20ng) significantly inhibited food intake at 60 and 120min post-injection, but a significant difference is not detected at 30min post-injection. Treatment with human and bovine insulin had no effect in this study. Finally, we compared the effect of ICV injection of chicken and porcine insulin on food consumption in starved chicks. The results of the present study indicate that chicken insulin has a higher potent ability to depress feeding behavior in the CNS compared with porcine, human, and bovine insulin. Therefore, it seems that the ranking in order of potency of the insulins on the anorexic effect in neonatal chicks is chicken insulin>porcine insulin>human insulin>bovine insulin. It is suggested that the 8th-10th amino acids of the A-chain and/or the C-terminal amino acid of the B-chain in insulin have an important role in bioactivity in the CNS with special reference to feeding behavior.

Charge Redistribution in Adenosylribosyl Transferase Caused by Substitution of a Single Amino Acid Residue

Dielectric measurements in the frequency range 10(5)-10(8) Hz were performed on wild-type (wt) adenosylribosyl transferase and a mutant enzyme. The analysis of the dielectric relaxation curve allowed the estimation of the hydrodynamic radius and of the electric dipole moment. The first parameter remained unchanged in wt and mutant protein. The dipole moment of the mutant, however, was significantly increased. Implications on the electrostatic interactions between enzyme and substrate are discussed.

Structural insights into phenylethanolamines high-affinity binding site in NR2B from binding and molecular modeling studies

BackgroundPhenylethanolamines selectively bind to NR2B subunit-containing N-methyl-D-aspartate-subtype of ionotropic glutamate receptors and negatively modulate receptor activity. To investigate the structural and functional properties of the ifenprodil binding domain on the NR2B protein, we have purified a soluble recombinant rat NR2B protein fragment comprising the first ~400 amino acid amino-terminal domain (ATD2B) expressed in E. coli. Spectral measurements on refolded ATD2B protein demonstrated specific binding to ifenprodil. We have used site-directed mutagenesis, circular dichroism spectroscopy and molecular modeling to obtain structural information on the interactions between critical amino acid residues and ifenprodil of our soluble refolded ATD2B proteins. Ligand-induced changes in protein structure were inferred from changes in the circular dichroism spectrum, and the concentration dependence of these changes was used to determine binding constants for ifenprodil and its analogues.ResultsLigand binding of ifenprodil, RO25,6981 and haloperidol on soluble recombinant ATD2B determined from circular dichroism spectroscopy yielded low-to-high micromolar equilibrium constants which concurred with functional IC50 measurement determined in heterologously expressed NR1/NR2B receptors in Xenopus oocytes. Amino acid residue substitutions of Asp101, Ile150 and Phe176 with alanine residue within the ATD2B protein altered the recombinant protein dissociation constants for ifenprodil, mirroring the pattern of their functional phenotypes. Molecular modeling of ATD2B as a clam-shell-like structure places these critical residues near a putative ligand binding site.ConclusionWe report for the first time biochemical measurements show that the functional measurements actually reflect binding to the ATD of NR2B subunit. Insights gained from this study help advance the theory that ifenprodil is a ligand for the ATD of NR2B subunit.