Polyproline II helical structure in protein unfolded states: lysine peptides revisited

The left-handed polyproline II (PPII) helix gives rise to a circular dichroism spectrum that is remarkably similar to that of unfolded proteins. This similarity has been used as the basis for the hypothesis that unfolded proteins possess considerable PPII helical content. It has long been known that homopolymers of lysine adopt the PPII helical conformation at neutral pH, presumably a result of electrostatic repulsion between side chains. It is shown here that a seven-residue lysine peptide also adopts the PPII conformation. In contrast with homopolymers of lysine, this short peptide is shown to retain PPII helical character under conditions in which side-chain charges are heavily screened or even neutralized. The most plausible explanation for these observations is that the peptide backbone favors the PPII conformation to maximize favorable interactions with solvent. These data are evidence that unfolded proteins do indeed possess PPII content, indicating that the ensemble of unfolded states is significantly smaller than is commonly assumed.     

Acyl group specificity at the active site of tetrahydridipicolinate N-succinyltransferase

Tetrahydrodipicolinate N-succinyltransferase (DapD) catalyzes the succinyl-CoA-dependent acylation of L-2-amino-6-oxopimelate to 2-N-succinyl-6-oxopimelate as part of the succinylase branch of the meso-diaminopimelate/lysine biosynthetic pathway of bacteria, blue-green algae, and plants. This pathway provides meso-diaminopimelate as a building block for cell wall peptidoglycan in most bacteria, and is regarded as a target pathway for antibacterial agents. We have solved the X-ray crystal structures of DapD in ternary complexes with pimelate/succinyl-CoA and L-2-aminopimelate with the nonreactive cofactor analog, succinamide-CoA. These structures define the binding conformation of the cofactor succinyl group and its interactions with the enzyme and place its thioester carbonyl carbon in close proximity to the nucleophilic 2-amino group of the acceptor, in support of a direct attack ternary complex mechanism. The acyl group specificity differences between homologous tetrahydrodipicolinate N-acetyl- and N-succinyltransferases can be rationalized with reference to at least three amino acids that interact with or give accessible active site volume to the cofactor succinyl group. These residues account at least in part for the substrate specificity that commits metabolic intermediates to either the succinylase or acetylase branches of the meso-diaminopimelate/lysine biosynthetic pathway.     

Generation of an Escherichia coli lysA targeted deletion mutant by double cross-over recombination for potential use in a bacterial growth-based lysine assay

AIMS: To generate a stable Escherichia coli lysine auxotroph for the lysine bioavailability assay. METHODS AND RESULTS: An E. coli lysine auxotrophic strain was constructed by deleting the entire lysA gene and replacing it with a gene that confers resistance to ampicillin (bla). The linear DNA contained 50 bp homologous sequence of upstream of lysA in one end and 50 bp of downstream of lysA in the other end. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The deltalysA::bla strain exhibited a linear response to lysine supplementation and can be used for quantifying lysine.     

Conformational study of N(epsilon)-(carboxymethyl)lysine adducts of recombinant gammaC-crystallin

N -(carboxymethyl)lysine, an advanced glycation end product, is present in the human lens. The effects of CML formation on protein conformation and stability were studied using the recombinant C-crystallin as a model. Conformational change was studied by spectroscopic measurements such as fluorescence and circular dichroism. Conformational stability was determined by unfolding with heat. The results indicated that no conformational change was observed due to CML formation, but conformational stability decreased. These observations can be explained in terms of the relatively stable structure of -crystallin, especially when compared with other crystallins. The lens nucleus is rich in -crystallin and its stable conformation can assist -crystallin sustained insults and remain soluble.     

精制白喉毒素脱毒工艺的改进

为了改进精制白喉毒素脱毒工艺中存在的时间长和损失大等问题,我们考虑进行脱毒过程中加入Ｌ－赖氨酸协同脱毒的试验,并以原工艺进行比较,结果使原工艺存在的问题均得到较好的解决。     

Nonsense and missense translational suppression in plant cells mediated by tRNALys

A nuclear tRNA^Lys gene from Arabidopsis thaliana was cloned and mutated so as to express tRNAs with altered anticodons which bind to a UAG nonsense (amber) codon and to the Arg (AGG), Asn (AAC,AAT), Gln (CAG) or Glu (GAG) codons. Concomitantly, a codon in the firefly luciferase gene for a functionally important Lys was altered to an amber codon, or to Arg, Asn, Gln, Glu, Thr and Trp codons, so as to construct reporter genes reliant upon incorporation of Lys. The altered tRNA^Lys and luciferase genes were introduced into Nicotiana benthamiana protoplasts and expression of the mutated tRNAs was verified by translational suppression of the mutant firefly luciferase genes. Expression of the amber suppressor tRNA _CUA ^Lys from non-replicative vectors promoted 10–40% suppression of the luciferase nonsense reporters while expression of the amber and missense tRNA^Lys suppressor genes from a geminivirus vector capable of replication promoted 30–80% suppression of the luciferase nonsense reporter and up to 10% suppression of the luciferase missense reporters with Arg, Asn, Gln and Glu codons.     

Improper protein trafficking contributes to artemisinin sensitivity in cells lacking the KDAC Rpd3p

Lysine deacetylases (KDACs) inhibitors may have therapeutic value in anti-malarial combination therapies with artemisinin. To evaluate connections between KDACs and artemisinin, Saccharomyces cerevisiae deletion mutants in KDAC genes were assayed. Deletion of RPD3, but not other KDAC genes, resulted in strong sensitivity to artemisinin, which was also observed in sit4Δ mutants with impaired endoplasmic reticulum (ER) to Golgi protein trafficking. Decreased accumulation of the transporters Pdr5p, Fur4p, and Tat2p was observed in rpd3Δ and sit4Δ cells. The unfolded protein response is induced in rpd3Δ cells consistent with retention of proteins in the ER. Disruption of protein trafficking appears to sensitize cells to artemisinin and targeting these pathways may be useful as part of artemisinin based anti-malarial therapy.     

New surface contacts formed upon reductive lysine methylation: Improving the probability of protein crystallization

Surface lysine methylation (SLM) is a technique for improving the rate of success of protein crystallization by chemically methylating lysine residues. The exact mechanism by which SLM enhances crystallization is still not clear. To study these mechanisms, and to analyze the conditions where SLM will provide the optimal benefits for rescuing failed crystallization experiments, we compared 40 protein structures containing N,N-dimethyl-lysine (dmLys) to a nonredundant set of 18,972 nonmethylated structures from the PDB. By measuring the relative frequency of intermolecular contacts (where contacts are defined as interactions between the residues in proximity with a distance of 3.5 Å or less) of basic residues in the methylated versus nonmethylated sets, dmLys-Glu contacts are seen more frequently than Lys-Glu contacts. Based on observation of the 10 proteins with both native and methylated structures, we propose that the increased rate of contact for dmLys-Glu is due to both a slight increase in the number of amine-carboxyl H-bonds and to the formation of methyl C–H···O interactions. By comparing the relative contact frequencies of dmLys with other residues, the mechanism by which methylation of lysines improves the formation of crystal contacts appears to be similar to that of Lys to Arg mutation. Moreover, analysis of methylated structures with the surface entropy reduction (SER) prediction server suggests that in many cases SLM of predicted SER sites may contribute to improved crystallization. Thus, tools that analyze protein sequences and mark residues for SER mutation may identify proteins with good candidate sites for SLM.     

A fast and efficient method for quantitative measurement of S-adenosyl-l-methionine-dependent methyltransferase activity with protein substrates

Modification of protein residues by S-adenosyl-l-methionine (AdoMet)-dependent methyltransferases impacts an array of cellular processes. Here we describe a new approach to quantitatively measure the rate of methyl transfer that is compatible with using protein substrates. The method relies on the ability of reverse-phase resin packed at the end of a pipette tip to quickly separate unreacted AdoMet from radiolabeled protein products. Bound radiolabeled protein products are eluted directly into scintillation vials and counted. In addition to decreasing analysis time, the sensitivity of this protocol allows the determination of initial rate data. The utility of this protocol was shown by generating a Michaelis-Menten curve for the methylation of heterogeneous nuclear ribonucleoprotein K (hnRNP K) protein by human protein arginine methyltransferase 1, variant 1 (hPRMT1v1), in just over 1 h. An additional advantage of this assay is the more than 3000-fold reduction in radioactive waste over existing protocols.