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Results for “Amber codon scanning”

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6 recordsLinked to original sources

PubMed2026

Library-based, multiplexed strategy for mapping protein interaction networks via crosslinking.

BACKGROUND: Protein-protein interactions are fundamental to cellular function, yet resolving their interaction interfaces and dynamic behaviors in native biological contexts remains challenging, particularly for weak or transient interactions. Crosslinking strategies based on noncanonical amino acids offer an effective means to capture such interactions; however, traditional single-site incorporation provides limited coverage and may overlook critical interaction hotspots. RESULTS: By employing a mutagenesis library, multiple interaction partners and cross-linking sites of a target protein can be simultaneously screened in a single experiment, without prior knowledge of its precise structural or functional features, enabling effective and unbiased analysis of its interaction network. In this study, we constructed an amber codon-scanning mutagenesis library of PSMD10, facilitating independent incorporation of the photocrosslinking ncAA p-azido-phenylalanine at multiple distinct residues. This approach allowed us to systematically interrogate and precisely map potential interaction regions across the protein surface. Coupled with crosslinking mass spectrometry, we identified multiple residues involved in intermolecular interactions, as well as previously unreported interaction partners, including T2FA, TBA1C, and ATRIP. CONCLUSIONS: These findings expand our understanding of PSMD10-associated proteasome interactome, demonstrate a multiplexed strategy for in situ mapping of protein interaction interfaces with broad coverage, and offer a valuable platform for developing therapeutics that target protein-protein interactions.

Protein Interaction Mapping↗

PubMed2002

Monitoring mis-acylated tRNA suppression efficiency in mammalian cells via EGFP fluorescence recovery.

A reporter assay was developed to detect and quantify nonsense codon suppression by chemically aminoacylated tRNAs in mammalian cells. It is based on the cellular expression of the enhanced green fluorescent protein (EGFP) as a reporter for the site-specific amino acid incorporation in its sequence using an orthogonal suppressor tRNA derived from Escherichia coli. Suppression of an engineered amber codon at position 64 in the EGFP run-off transcript could be achieved by the incorporation of a leucine via an in vitro aminoacylated suppressor tRNA. Microinjection of defined amounts of mutagenized EGFP mRNA and suppressor tRNA into individual cells allowed us to accurately determine suppression efficiencies by measuring the EGFP fluorescence intensity in individual cells using laser-scanning confocal microscopy. Control experiments showed the absence of natural suppression or aminoacylation of the synthetic tRNA by endogenous aminoacyl-tRNA synthetases. This reporter assay opens the way for the optimization of essential experimental parameters for expanding the scope of the suppressor tRNA technology to different cell types.

Acylation↗

PubMed2002

Essential hydrophilic carboxyl-terminal regions including cysteine residues of the yeast stretch-activated calcium-permeable channel Mid1.

The yeast Saccharomyces cerevisiae MID1 gene encodes a stretch-activated Ca(2+)-permeable nonselective cation channel composed of 548 amino acid residues. A physiological role of the Mid1 channel is known to maintain the viability of yeast cells exposed to mating pheromone, but its structural basis remains to be clarified. To solve this problem, we identified the mutation sites of mid1 mutant alleles generated by in vivo ethyl methanesulfonate mutagenesis and found that two mid1 alleles have nonsense mutations at the codon for Trp(441), generating a truncated Mid1 protein lacking two-thirds of the intracellular carboxyl-terminal region from Asn(389) to Thr(548). In vitro random mutagenesis with hydroxylamine also showed that the carboxyl-terminal region is essential. To identify the functional portion of the carboxyl-terminal region in detail, we performed a progressive carboxyl-terminal truncation followed by functional analyses and found that the truncated protein produced from the mid1 allele bearing the amber mutation at the codon for Phe(522) (F522Am) complemented the mating pheromone-induced death phenotype of the mid1 mutant and increased its Ca(2+) uptake activity to a wild-type level, whereas N521Am did not. This result indicates that the carboxyl-terminal domain spanning from Asn(389) to Asn(521) is required for Mid1 function. Interestingly, this domain is cysteine-rich, and alanine-scanning mutagenesis revealed that seven out of 10 cysteine residues are unexchangeable. These results clearly indicate that the carboxyl-terminal domain including the cysteine residues is important for Mid1 function.

Alanine↗

PubMed2000

Substrate-binding clusters of the K+-transporting Kdp ATPase of Escherichia coli investigated by amber suppression scanning mutagenesis.

The Kdp-ATPase of Escherichia coli is a four-subunit P-type ATPase that accumulates K(+) with high affinity and specificity. Residues clustered in four regions of the KdpA subunit of Kdp were implicated as critical for K(+) binding from the analysis of mutants with reduced affinity for K(+) (Buurman, E., Kim, K.-T., and Epstein, W. (1995) J. Biol. Chem. 270, 6678-6685). K(+) binding by this pump has been analyzed in detail by site-directed mutagenesis. We have examined 83 of the 557 residues in KdpA, from 11 to 34 residues in each of four binding clusters known to affect K(+) binding. Amber mutations were constructed in a plasmid carrying the kdpFABC structural genes. Transferring these plasmids to 12 suppressor strains, each inserting a different amino acid at amber codons, created 12 different substitutions at the mutated sites. This study delineates the four clusters and confirms that they are important for K(+) affinity but have little effect on the rate of transport. At only 21 of the residues studied did at least three substitutions alter affinity for K(+), an indication that a residue is in or very near a K(+) binding site. At many residues lysine was the only substitution that altered its affinity. The effect of lysine is most likely a repulsive effect of this cationic residue on K(+) and thus reflects the effective distance between a residue and the site of binding or passage of K(+) in KdpA. Once a crystallographic structure of Kdp is available, this measure of effective distance will help identify the path of K(+) as it moves through the KdpA subunit to cross the membrane.

Adenosine Triphosphatases↗

PubMed1996

High resolution analysis of the readthrough domain of beet necrotic yellow vein virus readthrough protein: a KTER motif is important for efficient transmission of the virus by Polymyxa betae.

The 5'-terminal cistron of beet necrotic yellow vein furovirus RNA 2 encodes the 21 kDa major viral coat protein and terminates with an amber stop codon which can undergo suppression to give rise to a 75 kDa readthrough (RT) protein referred to as P75. P75 is a minor component of virions and the 54 kDa RT domain following the coat protein sequence is important both for virus assembly and transmission by the fungal vector Polymyxa betae. To better define the regions of the RT domain involved in these two steps, RNA 2 transcripts encoding different in-frame RT domain deletion mutants were tested for their ability to form virions when inoculated to plants with the other viral RNAs and to be fungus-transmitted. All deletions in the N-terminal half of the RT domain interfered with virus assembly and partially or completely inhibited fungus transmission. A 4 1 1 nucleotide deletion within the C-terminal half of the RT domain did not inhibit assembly but blocked fungus transmission of the virus. Alanine scanning mutagenesis within the aforesaid 4 1 1 nucleotide subdomain identified a peptide motif (KTER) which is important for the fungus transmission process.

Base Sequence↗

PubMed1996

Expression of the barley stripe mosaic virus RNA beta "triple gene block".

Genomic RNA beta of barley strip mosaic virus (BSMV) contains four defined open reading frames (ORFs). These include the coat protein (beta a) and a "triple gene block" consisting of the beta b, beta c, and beta d ORFs that overlap one another. Two subgenomic beta RNAs (sgRNA beta 1 and sgRNA beta 2) with sizes of 2.5 and 0.96 kb were identified in BSMV-infected protoplasts, and their transcription initiation sites were mapped to nucleotides 789 and 2327, respectively, of RNA beta by primer extension experiments. In a cell-free wheat germ translation system, genomic RNA beta served as a mRNA only for the 22-kDa coat protein, and sgRNA beta 1 directed synthesis of only the 58-kDA beta b protein. However, with sgRNA beta 2, three proteins with sizes of 14, 17, and 23 kDa were synthesized. Both the 14- and the 23-kDa proteins were recognized by the beta d antibodies in vitro and in vivo. These results demonstrated that the 14-kDa protein was encoded by the beta d ORF and suggested that the 23-kDa protein, designated beta d', is a readthrough product of the amber stop codon of the beta d ORF. Mutagenesis of sgRNA beta 2 revealed that the 17-kDa protein was a product of the beta c ORF. Expression of sgRNA beta 1 and sgRNA beta 2 was also investigated with the chloramphenicol acetyl transferase (CAT) reporter gene in protoplasts coinfected with RNAs alpha and gamma plus chimeric RNA beta derivatives containing the CAT gene in-frame with the beta b, beta c, beta d, or beta d' ORFs. Elimination of the sgRNA beta 1 promoter abolished CAT expression from the beta b-CAT chimeric RNA, and removal of the sgRNA beta 2 promoter prevented CAT expression from the beta c-CAT, beta d-CAT, and beta d'-CAT chimeric RNAs. Taken together, these results demonstrate that the BSMV coat protein is the sole translation product of the genomic RNA beta, whereas sgRNA beta 1 serves as a messenger for translation of the beta b protein, and sgRNA beta 2 functions as a messenger for translation of beta c and beta d and the newly discovered beta d' protein. Additional mutagenesis experiments indicate that beta c is translated by a leaky scanning mechanism.

Base Sequence↗