The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Dafydd D Jones - One of the best experts on this subject based on the ideXlab platform.
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structural and dynamic changes associated with beneficial engineered single Amino Acid Deletion mutations in enhanced green fluorescent protein
Acta Crystallographica Section D-biological Crystallography, 2014Co-Authors: James A J Arpino, Pierre J Rizkallah, Dafydd D JonesAbstract:Single-Amino-Acid Deletions are a common part of the natural evolutionary landscape but are rarely sampled during protein engineering owing to limited and prejudiced molecular understanding of mutations that shorten the protein backbone. Single-Amino-Acid Deletion variants of enhanced green fluorescent protein (EGFP) have been identified by directed evolution with the beneficial effect of imparting increased cellular fluorescence. Biophysical characterization revealed that increased functional protein production and not changes to the fluorescence parameters was the mechanism that was likely to be responsible. The structure EGFPD190Δ containing a Deletion within a loop revealed propagated changes only after the deleted residue. The structure of EGFPA227Δ revealed that a `flipping' mechanism was used to adjust for residue Deletion at the end of a β-strand, with Amino Acids C-terminal to the Deletion site repositioning to take the place of the deleted Amino Acid. In both variants new networks of short-range and long-range interactions are generated while maintaining the integrity of the hydrophobic core. Both Deletion variants also displayed significant local and long-range changes in dynamics, as evident by changes in B factors compared with EGFP. Rather than being detrimental, Deletion mutations can introduce beneficial structural effects through altering core protein properties, folding and dynamics, as well as function.
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investigating protein structural plasticity by surveying the consequence of an Amino Acid Deletion from tem 1 β lactamase
FEBS Letters, 2007Co-Authors: Alan M Simm, Amy Joy Baldwin, Kathy Busse, Dafydd D JonesAbstract:While the Deletion of an Amino Acid is a common mutation observed in nature, it is generally thought to be disruptive to protein structure. Using a directed evolution approach, we find that the enzyme TEM-1 β-lactamase was broadly tolerant to the Deletion mutations sampled. Circa 73% of the variants analysed retained activity towards ampicillin, with Deletion mutations observed in helices and strands as well as regions important for structure and function. Several Deletion variants had enhanced activity towards ceftazidime compared to the wild-type TEM-1 demonstrating that removal of an Amino Acid can have a beneficial outcome.
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triplet nucleotide removal at random positions in a target gene the tolerance of tem 1 β lactamase to an Amino Acid Deletion
Nucleic Acids Research, 2005Co-Authors: Dafydd D JonesAbstract:The Deletion of Amino Acids is one of the evolutionary mechanisms by which nature adapts the function of proteins. A simple method has been developed that mimics this event in vitro by introducing a Deletion of exactly three nucleotides at random positions in a target gene. The method involved the engineering of the mini-Mu transposon to introduce a recognition sequence for the restriction enzyme MlyI. The new transposon, MuDel, was capable of efficient insertion into a target DNA sequence. To determine the efficacy of the method, the bla gene that encodes the TEM-1 AŸ-lactamase was used as the target and a small library containing 22 different sequence variants was created. Of these 22 variants, 8 were identified that conferred resistance to ampicillin on Escherichia coli. Each of the TEM-1 variants possessed a distinct ampicillin minimum inhibitory concentration, ranging from 500 to >10 000 µg/ml. Sequence analysis revealed that active TEM-1 variants contained Deletions not just in loops but also helices, and included regions known to be involved in catalysis, antibiotic resistance and inhibitor binding. This new technology is transferable to most genes, permitting an extensive analysis of Deletion mutations on protein function.
Clifford H Lane - One of the best experts on this subject based on the ideXlab platform.
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Amino Acid Deletion at codon 67 and thr to gly change at codon 69 of human immunodeficiency virus type 1 reverse transcriptase confer novel drug resistance profiles
Journal of Virology, 2001Co-Authors: Tomozumi Imamichi, Michael A Murphy, Hiromi Imamichi, Clifford H LaneAbstract:The potential roles of an Amino Acid Deletion at codon 67 (Δ67) and a Thr-to-Gly change at codon 69 (T69G) in the reverse transcriptase of human immunodeficiency virus (HIV) type 1 in drug sensitivity and relative replication fitness were studied. Our results suggest that the Δ67 and T69G changes can be categorized as mutations associated with multidrug resistance. The combination of both mutations with an L74I change (Δ67+T69G/L74I) leads to a novel 3′-azido-3′-deoxythymidine resistance motif and compensates for impaired HIV replication.
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relative replication fitness of a high level 3 azido 3 deoxythymidine resistant variant of human immunodeficiency virus type 1 possessing an Amino Acid Deletion at codon 67 and a novel substitution thr gly at codon 69
Journal of Virology, 2000Co-Authors: Tomozumi Imamichi, Hiromi Imamichi, Steve C Berg, Juan C Lopez, Julia A Metcalf, Judith Falloon, Clifford H LaneAbstract:The combination of an Amino Acid Deletion at codon 67 (Δ67) and Thr-to-Gly change at codon 69 (T69G) in the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) is associated with high-level resistance to multiple RT inhibitors. To determine the relative contributions of the Δ67 and T69G mutations on viral fitness, we performed a series of studies of HIV replication using recombinant variants. A high-level 3′-azido-3′-deoxythymidine (AZT)-resistant variant containing Δ67 plus T69G/K70R/L74I/K103N/T215F/K219Q in RT replicated as efficiently as wild-type virus (Wt). In contrast, the construct without Δ67 exhibited impaired replication (23% of growth of Wt). A competitive fitness study failed to reveal any differences in replication rates between the Δ67+T69G/K70R/L74I/K103N/T215F/K219Q mutant and Wt. Evaluation of proviral DNA sequences over a 3-year period in a patient harboring the multiresistant HIV revealed that the T69G mutation emerged in the context of a D67N/K70R/T215F/K219Q mutant backbone prior to appearance of the Δ67 Deletion. To assess the impact of this stepwise accumulation of mutations on viral replication, a series of recombinant variants was constructed and analyzed for replication competence. The T69G mutation was found to confer 2′,3′-dideoxyinosine resistance at the expense of fitness. Subsequently, the development of the Δ67 Deletion led to a virus with improved replication and high-level AZT resistance.
Yusun Chang - One of the best experts on this subject based on the ideXlab platform.
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epstein barr virus latent membrane protein 1 structure and functions
Journal of Biomedical Science, 2003Co-Authors: Yusun ChangAbstract:The Epstein-Barr virus latent membrane protein (LMP) 1 is a versatile protein that has profound effects on target cells through its effect on constitutive cellular proteins, e.g. TRAFs, TRADD, RIP, JAK3, BRAM1, and p85. LMP1 can stimulate or inhibit signaling pathways, resulting in transformation of rodent fibroblast cell lines, blockade of differentiation in epithelial cells, upregulation of antiapoptotic proteins, production of cytokines, upregulation of cell surface markers, upregulation of DNA methyltransferase activity, and downregulation of cell adhesion molecules and cyclin-dependent kinases. Overall, this results in greater transformation and survival in LMP1-expressing cells. Within nasopharyngeal carcinoma biopsy tissues, a naturally occurring LMP1 variant has been identified as having a 10-Amino Acid Deletion in the C-terminus that seems to confer greater transformation potential than non-deleted LMP1. The role of LMP1 as a viral oncogene and its interaction with cellular factors are discussed.
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effect of a 10 Amino Acid Deletion on the oncogenic activity of latent membrane protein 1 of epstein barr virus
Oncogene, 1996Co-Authors: Yusun Chang, Shihtung LiuAbstract:A previous study has shown that the BNLF1 of Epstein-Barr virus (EBV), isolated from a nasopharyngeal carcinoma biopsy (BNLF1-1510), was able to transform Balb/3T3 cells. On the other hand, BNLF1 of a prototype virus B95-8 (BNLF1-958) was not transforming unless the gene was transcribed from a strong promoter. In this study, we have generated chimeric BNLF1 by exchanging the DNA fragments between BNLF1-1510 and BNLF1-958 and examined their expression and transformation ability in Balb/3T3 cells. Results showed that transformation of Balb/3T3 cells by BNLF1-1510 was not due to the excessive expression of the gene. Transfection of Balb/3T3 cells with chimeric BNLF1 showed that the genes with 3' 453 bp sequence of BNLF1-1510 were oncogenic to the cells. Study also revealed that changing the numbers of the 33 bp repeats in the 3' region of the two BNLF1s did not affect the transformation characteristics. On the other hand, Deletion of a 30 bp sequence of BNLF1-958, which is absent in BNLF1-1510, changed the gene from non-oncogenic to oncogenic and insertion of this 30 bp sequence into BNLF1-1510 abolished the transformation ability. BNLF1 without this 30 bp sequence was also found in the tumours of other EBV-related neoplastic disease, suggesting that absence of this 30 bp sequence in BNLF1 may be associated with the oncogenesis of these diseases.
H R Kaback - One of the best experts on this subject based on the ideXlab platform.
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functional estimation of loop helix boundaries in the lactose permease of escherichia coli by single Amino Acid Deletion analysis
Biochemistry, 2001Co-Authors: C D Wolin, H R KabackAbstract:Mutants with single Amino Acid Deletions in the loops of lactose permease retain activity, while mutants with single Deletions in transmembrane helices are inactive, and the loop−helix boundaries of helices IV, V, VII, VIII, and IX have been approximated functionally by the systematic Deletion of single residues [Wolin, C. D., and Kaback, H. R. (1999) Biochemistry 38, 8590−8597]. The experimental approach is applied here to the remainder of the permease. Periplasmic and cytoplasmic loop−helix boundaries for helices I, II, X, XI, and XII and the cytoplasmic boundary of helix III are in reasonable agreement with structural predictions. In contrast, the periplasmic end of helix III appears to be five to eight residues further into the transmembrane domain than predicted. Taken together with the previous findings, the analysis estimates that 11 of the 12 transmembrane helices have an average length of 21 residues. Surprisingly, Deletion analysis of loop V/VI, helix VI, and loop VI/VII does not yield an activi...
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functional estimation of loop helix boundaries in the lactose permease of escherichia coli by single Amino Acid Deletion analysis
Biochemistry, 2001Co-Authors: C D Wolin, H R KabackAbstract:Mutants with single Amino Acid Deletions in the loops of lactose permease retain activity, while mutants with single Deletions in transmembrane helices are inactive, and the loop--helix boundaries of helices IV, V, VII, VIII, and IX have been approximated functionally by the systematic Deletion of single residues [Wolin, C. D., and Kaback, H. R. (1999) Biochemistry 38, 8590-8597]. The experimental approach is applied here to the remainder of the permease. Periplasmic and cytoplasmic loop-helix boundaries for helices I, II, X, XI, and XII and the cytoplasmic boundary of helix III are in reasonable agreement with structural predictions. In contrast, the periplasmic end of helix III appears to be five to eight residues further into the transmembrane domain than predicted. Taken together with the previous findings, the analysis estimates that 11 of the 12 transmembrane helices have an average length of 21 residues. Surprisingly, Deletion analysis of loop V/VI, helix VI, and loop VI/VII does not yield an activity profile typical of the rest of the protein, as individual Deletion of only three residues in this region abolishes activity. Thus, transmembrane domain VI which is probably on the periphery of the 12-helix bundle may make few functionally important contacts.
Tomozumi Imamichi - One of the best experts on this subject based on the ideXlab platform.
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Amino Acid Deletion at codon 67 and thr to gly change at codon 69 of human immunodeficiency virus type 1 reverse transcriptase confer novel drug resistance profiles
Journal of Virology, 2001Co-Authors: Tomozumi Imamichi, Michael A Murphy, Hiromi Imamichi, Clifford H LaneAbstract:The potential roles of an Amino Acid Deletion at codon 67 (Δ67) and a Thr-to-Gly change at codon 69 (T69G) in the reverse transcriptase of human immunodeficiency virus (HIV) type 1 in drug sensitivity and relative replication fitness were studied. Our results suggest that the Δ67 and T69G changes can be categorized as mutations associated with multidrug resistance. The combination of both mutations with an L74I change (Δ67+T69G/L74I) leads to a novel 3′-azido-3′-deoxythymidine resistance motif and compensates for impaired HIV replication.
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relative replication fitness of a high level 3 azido 3 deoxythymidine resistant variant of human immunodeficiency virus type 1 possessing an Amino Acid Deletion at codon 67 and a novel substitution thr gly at codon 69
Journal of Virology, 2000Co-Authors: Tomozumi Imamichi, Hiromi Imamichi, Steve C Berg, Juan C Lopez, Julia A Metcalf, Judith Falloon, Clifford H LaneAbstract:The combination of an Amino Acid Deletion at codon 67 (Δ67) and Thr-to-Gly change at codon 69 (T69G) in the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) is associated with high-level resistance to multiple RT inhibitors. To determine the relative contributions of the Δ67 and T69G mutations on viral fitness, we performed a series of studies of HIV replication using recombinant variants. A high-level 3′-azido-3′-deoxythymidine (AZT)-resistant variant containing Δ67 plus T69G/K70R/L74I/K103N/T215F/K219Q in RT replicated as efficiently as wild-type virus (Wt). In contrast, the construct without Δ67 exhibited impaired replication (23% of growth of Wt). A competitive fitness study failed to reveal any differences in replication rates between the Δ67+T69G/K70R/L74I/K103N/T215F/K219Q mutant and Wt. Evaluation of proviral DNA sequences over a 3-year period in a patient harboring the multiresistant HIV revealed that the T69G mutation emerged in the context of a D67N/K70R/T215F/K219Q mutant backbone prior to appearance of the Δ67 Deletion. To assess the impact of this stepwise accumulation of mutations on viral replication, a series of recombinant variants was constructed and analyzed for replication competence. The T69G mutation was found to confer 2′,3′-dideoxyinosine resistance at the expense of fitness. Subsequently, the development of the Δ67 Deletion led to a virus with improved replication and high-level AZT resistance.