The "one gene = one protein" model for understanding the genome is well-known. But, as ... more The "one gene = one protein" model for understanding the genome is well-known. But, as with most biological "rules", it has its exceptions. The viral family that brings polio, the common cold and foot-and-mouth disease translates its entire genome into one long "polyprotein". The study of this translation strategy is bringing to light new targets for fighting disease and new techniques for the biotech industry.
The 2A/2B cleavage of aphtho- and cardiovirus 2A polyproteins is mediated by their 2A proteins &#... more The 2A/2B cleavage of aphtho- and cardiovirus 2A polyproteins is mediated by their 2A proteins 'cleaving' at their own C termini. We have analysed this activity using artificial reporter polyprotein systems comprising green fluorescent protein (GFP) linked via foot-and-mouth disease virus (FMDV) 2A to beta-glucuronidase (GUS) -- forming a single, long, open reading frame. Analysis of the distribution of radiolabel showed a high proportion of the in vitro translation products (approximately 90%) were in the form of the 'cleavage' products GUS and [GFP2A]. Alternative models have been proposed to account for the 'cleavage' activity: proteolysis by a host-cell proteinase, autoproteolysis or a translational effect. To investigate the mechanism of this cleavage event constructs encoding site-directed mutant and naturally occurring '2A-like' sequences were used to program in vitro translation systems and the gel profiles analysed. Analysis of site-directed ...
The 2A region of the aphthovirus foot-and-mouth disease virus (FMDV) polyprotein is only 18 aa lo... more The 2A region of the aphthovirus foot-and-mouth disease virus (FMDV) polyprotein is only 18 aa long. A 'primary' intramolecular polyprotein processing event mediated by 2A occurs at its own C terminus. FMDV 2A activity was studied in artificial polyproteins in which sequences encoding reporter proteins flanked the 2A sequence such that a single, long, open reading frame was created. The self-processing properties of these artificial polyproteins were investigated and the co-translational 'cleavage' products quantified. The processing products from our artificial polyprotein systems showed a molar excess of 'cleavage' product N-terminal of 2A over the product C-terminal of 2A. A series of experiments was performed to characterize our in vitro translation systems. These experiments eliminated the translational or transcriptional properties of the in vitro systems as an explanation for this imbalance. In addition, the processing products derived from a control c...
The primary 2A/2B polyprotein cleavage of aphtho-and cardioviruses is mediated by their 2A protei... more The primary 2A/2B polyprotein cleavage of aphtho-and cardioviruses is mediated by their 2A proteins cleaving C-terminally. Whilst the aphthovirus 2A region is only 16 aa (possibly 18 aa) long, the cardiovirus 2A protein is some 150 aa. We have previously shown that foot-and-mouth disease virus (FMDV) 2A is able to mediate cleavage in an artificial (chloramphenicol acetyltransferase/FMDV 2A/beta-glucuronidase [CAT-2A-GUS]) polyprotein system devoid of any other FMDV sequences with high (approximately 85%), although not complete, cleavage. In this paper we show that insertion of upstream FMDV capsid protein 1 D sequences increases the activity. In addition, we have demonstrated that the cardiovirus Theiler's murine encephalomyelitis virus(TME) 2A protein, when linked to GUS in a single ORF, is able to cleave at its own C terminus with high efficiency--if not completely. The C-terminal 19 aa of TME 2A, together with the N-terminal proline residue of protein 2B, were inserted into t...
2A is an oligopeptide sequence mediating a ribosome 'skipping' effect, producing an appar... more 2A is an oligopeptide sequence mediating a ribosome 'skipping' effect, producing an apparent 'cleavage' of polyproteins. First identified and characterized in picornaviruses, '2A-like' sequences are found in other mammalian viruses and a wide range of insect viruses. Databases were analysed using a motif conserved amongst 2A/2A-like sequences. The newly identified 2A-like sequences (30 aa) were inserted into a reporter polyprotein
ABSTRACT A strategy based upon removing the requirement for all of the carbonyl dipoles to align ... more ABSTRACT A strategy based upon removing the requirement for all of the carbonyl dipoles to align at the same time in the transition state leading to the cyclisation of N-[(2S)-2-chloropropionyl]-(2S)-Pro-(2R)-Ala-(2S,4S)-4-thioPro-OMe to a Zimm-Bragg type α-helix peptide intitator template was successful. Each amide bond of the 12-membered macrocyclic template existed in the trans-rotomeric form. Derivatives of the template were prepared by extending the C-terminus and these were characterised by NMR spectroscopy and restrained simulated annealing. In deuterochloroform solution at low temperature, separate sets of NMR signals were observed for two rapidly interconverting helical conformational isomers of the thioether macrocycle which possessed an appended trialkylammonium ion. A similar time-averaged conformation was also observed in aqueous solution. At −80 °C in d2-dichloromethane the rate of conformational exchange was slowed sufficiently to obtain resonance assignments and NOE data separately for each isomer. In the minor isomer (40%), the four carbonyl oxygen hydrogen-bond acceptors of the template are aligned in an α-helical conformation and in the major conformer the Pro2 carbonyl dipole was anti-aligned with the other three dipoles. Thus, the conformers differ in the orientation of one carbonyl group. Molecular modelling calculations showed that the minor isomer was stabilised by coulombic interactions between the trialkylammonium salt and the carbonyl group dipole moments.
Journal of the Chemical Society, Perkin Transactions 1, 1998
ABSTRACT Recently, we designed 12-membered macrocyclic template caps to entrain peptides into α-h... more ABSTRACT Recently, we designed 12-membered macrocyclic template caps to entrain peptides into α-helical structures, based on the covalent connection of the first and fourth residues of proline containing tetrapeptides. It was not possible to complete the synthesis of the templates from the acyclic precursors and it appeared that the generation of large molecular dipoles, caused by aligning the carbonyl groups, prevented reaction. While this work was in progress, Kemp’s group published the structure of a 12-membered macrocyclic triproline template designed to initiate an α-helix that was very similar in structure to one of our own targets. However, the compound failed to cyclise in a conformation required for α-helix initiation and one or more carboxamide dipoles were not aligned. Here we provide a detailed conformational analysis of the system and test two methods for forcing the acyclic precursor into the macrocyclic conformation required for helix initiation. The first is the destabilisation of unwanted conformations in the transition state for cyclisation, and the second is the stabilisation of the favoured transition state structure through the introduction of a hydrogen-bonding interaction. Both strategies were unsuccessful and the reasons are discussed. A successful strategy which does not require the carbonyl dipoles to align in the transition state is presented in the following paper.
The "one gene = one protein" model for understanding the genome is well-known. But, as ... more The "one gene = one protein" model for understanding the genome is well-known. But, as with most biological "rules", it has its exceptions. The viral family that brings polio, the common cold and foot-and-mouth disease translates its entire genome into one long "polyprotein". The study of this translation strategy is bringing to light new targets for fighting disease and new techniques for the biotech industry.
The 2A/2B cleavage of aphtho- and cardiovirus 2A polyproteins is mediated by their 2A proteins &#... more The 2A/2B cleavage of aphtho- and cardiovirus 2A polyproteins is mediated by their 2A proteins 'cleaving' at their own C termini. We have analysed this activity using artificial reporter polyprotein systems comprising green fluorescent protein (GFP) linked via foot-and-mouth disease virus (FMDV) 2A to beta-glucuronidase (GUS) -- forming a single, long, open reading frame. Analysis of the distribution of radiolabel showed a high proportion of the in vitro translation products (approximately 90%) were in the form of the 'cleavage' products GUS and [GFP2A]. Alternative models have been proposed to account for the 'cleavage' activity: proteolysis by a host-cell proteinase, autoproteolysis or a translational effect. To investigate the mechanism of this cleavage event constructs encoding site-directed mutant and naturally occurring '2A-like' sequences were used to program in vitro translation systems and the gel profiles analysed. Analysis of site-directed ...
The 2A region of the aphthovirus foot-and-mouth disease virus (FMDV) polyprotein is only 18 aa lo... more The 2A region of the aphthovirus foot-and-mouth disease virus (FMDV) polyprotein is only 18 aa long. A 'primary' intramolecular polyprotein processing event mediated by 2A occurs at its own C terminus. FMDV 2A activity was studied in artificial polyproteins in which sequences encoding reporter proteins flanked the 2A sequence such that a single, long, open reading frame was created. The self-processing properties of these artificial polyproteins were investigated and the co-translational 'cleavage' products quantified. The processing products from our artificial polyprotein systems showed a molar excess of 'cleavage' product N-terminal of 2A over the product C-terminal of 2A. A series of experiments was performed to characterize our in vitro translation systems. These experiments eliminated the translational or transcriptional properties of the in vitro systems as an explanation for this imbalance. In addition, the processing products derived from a control c...
The primary 2A/2B polyprotein cleavage of aphtho-and cardioviruses is mediated by their 2A protei... more The primary 2A/2B polyprotein cleavage of aphtho-and cardioviruses is mediated by their 2A proteins cleaving C-terminally. Whilst the aphthovirus 2A region is only 16 aa (possibly 18 aa) long, the cardiovirus 2A protein is some 150 aa. We have previously shown that foot-and-mouth disease virus (FMDV) 2A is able to mediate cleavage in an artificial (chloramphenicol acetyltransferase/FMDV 2A/beta-glucuronidase [CAT-2A-GUS]) polyprotein system devoid of any other FMDV sequences with high (approximately 85%), although not complete, cleavage. In this paper we show that insertion of upstream FMDV capsid protein 1 D sequences increases the activity. In addition, we have demonstrated that the cardiovirus Theiler's murine encephalomyelitis virus(TME) 2A protein, when linked to GUS in a single ORF, is able to cleave at its own C terminus with high efficiency--if not completely. The C-terminal 19 aa of TME 2A, together with the N-terminal proline residue of protein 2B, were inserted into t...
2A is an oligopeptide sequence mediating a ribosome 'skipping' effect, producing an appar... more 2A is an oligopeptide sequence mediating a ribosome 'skipping' effect, producing an apparent 'cleavage' of polyproteins. First identified and characterized in picornaviruses, '2A-like' sequences are found in other mammalian viruses and a wide range of insect viruses. Databases were analysed using a motif conserved amongst 2A/2A-like sequences. The newly identified 2A-like sequences (30 aa) were inserted into a reporter polyprotein
ABSTRACT A strategy based upon removing the requirement for all of the carbonyl dipoles to align ... more ABSTRACT A strategy based upon removing the requirement for all of the carbonyl dipoles to align at the same time in the transition state leading to the cyclisation of N-[(2S)-2-chloropropionyl]-(2S)-Pro-(2R)-Ala-(2S,4S)-4-thioPro-OMe to a Zimm-Bragg type α-helix peptide intitator template was successful. Each amide bond of the 12-membered macrocyclic template existed in the trans-rotomeric form. Derivatives of the template were prepared by extending the C-terminus and these were characterised by NMR spectroscopy and restrained simulated annealing. In deuterochloroform solution at low temperature, separate sets of NMR signals were observed for two rapidly interconverting helical conformational isomers of the thioether macrocycle which possessed an appended trialkylammonium ion. A similar time-averaged conformation was also observed in aqueous solution. At −80 °C in d2-dichloromethane the rate of conformational exchange was slowed sufficiently to obtain resonance assignments and NOE data separately for each isomer. In the minor isomer (40%), the four carbonyl oxygen hydrogen-bond acceptors of the template are aligned in an α-helical conformation and in the major conformer the Pro2 carbonyl dipole was anti-aligned with the other three dipoles. Thus, the conformers differ in the orientation of one carbonyl group. Molecular modelling calculations showed that the minor isomer was stabilised by coulombic interactions between the trialkylammonium salt and the carbonyl group dipole moments.
Journal of the Chemical Society, Perkin Transactions 1, 1998
ABSTRACT Recently, we designed 12-membered macrocyclic template caps to entrain peptides into α-h... more ABSTRACT Recently, we designed 12-membered macrocyclic template caps to entrain peptides into α-helical structures, based on the covalent connection of the first and fourth residues of proline containing tetrapeptides. It was not possible to complete the synthesis of the templates from the acyclic precursors and it appeared that the generation of large molecular dipoles, caused by aligning the carbonyl groups, prevented reaction. While this work was in progress, Kemp’s group published the structure of a 12-membered macrocyclic triproline template designed to initiate an α-helix that was very similar in structure to one of our own targets. However, the compound failed to cyclise in a conformation required for α-helix initiation and one or more carboxamide dipoles were not aligned. Here we provide a detailed conformational analysis of the system and test two methods for forcing the acyclic precursor into the macrocyclic conformation required for helix initiation. The first is the destabilisation of unwanted conformations in the transition state for cyclisation, and the second is the stabilisation of the favoured transition state structure through the introduction of a hydrogen-bonding interaction. Both strategies were unsuccessful and the reasons are discussed. A successful strategy which does not require the carbonyl dipoles to align in the transition state is presented in the following paper.
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Papers by Martin Ryan