Final Report Summary - DGAPA76 (The production, crystallisation, and structure determination and analysis of membrane proteins involved in the virulence of Pseudomonas aeruginosa)
General Progress Of The Project
Modification to the project plans
It was decided to maximise the success rate of this project to run in parallel with three other proteins (HasD, HasE, HasF) that are highly homologous to the project target proteins AprD, AprE and AprF (see initial project proposal part B1. 1. 3 Protease secretion systems in P. aeruginosa). This has little to none impact on the speed of the milestones in the initial steps as cloning can be done in parallel without any impairment. HasD has 62. 4 % sequence identity to target AprD, HasE has 52. 1 % sequence identity to target AprE and HasF has 56. 2 % sequence identity to target AprF
Milestones reached (Part B4.
3)
-Training in target design, cloning, membrane protein expression, purification and strategy.
-Cloning for some but not all the target proteins
-Test expressions for two target constructs (one proteins)
-Trainig in membrane protein biochemistry
-Supervision of graduate students
-New research and method development on crystal centring using X-ray diffraction and multi-crystal data collection
Success of the project
Unfortunately the main final objective of obtaining structural work on the secretion systems in Pseudomonas was not successful. This was mainly due to the short time the project run (~12 months) although significant progress had been made during that period. The biggest sucessed obtained during the course of the project were related to work that was already in progress before the fellowship started but that in many ways was related to the fellowhship project in terms of techniques, capabilities and training in membrane proteins. It should be also of note that obtaining structural information in membrane proteins is a task often very long and that tipicaly it is needed ~2 years of work before a significant results is obtained.
Althgough only 1 year of his research was run under European Union funding the reality is that the fellow spend 3 years of his carreer in Ireland after having moved from Portugal. During this period he gained skills in areas out of his area of expertise at the time of the move and also provided skill transfers to the research group he joined. In addition to this the fellow also got involved in front field research in membrane protein structural biology as the high impact publications show. This meant that all 4 training aims (See proposal part B2.
1) were reached resulting in a major leap on his carrer progression allowing him to be one of the world leaders in membrane protein single crystal diffraction particularly when applied to lipid cubic phase grown crystals. This was clearly an asset for his next job.
brief Summary Of Work Performed And Results
Work Performed:
Pseudomonas targets
A strategy with 18 constructs was devised that included C-and N-terminal histidine TAGs for AprD, AprE and for homologous proteins HasD, HasE. For AprD and HasD the ABC binding cassette proteins. The target clones included the full length but also the two individual domains as separate targets. For AprF/HasF C-terminal histidine TAG constructs were targeted with and without signal peptide because this would allow b-barrel refolding if needed a technique that the lab is proficient. Cloning of the 18 constructs progressed and test expressions on postive results performed.
Training, student supervision, enhancement of collaboration of European researchers
-Continuous co-supervision of doctoral students Nicole Hoefer and Joseph Lyons on their research projects.
-Collaboration with beamline staff at GMCA, APS, Argonne, IL, USA on the fluorescence rastering techniques to locate and centre crystals.
-Further development on the technique to harvest in meso crystals from glass plates
Results
Pseudomonas targets
From PA genomic DNA, PCR reactions were performed using appropriate primers to clone the 18 target constructs. The high GC content of PA constructs required PCR debugging but apparently 10 out of the 18 constructs were cloned after several rounds of cloning. From these and after external confirmation of DNA sequence target AprF was inserted in Topo-cloning vectors pET151/D-TOPO and pET101/D-TOPO for expression. Test expressions for both AprF constructs, with and without signal peptide, showed positive results on small scale test expressions at different E. coli grown temperatures. The next step would be to perform crystallisation experiments to try and obtain crystals that would allow structural elucidation. This last step was not performed due to fellow resignation
Training, student supervision, enhancement of collaboration of European researchers
-Membrane structures for a peptide and a large multi-subunit protein complex were determined following the work by Mrs Hoefer and Mr. Lyons, respectively. The close graduate student supervision in addition with developments in crystal centering, data collection, data reduction and structural refinement were key to the success of these projects.
-A technique named fluorescence rastering was developed to locate protein crystals using X-ray Fluorescence. This was was done in collaboration with the staff at the Argonne National Laboratory.
-The technique of harvesting crystals from sandwich glass plates improved of the course of 2 years was thought to all group members and become routine for the whole lab.
-Skill and tech-transfer to the highly reputed lab of Prof. Brian Kobilka in crystal harvesting, crystal centrering, data collection on multiple crystals resulted in the structure of a Gpcr target
Modification to the project plans
It was decided to maximise the success rate of this project to run in parallel with three other proteins (HasD, HasE, HasF) that are highly homologous to the project target proteins AprD, AprE and AprF (see initial project proposal part B1. 1. 3 Protease secretion systems in P. aeruginosa). This has little to none impact on the speed of the milestones in the initial steps as cloning can be done in parallel without any impairment. HasD has 62. 4 % sequence identity to target AprD, HasE has 52. 1 % sequence identity to target AprE and HasF has 56. 2 % sequence identity to target AprF
Milestones reached (Part B4.
3)
-Training in target design, cloning, membrane protein expression, purification and strategy.
-Cloning for some but not all the target proteins
-Test expressions for two target constructs (one proteins)
-Trainig in membrane protein biochemistry
-Supervision of graduate students
-New research and method development on crystal centring using X-ray diffraction and multi-crystal data collection
Success of the project
Unfortunately the main final objective of obtaining structural work on the secretion systems in Pseudomonas was not successful. This was mainly due to the short time the project run (~12 months) although significant progress had been made during that period. The biggest sucessed obtained during the course of the project were related to work that was already in progress before the fellowship started but that in many ways was related to the fellowhship project in terms of techniques, capabilities and training in membrane proteins. It should be also of note that obtaining structural information in membrane proteins is a task often very long and that tipicaly it is needed ~2 years of work before a significant results is obtained.
Althgough only 1 year of his research was run under European Union funding the reality is that the fellow spend 3 years of his carreer in Ireland after having moved from Portugal. During this period he gained skills in areas out of his area of expertise at the time of the move and also provided skill transfers to the research group he joined. In addition to this the fellow also got involved in front field research in membrane protein structural biology as the high impact publications show. This meant that all 4 training aims (See proposal part B2.
1) were reached resulting in a major leap on his carrer progression allowing him to be one of the world leaders in membrane protein single crystal diffraction particularly when applied to lipid cubic phase grown crystals. This was clearly an asset for his next job.
brief Summary Of Work Performed And Results
Work Performed:
Pseudomonas targets
A strategy with 18 constructs was devised that included C-and N-terminal histidine TAGs for AprD, AprE and for homologous proteins HasD, HasE. For AprD and HasD the ABC binding cassette proteins. The target clones included the full length but also the two individual domains as separate targets. For AprF/HasF C-terminal histidine TAG constructs were targeted with and without signal peptide because this would allow b-barrel refolding if needed a technique that the lab is proficient. Cloning of the 18 constructs progressed and test expressions on postive results performed.
Training, student supervision, enhancement of collaboration of European researchers
-Continuous co-supervision of doctoral students Nicole Hoefer and Joseph Lyons on their research projects.
-Collaboration with beamline staff at GMCA, APS, Argonne, IL, USA on the fluorescence rastering techniques to locate and centre crystals.
-Further development on the technique to harvest in meso crystals from glass plates
Results
Pseudomonas targets
From PA genomic DNA, PCR reactions were performed using appropriate primers to clone the 18 target constructs. The high GC content of PA constructs required PCR debugging but apparently 10 out of the 18 constructs were cloned after several rounds of cloning. From these and after external confirmation of DNA sequence target AprF was inserted in Topo-cloning vectors pET151/D-TOPO and pET101/D-TOPO for expression. Test expressions for both AprF constructs, with and without signal peptide, showed positive results on small scale test expressions at different E. coli grown temperatures. The next step would be to perform crystallisation experiments to try and obtain crystals that would allow structural elucidation. This last step was not performed due to fellow resignation
Training, student supervision, enhancement of collaboration of European researchers
-Membrane structures for a peptide and a large multi-subunit protein complex were determined following the work by Mrs Hoefer and Mr. Lyons, respectively. The close graduate student supervision in addition with developments in crystal centering, data collection, data reduction and structural refinement were key to the success of these projects.
-A technique named fluorescence rastering was developed to locate protein crystals using X-ray Fluorescence. This was was done in collaboration with the staff at the Argonne National Laboratory.
-The technique of harvesting crystals from sandwich glass plates improved of the course of 2 years was thought to all group members and become routine for the whole lab.
-Skill and tech-transfer to the highly reputed lab of Prof. Brian Kobilka in crystal harvesting, crystal centrering, data collection on multiple crystals resulted in the structure of a Gpcr target