Final Report Summary - CHEMOTAXISNMR (Invisible Protein States in Bacterial Chemotaxis: a relaxation dispersion NMR study.)
Summary description of the project objectives:
The major challenge of this proposal was to determine, at the residue-specific level, the factors governing the subtle differences in activity and behaviour of the 6 highly homologous CheYs from R. sphaeroides. The specific objectives to be addressed were:
- To determine the solution structure of several CheYs in their inactive and active states and in particular to define the differences in conformation that exist between the inactive and active forms of the CheYs.
- To study the dynamics of the inactive and active forms of several CheYs.
- To characterise invisible, ‘active-like’ conformations that exist in the unphosphorylated inactive CheYs using relaxation dispersion NMR method.
- To validate in-vivo all the results extracted from the NMR experiments.
Work performed since the beginning of the project including a description of the main results achieved so far:
- Preliminary study: Detailed study on how conditions (Mg2+ and BeF3 concentrations, pH) affect the structure and function of the CheY3 protein.
Under conditions where CheY3 is inactive (no BeF3 bound) and at low pH we have detected one minor and one major protein conformation and upon pH increase these populations are inverted and finally only one species is present. We have observed that only at high pH is Mg2+ required for the protein to bind BeF3, a phosphorylation mimic, and the affinity for Mg2+ is considerably higher than at low pH.
- Objective 1: Determination of the solution structure of several CheYs in their inactive and active states and in particular to define the differences in conformation that exist between the inactive and active forms of the CheYs.
The NMR spectra of CheY3 and CheY6 in their inactive and active states have been assigned using triple-resonance NMR methods. Information about secondary structure in the proteins has been obtained from analysis of 1H, 13C and 15N chemical shifts using the TALOS approach, as well as from CD experiments. Distance-dependent through-space NOEs derived from 15N- and 13C-edited 3D NOESY experiments have been used to define the local environment of residues. RDC experiments have been performed to provide structural restrains including RDC for aromatics residues. The available X-ray structures of CheY3 and CheY6 have been used as the starting point for the analysis of these NMR data and to derive solution structures in the both inactive and active states.
- Objective 2: Dynamics study of the inactive and active forms of several CheYs.
Previous studies of E. coli CheY indicate a relatively rigid alpha5/beta5 structure. Evidence from the X-ray structure of CheY6/CheA3 indicates that the 10-residue loop insertion in CheY6 may be flexible. 15N NMR relaxation experiments (heteronuclear NOE) have been used to characterise the dynamics of CheY6 in its inactive and active states, to define the dynamics of the loop insertion and any differences in dynamics between the two states. For comparison, 15N relaxation data for CheY3, which lacks this loop, have also been collected.
- Objective 3: Characterisation of invisible, ‘active-like’ conformations in inactive CheY3 and CheY6.
We have used CPMG-based relaxation dispersion methods to study the low-populated excited states that exist in CheY3 and CheY6 and give rise to the Rex exchange contributions on slower timescales. We have collected experiments using multiple frequencies on the available spectrometers operating at 500, 750 and 950 MHz. We have identified residues in CheY3 and CheY6 which undergo millisecond to microsecond processes which involve excited states with altered 15N chemical shifts. In this study differences in the solution state behaviour of CheY3 and CheY6 in these novel relaxation dispersion experiments have pointed out important differences of functional significance between these CheYs from R. sphaeroides.
- Objective 4: In vivo evaluation of the studied systems to correlate with the in vitro NMR results.
These studies have been performed by a PhD student co-supervised by Dr Lorena Varela. The 10-residue loop from CheY6 has been deleted and the functional consequences of this assessed in vivo. The evaluation of the functionality of the proteins studied by NMR has been assessed by innovative in vivo techniques consisting in overexpressing the proteins cloned in a vector containing CFP or YFP (fluorescent markers) in-frame downstream of the insertion site, producing proteins tagged fluorescently at the C terminus. These plasmids have been propagated in E. coli and conjugated into R. sphaeroides. Isopropyl β-D-1 thiogalactopyranoside has been used to express the protein in both R. sphaeroides and E. coli, and the resultant strains have been observed using fluorescence microscopy using the available Nikon TE200 microscope and CFP and YFP filter set (Chroma), and for short-time interval imaging (0.1-s and 1-min periods) the OMX v2 (Applied Precision) microscope has been used. The in vivo motility of the fluorescent bacteria has determined the effect of the studied proteins in the chemotaxis process.
Expected final results and their potential impact and use (including the socio-economic impact and the wider societal implications of the project so far).
We are in the process of calculating the solution structure of the CheY3 and CheY6 proteins in the absence (inactive) and presence (active) of BeF3, by using all the data collected:
- Assigned 1H, 13C and 15N chemical shifts.
- Distance-dependent through-space NOEs derived from 15N- and 13C-edited 3D NOESY experiments.
- Residual dipolar couplings which provide information about long-range structure within a protein.
The available X-ray structures of CheY3 and CheY6 are being used as the starting point for the analysis of these NMR data and the latter are being used to derive solution structures in the both the inactive and active states.
We are also in the process of extracting important conclusions about the chemical shifts obtained from the set of CPMG relaxation data and using it to define the conformation of the ‘invisible’ excited states.
This project has made advances in two highly relevant areas, chemotaxis, which is extremely important for the virulence of bacteria, and allostery, which is a common mechanism in proteins. The results of this project inform both of these areas and thus lead to improved knowledge and understanding in those important fields in Europe and around the globe. In addition, the nature of the project is very multidisciplinary, as it has been conceived as a collaboration between two groups specialist in different areas. The relevance of this project relies on valuable structural and dynamic information that has been obtained from NMR spectroscopy and that has been combined with the in vivo information extracted from fluorescence microscopy. This joined approach has not previously been carried out, and is helping to resolve outstanding biological questions with high pharmacological and industrial applicability (i.e. design of new antibiotics, improvement of bacterial strains used in industry, etc.). Thus it is envisaged that our studies will make a significant contribution to the field, benefiting the bacterial motility and behaviour research community with the understanding of the molecular processes governing chemotaxis.
The major challenge of this proposal was to determine, at the residue-specific level, the factors governing the subtle differences in activity and behaviour of the 6 highly homologous CheYs from R. sphaeroides. The specific objectives to be addressed were:
- To determine the solution structure of several CheYs in their inactive and active states and in particular to define the differences in conformation that exist between the inactive and active forms of the CheYs.
- To study the dynamics of the inactive and active forms of several CheYs.
- To characterise invisible, ‘active-like’ conformations that exist in the unphosphorylated inactive CheYs using relaxation dispersion NMR method.
- To validate in-vivo all the results extracted from the NMR experiments.
Work performed since the beginning of the project including a description of the main results achieved so far:
- Preliminary study: Detailed study on how conditions (Mg2+ and BeF3 concentrations, pH) affect the structure and function of the CheY3 protein.
Under conditions where CheY3 is inactive (no BeF3 bound) and at low pH we have detected one minor and one major protein conformation and upon pH increase these populations are inverted and finally only one species is present. We have observed that only at high pH is Mg2+ required for the protein to bind BeF3, a phosphorylation mimic, and the affinity for Mg2+ is considerably higher than at low pH.
- Objective 1: Determination of the solution structure of several CheYs in their inactive and active states and in particular to define the differences in conformation that exist between the inactive and active forms of the CheYs.
The NMR spectra of CheY3 and CheY6 in their inactive and active states have been assigned using triple-resonance NMR methods. Information about secondary structure in the proteins has been obtained from analysis of 1H, 13C and 15N chemical shifts using the TALOS approach, as well as from CD experiments. Distance-dependent through-space NOEs derived from 15N- and 13C-edited 3D NOESY experiments have been used to define the local environment of residues. RDC experiments have been performed to provide structural restrains including RDC for aromatics residues. The available X-ray structures of CheY3 and CheY6 have been used as the starting point for the analysis of these NMR data and to derive solution structures in the both inactive and active states.
- Objective 2: Dynamics study of the inactive and active forms of several CheYs.
Previous studies of E. coli CheY indicate a relatively rigid alpha5/beta5 structure. Evidence from the X-ray structure of CheY6/CheA3 indicates that the 10-residue loop insertion in CheY6 may be flexible. 15N NMR relaxation experiments (heteronuclear NOE) have been used to characterise the dynamics of CheY6 in its inactive and active states, to define the dynamics of the loop insertion and any differences in dynamics between the two states. For comparison, 15N relaxation data for CheY3, which lacks this loop, have also been collected.
- Objective 3: Characterisation of invisible, ‘active-like’ conformations in inactive CheY3 and CheY6.
We have used CPMG-based relaxation dispersion methods to study the low-populated excited states that exist in CheY3 and CheY6 and give rise to the Rex exchange contributions on slower timescales. We have collected experiments using multiple frequencies on the available spectrometers operating at 500, 750 and 950 MHz. We have identified residues in CheY3 and CheY6 which undergo millisecond to microsecond processes which involve excited states with altered 15N chemical shifts. In this study differences in the solution state behaviour of CheY3 and CheY6 in these novel relaxation dispersion experiments have pointed out important differences of functional significance between these CheYs from R. sphaeroides.
- Objective 4: In vivo evaluation of the studied systems to correlate with the in vitro NMR results.
These studies have been performed by a PhD student co-supervised by Dr Lorena Varela. The 10-residue loop from CheY6 has been deleted and the functional consequences of this assessed in vivo. The evaluation of the functionality of the proteins studied by NMR has been assessed by innovative in vivo techniques consisting in overexpressing the proteins cloned in a vector containing CFP or YFP (fluorescent markers) in-frame downstream of the insertion site, producing proteins tagged fluorescently at the C terminus. These plasmids have been propagated in E. coli and conjugated into R. sphaeroides. Isopropyl β-D-1 thiogalactopyranoside has been used to express the protein in both R. sphaeroides and E. coli, and the resultant strains have been observed using fluorescence microscopy using the available Nikon TE200 microscope and CFP and YFP filter set (Chroma), and for short-time interval imaging (0.1-s and 1-min periods) the OMX v2 (Applied Precision) microscope has been used. The in vivo motility of the fluorescent bacteria has determined the effect of the studied proteins in the chemotaxis process.
Expected final results and their potential impact and use (including the socio-economic impact and the wider societal implications of the project so far).
We are in the process of calculating the solution structure of the CheY3 and CheY6 proteins in the absence (inactive) and presence (active) of BeF3, by using all the data collected:
- Assigned 1H, 13C and 15N chemical shifts.
- Distance-dependent through-space NOEs derived from 15N- and 13C-edited 3D NOESY experiments.
- Residual dipolar couplings which provide information about long-range structure within a protein.
The available X-ray structures of CheY3 and CheY6 are being used as the starting point for the analysis of these NMR data and the latter are being used to derive solution structures in the both the inactive and active states.
We are also in the process of extracting important conclusions about the chemical shifts obtained from the set of CPMG relaxation data and using it to define the conformation of the ‘invisible’ excited states.
This project has made advances in two highly relevant areas, chemotaxis, which is extremely important for the virulence of bacteria, and allostery, which is a common mechanism in proteins. The results of this project inform both of these areas and thus lead to improved knowledge and understanding in those important fields in Europe and around the globe. In addition, the nature of the project is very multidisciplinary, as it has been conceived as a collaboration between two groups specialist in different areas. The relevance of this project relies on valuable structural and dynamic information that has been obtained from NMR spectroscopy and that has been combined with the in vivo information extracted from fluorescence microscopy. This joined approach has not previously been carried out, and is helping to resolve outstanding biological questions with high pharmacological and industrial applicability (i.e. design of new antibiotics, improvement of bacterial strains used in industry, etc.). Thus it is envisaged that our studies will make a significant contribution to the field, benefiting the bacterial motility and behaviour research community with the understanding of the molecular processes governing chemotaxis.