Periodic Reporting for period 2 - RAMP (RAtionalising Membrane Protein crystallisation)
Período documentado: 2019-03-01 hasta 2021-08-31
Over the past two decades, advances in the crystallisation of soluble proteins, diffraction data collection and data analysis have made structure solution of soluble proteins almost routine. But crystallisation – remains a major bottleneck for membrane proteins. The additional challenge with membrane proteins is two-fold: first, the protein itself is hard to produce in large quantities and tends to be unstable. Second, the parameter space is even larger than for soluble proteins due to additional parameters: the detergents. The crystallisation process is thus complex and poorly understood.
Membrane proteins are currently crystallized by using brute-force screening to search this high-dimensional parameter space to find initial conditions, followed by trial-and-error optimisation to grow crystals suitable for diffraction studies. Though over 85% of drug targets are membrane proteins, fewer than 650 unique membrane protein structures have been determined. We urgently require better methods to crystallize membrane proteins. RAMP created a unique training network that brings together three strands: (1) development of a microfluidics-based technology to control membrane protein crystallisation, and the ability to sample the large parameter space of crystallisation conditions rapidly; (2) the introduction to membrane protein crystallisation optimisation of modelling of the phase diagram, a technique used with great success for soluble protein crystallisation; and (3) the application of these developments to medically and biologically important membrane protein targets.
RAMP also trained the students on two new and emerging techniques: 1) serial crystallographic methods. These are increasingly used at synchrotron sources as well as at rapidly developing ultra-bright free-electron laser sources, and require crystals in the 1-20 μm size to solve structure of previously intractable proteins. 2) Neutron protein crystallography, which requires large crystals (> 0.01 mm3). It is, however, the only way to visualise protons - important information for drug design.
Membrane proteins are currently crystallized by using brute-force screening to search this high-dimensional parameter space to find initial conditions, followed by trial-and-error optimisation to grow crystals suitable for diffraction studies. Though over 85% of drug targets are membrane proteins, fewer than 650 unique membrane protein structures have been determined. We urgently require better methods to crystallize membrane proteins. RAMP created a unique training network that brings together three strands: (1) development of a microfluidics-based technology to control membrane protein crystallisation, and the ability to sample the large parameter space of crystallisation conditions rapidly; (2) the introduction to membrane protein crystallisation optimisation of modelling of the phase diagram, a technique used with great success for soluble protein crystallisation; and (3) the application of these developments to medically and biologically important membrane protein targets.
RAMP also trained the students on two new and emerging techniques: 1) serial crystallographic methods. These are increasingly used at synchrotron sources as well as at rapidly developing ultra-bright free-electron laser sources, and require crystals in the 1-20 μm size to solve structure of previously intractable proteins. 2) Neutron protein crystallography, which requires large crystals (> 0.01 mm3). It is, however, the only way to visualise protons - important information for drug design.
In the first strand of the RAMP network, the UGA developed an easy and inexpensive way to fabricate microchips (Figure 1) that cover the whole pipeline from crystal growth to beam, eliminating the need for crystal handling prior to the diffraction experiment. The chips have been characterized for their mechanical and flow properties as well as for their transparency to X-rays. We also have a new flowing reservoir dialysis set-up for the crystallisation bench (Figure 2), which overcomes the problems of large crystal growth of membrane proteins for neutron protein crystallography. Soluble and membrane model proteins have been successfully crystallized in both setups. RAMP students used these crystallisation tools to control the size of grown crystals for serial synchrotron and neutron crystallography as well as the target membrane proteins in strand 3. At Surrey University, as part of strand 2, a model for mixing the precipitants has been established. Mixing dynamics affects both crystal size and diffraction quality of generated crystals. This work feeds back to strand 1, allowing better control of the kinetic pathways induced in the new fluidic tools.
Lipids are critical in stabilising membrane proteins during crystallisation. However, neither the in meso nor the HiLiDe method, have been fully investigated, and it therefore remains unclear why they are successful for some targets and unsuccessful for others. Rationalizing membrane protein crystallisation by the HiLiDe method has progressed at Aarhus. The HiLiDe method uses high concentrations of lipids and detergents to relipidate the protein prior to crystallisation and systematic screening of lipid-protein-detergent ratios. A phase diagram has been used to guide protein crystallisation with the HiLiDe method. Imperial College, the University of Leeds and Molecular Dimensions have collaborated on developing and testing a new high-throughput lipid screen, which can be commercialised and made available to all. The work demonstrates that for three test proteins the lipid screen is able to identify known lipids that stabilise these proteins, as well as others that were not previously known. It is useful for crystallisation trials and other biophysical techniques (Figure 3). The paper (Cecchetti et al. PLoS ONE, 16(7): e0254118 (2021)) was recognised as an important methodological advance by Faculty Opinions (https://facultyopinions.com/prime/740463020).
In strand 3 three universities studied new structures of membrane transporters. The University of Leeds crystallised integral membrane pyrophosphatases (mPPases), looking to understand potassium activation. The structure of the non-potassium dependent P. aerophilum mPPase (PaPPase), when compared with potassium-dependent pyrophosphatases, such as T. maritima (TmPPase) and V. radiata mPPases (VrPPase), highlights potential mechanistic differences. Imperial College London expressed and purified A. thaliana borate transporter as well as single-point AtBOR1 mutants. In collaboration with TCD Imperial also carried out LCP-FRAP of both AtBOR1 and UapA revealing good diffusion of both proteins in the LCP matrix. Finally, University Aarhus solved the structure of P-type ATPase SERCA1 at room temperature. This promising result demonstrates the feasibility to obtain crystals that may be suitable for future neutron diffraction experiments (Figure 4).
Lipids are critical in stabilising membrane proteins during crystallisation. However, neither the in meso nor the HiLiDe method, have been fully investigated, and it therefore remains unclear why they are successful for some targets and unsuccessful for others. Rationalizing membrane protein crystallisation by the HiLiDe method has progressed at Aarhus. The HiLiDe method uses high concentrations of lipids and detergents to relipidate the protein prior to crystallisation and systematic screening of lipid-protein-detergent ratios. A phase diagram has been used to guide protein crystallisation with the HiLiDe method. Imperial College, the University of Leeds and Molecular Dimensions have collaborated on developing and testing a new high-throughput lipid screen, which can be commercialised and made available to all. The work demonstrates that for three test proteins the lipid screen is able to identify known lipids that stabilise these proteins, as well as others that were not previously known. It is useful for crystallisation trials and other biophysical techniques (Figure 3). The paper (Cecchetti et al. PLoS ONE, 16(7): e0254118 (2021)) was recognised as an important methodological advance by Faculty Opinions (https://facultyopinions.com/prime/740463020).
In strand 3 three universities studied new structures of membrane transporters. The University of Leeds crystallised integral membrane pyrophosphatases (mPPases), looking to understand potassium activation. The structure of the non-potassium dependent P. aerophilum mPPase (PaPPase), when compared with potassium-dependent pyrophosphatases, such as T. maritima (TmPPase) and V. radiata mPPases (VrPPase), highlights potential mechanistic differences. Imperial College London expressed and purified A. thaliana borate transporter as well as single-point AtBOR1 mutants. In collaboration with TCD Imperial also carried out LCP-FRAP of both AtBOR1 and UapA revealing good diffusion of both proteins in the LCP matrix. Finally, University Aarhus solved the structure of P-type ATPase SERCA1 at room temperature. This promising result demonstrates the feasibility to obtain crystals that may be suitable for future neutron diffraction experiments (Figure 4).
RAMP provided interdisciplinary, inter-sectoral research training to the next generation of scientists, producing leaders who will be able to advance the field academically and be able to make leading contributions for drug discovery research.
We developed rational approaches for optimising on-chip protein crystallisation via chemical composition and temperature control. Combining transparent microfluidics and dialysis provided control over the experiment and reversible exploration of the crystallisation conditions. We have shown that in addition to diffusion, solute-driven convection drives mixing, reducing both the mixing time and the concentration gradients during mixing. Our use of a phase diagram as a guide for membrane protein crystallisation is promising. Mechanistic studies using new structures of membrane proteins have been carried out, mainly on integral membrane pyrophosphatases and SERCA. For SERCA we have shown it is feasible to obtain large crystals suitable for neutron crystallography.
The lipid screen developed by Imperial College London, University of Leeds and Molecular Dimensions will be commercialised, and funding is being sought to develop it further.
We developed rational approaches for optimising on-chip protein crystallisation via chemical composition and temperature control. Combining transparent microfluidics and dialysis provided control over the experiment and reversible exploration of the crystallisation conditions. We have shown that in addition to diffusion, solute-driven convection drives mixing, reducing both the mixing time and the concentration gradients during mixing. Our use of a phase diagram as a guide for membrane protein crystallisation is promising. Mechanistic studies using new structures of membrane proteins have been carried out, mainly on integral membrane pyrophosphatases and SERCA. For SERCA we have shown it is feasible to obtain large crystals suitable for neutron crystallography.
The lipid screen developed by Imperial College London, University of Leeds and Molecular Dimensions will be commercialised, and funding is being sought to develop it further.