100 kHz magic angle spinning for development of solid-state NMR methodology for probing protein dynamics

Lead Research Organisation: University of Warwick
Department Name: Chemistry

Abstract

Motion and change are essential features of living organisms and fundamentally important for many vital processes from protein folding and unfolding, ligand binding, signalling, allosteric regulation to enzymatic catalysis. Consequently, understanding motions at molecular level provides valuable insights into the phenomena involving change of structure both when they function as intended or when they malfunction. For example understanding how proteins misfold may help to fight debilitating diseases called amyloidoses that include Alzheimer's disease, type II diabetes or bovine spongiform encephalopathy more widely known as "mad cow" disease. Moreover, understanding motions that are intrinsically associated with signalling pathways may result in development of better drugs that target such pathways (most medicines work this way). Even development of practical environmentally friendly biobatteries and biofuel cells may be aided by knowledge of molecular motions as they make use of enzymes. Thus it is really important to devise ways to measure protein motions at atomic resolution.
To do that, in this project, we will develop a technique called nuclear magnetic resonance (NMR), which relies on the inherent magnetism of atomic nuclei. When placed in a strong magnetic field magnetic moments of nuclei align with the external field but this alignment may be changed by application of radio waves at specific frequencies. By measuring the associated frequencies one can learn about the relative position of atoms with respect to each other and how this position changes with time i.e. molecular motions. A very powerful aspect of this technique is that one can learn such information not only for a molecule overall but for specific atoms in it. In solid-state NMR, which is the primary method used in this project, the high resolution necessary to distinguish individual sites is enabled by a technique called magic angle spinning (MAS), which involves fast rotation of the sample around an axis inclined at an angle of 54.7 degrees to the external magnetic field. Recently introduced cutting edge instrumentation allows achieving spinning frequencies up to 100 000 revolutions per second. The centre of this project is the purchase of the first in the UK probe capable of 100 kHz MAS. The improved efficiency of MAS at such astounding frequencies makes possible designing new experiments that provide new analytical tools to access motions, e.g. site-specific 1H relaxation or highly sensitive 1H-detected relaxation measurements in fully protonated samples. The 100 kHz spinning removes a number of undesired effects obscuring the measurements of parameters reporting on molecular motions and thus allows a detailed view of protein motions to be obtained.
In this project we propose to develop a series of robust solid-state NMR spectroscopic methods that take advantage of the new 100 kHz spinning regime and will provide improved access to measuring of dynamic processes in proteins at atomic resolution and in a site-specific manner. In particular, we will focus on techniques that provide access to slow motions in the regime that is difficult to access by the solid-state NMR sister method - solution NMR. In addition, in order to improve practicality of the developed techniques we will optimise them for speed and sensitivity.

Planned Impact

Impact through training
Three PhD students will be trained in developing NMR methodology at 100 kHz MAS as a part of this research project. The transferable skills and unique analytical skills obtained as a result of participating in this research will facilitate progress of their main PhD projects and will improve their employability in biotech industry or academia.

Impact through collaborations with industry
The PI has established two industrial collaborations that will directly benefit from this project. The collaboration with Bruker involves developing methodology that helps to preserve viability of biological samples under the conditions of extremely fast magic angle spinning which is directly relevant to this project. In addition, the main project of one of the PhD students involves industrial collaboration with Pfizer on application of NMR relaxation based methods to facilitate chromatographic method development and probing stability of pharmaceutical formulations. This collaboration will be used as a platform to transfer the methodology developed in the context of fundamental biomolecular studies to practical industrial applications including optimisation of compound separation, characterisation of pharmaceutical formulations and drug development.

Impact on technology development
As this project involves efforts to maximise the utility of novel fast magic angle spinning technology it will lead to popularisation of such technology and provide motivation for further technological development. Currently, there are competing manufacturers developing such technology.

Societal and environmental impact through scientific progress
The tools developed as a result of this project will allow characterising in detail dynamic transformations of proteins that are implicated in their function. Consequently, such tools may aid understanding of many diseases involving dynamic changes of proteins and in a longer-term help finding a cure for them and thus contribute to enhancing quality of health and life of the society. For example, knowledge of dynamics of protein kinases should help in developing their effective inhibitors and thus aid treating certain forms of cancer. In general, dynamical factors need increasingly to be considered in addition to structural factors in order to design effective ligands and inhibitors of proteins and hence effective drugs. The tools developed in this project will facilitate this process. These tools can also contribute to understanding processes of protein misfolding that are at the core of diseases such as Alzheimer's or type II diabetes that are becoming an increasing burden on the UK and other health systems around the globe. Finding a cure for these diseases facilitated by research enabled by our results can have profound health and economic impact on the society.
Mobility is important for the function and stability of enzymes. Consequently, any processes involving use of enzymes can benefit from tools enabling detailed characterisation of protein motions: from industrially important immobilised enzymes to developing and perfecting environmentally friendly biobatteries and biofuel cells and facilitating rational synthetic biology approaches to produce new drugs difficult to synthesise by traditional chemical means.
In order to ensure that our results reach the right scientific audience we will use the traditional dissemination methods such as publications in international peer-reviewed journals and presentations at conferences as well as direct transfer of knowledge through collaborations with researchers working on the discussed above issues (see Case for Support and Pathways to Impact).

Publications

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Hoop CL (2016) Huntingtin exon 1 fibrils feature an interdigitated ß-hairpin-based polyglutamine core. in Proceedings of the National Academy of Sciences of the United States of America

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Lamley JM (2014) Solid-state NMR of a protein in a precipitated complex with a full-length antibody. in Journal of the American Chemical Society

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Lamley JM (2015) Intermolecular Interactions and Protein Dynamics by Solid-State NMR Spectroscopy. in Angewandte Chemie (Weinheim an der Bergstrasse, Germany)

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Lewandowski JR (2015) Protein dynamics. Direct observation of hierarchical protein dynamics. in Science (New York, N.Y.)

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Sternberg U (2018) H line width dependence on MAS speed in solid state NMR - Comparison of experiment and simulation. in Journal of magnetic resonance (San Diego, Calif. : 1997)

 
Description During the tenure of this grant we have developed a number of solid-state NMR methods that allow to probe structure and dynamics of biomolecules at atomic resolution. In particular, we developed a range of methods using proton detection, which enable working with very small samples. The ability to work with small samples is very important for applicability of solid-state NMR because often biological samples are difficult to be produced in large quantities required for more standard approaches. The majority of the methods concern new ways of probing molecular motions or how the structure molecules changes in time. We have developed methods that probe in a site-specific manner motions occurring on timescales from ps to ms based on measurements of 1H, 15N and 13C sites. Knowledge of molecular motions is important for understanding of many biological processes and can aid finding solutions for when such processes malfunction (as in the case of diseases) or can aid exploiting such processes. For example, we now employ the developed methodology to understand molecular factories found in nature, which produce antibiotics. We starting employing the gained understanding to modify such systems in hope to produce new antibiotics not found in nature.
Exploitation Route This grant has enabled introduction of the new 100 kHz magic angle spinning instrumentation into UK. The experience obtain as a result of this project has enabled out group to aid other potential users to both appreciate the potential benefits of employing this new instrumentation and to adopt it. For example, we were able to advise the National UK 850 MHz Solid-state NMR Facility on acquisition of instrumentation with similar capability. As a result the facility has currently a new 0.7 mm probe capable of spinning to 111 kHz that can be used by the entire UK solid-state NMR user base. We continue advise the users and the facility manager on implementation of this instrumentation. For example, recently we have started collaboration with another user to apply this methodology to studying plants. We have also established a collaborative facility with Medicines Discovery Catapult, which aims to introduce this methodology to SMEs to support their drug development activities. The scientific results of this project have been and continue to be disseminated through standard ways such as scientific publications and presentations at conferences.
Sectors Agriculture, Food and Drink,Chemicals,Education,Energy,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description The methodology developed during this project has provided basis for establishing a collaboration with a pharmaceutical company to work on rational development of a novel antibiotic into a viable drug. This should lead to enhancing of health. In addition, the expertise developed during the project has contributed to establishing a collaborative facility with Medicines Discovery Catapult to provide access to fast spinning solid-state NMR in the context of drug development.
First Year Of Impact 2017
Sector Pharmaceuticals and Medical Biotechnology
Impact Types Societal,Economic

 
Description BBSRC Responsive Mode
Amount £730,455 (GBP)
Funding ID BB/R010218/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 03/2018 
End 02/2021
 
Description ERC Starting Grant
Amount € 1,999,044 (EUR)
Funding ID 639907 
Organisation European Research Council (ERC) 
Sector Public
Country Belgium
Start 05/2015 
End 04/2020
 
Description Elucidating and exploiting docking domain-mediated carrier protein recognition in natural product megasynthetases
Amount £742,035 (GBP)
Funding ID BB/R010218/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 03/2018 
End 02/2021
 
Description Gates Foundation Grant
Amount $750,000 (USD)
Funding ID OPP1160394 
Organisation Bill and Melinda Gates Foundation 
Sector Charity/Non Profit
Country United States
Start 10/2016 
End 12/2017
 
Description INTEGRATE AMR Pump Priming Fund
Amount £13,000 (GBP)
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 03/2017 
End 06/2017
 
Description Access to fast spinning methodology for SMEs in drug discovery 
Organisation Medicines Discovery Catapult
Country United Kingdom 
Sector Private 
PI Contribution We provide access and expertise in fast magic angle spinning solid-state NMR to SMEs in the context of drug discovery projects.
Collaborator Contribution Purchased a 0.7mm magic angle spinning NMR probe to be used for the projects. Provide project management.
Impact Pilot project on characterisation of a docking domain in non-ribosomal peptide synthesase involved in biosynthesis of antibiotic tyrocidine. https://md.catapult.org.uk/case-studies/protein-interactions-in-non-ribosomal-peptide-synthetases-nrpss/
Start Year 2017
 
Description Development of fast magic angle spinning instrumentation 
Organisation Tallinn University of Technology
Department Technomedicum
Country Estonia 
Sector Academic/University 
PI Contribution Evaluated and provided feedback about various generations of 0.8 mm MAS probe for application on biomolecules.
Collaborator Contribution Provide experimental 0.8mm MAS probe.
Impact (1) Lamley, J. M.; Iuga, D.; Öster, C.; Sass, H.-J.; Rogowski, M.; Oss, A.; Past, J.; Reinhold, A.; Grzesiek, S.; Samoson, A.; Lewandowski, J. R. J. Am. Chem. Soc. 2014, 136 (48), 16800.
Start Year 2012
 
Description Dynamics of b1AR receptor 
Organisation University of Basel
Department Biozentrum Basel
Country Switzerland 
Sector Academic/University 
PI Contribution We performed pilot study of feasibility of probing dynamics of b1AR receptor by solid-state NMR.
Collaborator Contribution Provided isotopically labelled samples.
Impact na
Start Year 2019
 
Description Fast magic angle spinning methods based characterisation of Abeta oligomers with curcumin 
Organisation Luleå University of Technology
Department Department of Civil, Environmental and Natural Resources Engineering
Country Sweden 
Sector Academic/University 
PI Contribution 1H-detected experiments relying on fast magic angle spinning were applied to study interactions of Abeta oligomers with curcumin.
Collaborator Contribution Provided isotopically labeled samples.
Impact Initial spectra of Abeta oligomers were recorded in the presence and absence of curcumin.
Start Year 2012
 
Description Medicines Discovery Catapult 
Organisation Medicines Discovery Catapult
Country United Kingdom 
Sector Private 
PI Contribution Established a collaborative facility with Medicines Discovery Catapult providing expertise on solid-state NMR to facilitate R&D for SMEs.
Collaborator Contribution Purchased a 0.7 mm probe. Funded a facility manager partially embedded in Lewandowski group.
Impact Establishing the facility with one call for collaborations so far.
Start Year 2017
 
Description Microtubules binding proteins 
Organisation University of Leicester
Country United Kingdom 
Sector Academic/University 
PI Contribution Performed pilot solid-state NMR measurements on protein samples.
Collaborator Contribution Provided samples.
Impact Preliminary measurements.
Start Year 2018
 
Description Solid-state NMR of multidomain proteins 
Organisation University of Patras
Department Department of Environmental & Natural Resources Management
Country Greece 
Sector Academic/University 
PI Contribution Solid-state NMR of a multidomain protein
Collaborator Contribution Supply samples.
Impact No outputs yet.
Start Year 2016
 
Description Structural investigations of PPM synthase 
Organisation University of Warwick
Department Department of Economics
Country United Kingdom 
Sector Academic/University 
PI Contribution Working towards structure determination of a PPM synthase
Collaborator Contribution Produce samples. Perform biochemical essays.
Impact No outputs so far.
Start Year 2016
 
Description Structures of antibiotic-lipid II complexes 
Organisation University of Warwick
Country United Kingdom 
Sector Academic/University 
PI Contribution We have used combination of solution and solid-state NMR (including 100 kHz spinning methodology) to solve structures of antibiotics in complexes with lipid II to inform rational drug development efforts.
Collaborator Contribution Synthesize lipid II.
Impact No outcomes yet. Publication in preparation.
Start Year 2015
 
Description Using of solvent Paramagnetic Relaxation Enhancements (PREs) in solid state 
Organisation Technical University of Munich
Country Germany 
Sector Academic/University 
PI Contribution Designed and performed NMR measurements on a range of systems.
Collaborator Contribution Provided expertise on computational protocols for using PREs for structure calculation.
Impact 10.1021/jacs.7b03875
Start Year 2015
 
Description ZapA 
Organisation University of Warwick
Department School of Life Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Started investigation of protein involved in bacterial wall biosynthesis by solution and solid-state NMR.
Collaborator Contribution Help with protein production and background data and expertise.
Impact no outputs yet
Start Year 2016
 
Description Analytical Science Networking and Partnership 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact We were involved in a workshop involving representatives from several companies (e.g. AstraZeneca, JEOL, Bruker, Syngenta, Pfizer, Unilever, Lubrizol, Linear Diagnostics etc.) to explore potential collaborations. One of the purposes of these workshops was to familiarize the representatives of the industry with our scientific capabilities and for the representatives of the industry to present us with problems they would like to tackle.
Year(s) Of Engagement Activity 2015,2016
URL http://www2.warwick.ac.uk/fac/sci/mas/aboutmascdt/
 
Description Info session with Mahidol University representatives 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Schools
Results and Impact A group of the representatives from Mahidol University, Bangkok, Thailand met with a number researchers to explore possibility of establishing links between Mahidol University and University of Warwick. We have presented our work and participated in a discussion about potential collaborations.
Year(s) Of Engagement Activity 2015
 
Description Organisation of an Alpine Conference on Magnetic Resonance in Solids 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact About 200 scientists at different stages of their careers participated in this conference leading to exchange of expertise.
Year(s) Of Engagement Activity 2019
URL https://alpine-conference.org/
 
Description Pfizer visit 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact One of my PhD students, Azzedine Dabo, gave a talk during collaborative visit to Pfizer campus in Sandwich, which increased interest in further collaborations.
Year(s) Of Engagement Activity 2015
 
Description Think Science - lecture 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact 150 students participated in a lecture about science and encouraging them to go into science. 4 high school teachers participated in discussion on UCAS admissions process.
Year(s) Of Engagement Activity 2016
 
Description Workshop on potential collaborations with Pfizer 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Several representatives from Pfizer gave a series of presentations followed by a discussion on establishing links.
Year(s) Of Engagement Activity 2015