Solution structure by NMR spectroscopy

Lead Research Organisation: MRC Laboratory of Molecular Biology

Abstract

A fundamentally important aspect of research in molecular biology has always been the determination of detailed three-dimensional structures of biologically important molecules such as proteins, DNA and RNA. Knowledge of such structures, and particularly of how they interact with one another, is an essential prerequisite to understanding how these molecules function in living organisms, from which can follow breakthroughs in understanding and treating disease. Magnetic resonance spectroscopy (MRS), X-ray crystallography and electron cryo-microscopy (EM) are the main methods used to determine structures. X-ray crystallography and, for larger systems, EM are very efficient tools for obtaining detailed and accurate structures, but MRS has the advantage that it works in solution, so the molecule is seen in an environment more similar to that in an organism. This can be especially important when studying interactions between molecules. The work in my group, much of which involves collaborations with several other groups at LMB, focuses on determining solution structures and characterizing interactions for proteins involved in a wide variety of biologically and medically important areas, including DNA repair, vesicle trafficking, and regulation of gene expression.

Technical Summary

We will use NMR spectroscopy, and where appropriate X-ray crystallography, to determine the structures of a number of proteins and their complexes with DNA, with peptides and with other proteins. Where possible, high-resolution structures of the relevant complexes will be determined directly using extensive NOE measurements; in the cases of some weaker complexes (e.g. those of proteins with small peptides derived from the sequence of a larger binding partner), ligand footprinting will be carried out using a combination of chemical shift titrations, cross-saturation experiments and, where possible, intermolecular NOE measurements.
A key component of the work will be a study of human poly-ADPribose polymerase-1 (PARP-1); this protein detects and signals the presence of DNA single-strand breaks so as to initiate repair, and it is an important target in cancer therapy. NMR is particularly well-suited to characterise the intermolecular and interdomain interactions that underpin PARP-1’s molecular mechanism of action upon DNA recognition. Other targets for study will be selected from amongst the projects ongoing in other groups at LMB and CIMR, and will be undertaken as collaborations. In particular, protein structures and protein-peptide interactions relevant to vesicle trafficking and intracellular immunity will be studied, among others. Solution structures of a number of protein domains and their complexes will be determined.

Publications

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publication icon
Argentaro A (2007) Structural consequences of disease-causing mutations in the ATRX-DNMT3-DNMT3L (ADD) domain of the chromatin-associated protein ATRX. in Proceedings of the National Academy of Sciences of the United States of America

 
Description AICR Research Grant
Amount £95,682 (GBP)
Organisation Association for International Cancer Research 
Sector Charity/Non Profit
Country United Kingdom
Start 08/2010 
End 07/2012
 
Description Boehringer Ingelheim Fonds Scholarship
Amount € 57,600 (EUR)
Organisation Boehringer Ingelheim 
Sector Private
Country Germany
Start 11/2018 
End 10/2020
 
Description Boehringer PhD Studentship
Amount £50,520 (GBP)
Organisation Boehringer Ingelheim 
Sector Private
Country Germany
Start 10/2006 
End 09/2009
 
Description LMB/AZ Blue Sky
Amount £315,000 (GBP)
Funding ID BSF22 
Organisation AstraZeneca 
Department Astra Zeneca
Sector Private
Country United States
Start 08/2017 
End 08/2020
 
Description Ahel group 
Organisation University of Manchester
Department Cancer Research UK Manchester Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution Our collaborators initiated the project and carried out biological experiments, we determined structures and charaterised ligand binding by NMR spectroscopy and other biophysical techniques.
Collaborator Contribution Intellectual input
Impact Joint publication
Start Year 2007
 
Description Boulton group 
Organisation Cancer Research UK
Department Cancer Research UK London Research Institute (LRI)
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Our collaborators initiated the project and carried out biological experiments, we will determine structures and carry out further biophysical experiments.
Collaborator Contribution Initiated the project, and supplied materials and intellectual input
Impact None so far
Start Year 2014
 
Description Bruker 
Organisation Bruker Corporation
Department Bruker BioSpin
Country Germany 
Sector Private 
PI Contribution Together with the Nagai group at LMB, we prepared protein samples for structure determination. These included protein samples in which native zinc ions had been replaced by 113-Cd ions. The structure determination was carried out by us, using a combination of NMR data collected by us and by Bruker.
Collaborator Contribution Our partners at Bruker collected 113-Cd NMR data that formed an important part of the input to the structure determination of the protein studied (Bud31p).
Impact Joint publication
Start Year 2012
 
Description Higgs group 
Organisation Medical Research Council (MRC)
Department MRC Molecular Haematology Unit
Country United Kingdom 
Sector Public 
PI Contribution Our collaborators initiated the project and carried out biological experiments, we determined structures and charaterised ligand binding by NMR spectroscopy and other biophysical techniques.
Collaborator Contribution Intellectual input
Impact joint publication
Start Year 2006
 
Description James group 
Organisation Medical Research Council (MRC)
Department MRC Laboratory of Molecular Biology (LMB)
Country United Kingdom 
Sector Public 
PI Contribution Our collaborators initiated the project and carried out biological experiments, we are characterising ligand binding and conformational changes by NMR spectroscopy.
Collaborator Contribution Our partners initiated the project and carried out biological and other structural experiments.
Impact None so far
Start Year 2015
 
Description Kay group 
Organisation Medical Research Council (MRC)
Department MRC Laboratory of Molecular Biology (LMB)
Country United Kingdom 
Sector Public 
PI Contribution Our collaborators initiated the project and carried out biological experiments, we determined structures.
Collaborator Contribution Intellectual input and materials (samples for structural characterisation)
Impact Joint publication
Start Year 2006
 
Description Nagai group 
Organisation Medical Research Council (MRC)
Department MRC Laboratory of Molecular Biology (LMB)
Country United Kingdom 
Sector Public 
PI Contribution Our collaborators initiated the project and carried out biological experiments, we determined structures.
Collaborator Contribution Intellectual input and materials (samples for structural characterisation)
Impact Joint publication
Start Year 2006
 
Description Owen group 
Organisation University of Cambridge
Department Cambridge Institute for Medical Research (CIMR)
Country United Kingdom 
Sector Academic/University 
PI Contribution Our collaborators initiated the project and carried out biological experiments, we will determine structures and carry out further biophysical and biological experiments.
Collaborator Contribution Intellectual input and materials (samples for structural characterisation)
Impact None so far
Start Year 2012
 
Description Pascal group (Montreal) 
Organisation University of Montreal
Country Canada 
Sector Academic/University 
PI Contribution Both partners are contributing expertise and intellectual input as well as contributing to the writing of joint papers. Experimental contributions from my group comprise mainly NMR structural analyses of fragments and subcomplexes of protein and protein mutants with DNA, as well as biophysical and biochemical characterisations of these species.
Collaborator Contribution We are collaborating on the DNA-recognition and activation of the enzyme human PARP-1. Both partners are contributing expertise and intellectual input as well as contributing to the writing of joint papers. Experimental contributions from my partners comprise further biophysical and biochemical characterisations of fragments and subcomplexes of protein and protein mutants with DNA, as well as in vivo characterisations of these species.
Impact Joint publication
Start Year 2015
 
Description Pascal group (Philadelphia) 
Organisation Thomas Jefferson University
Country United States 
Sector Academic/University 
PI Contribution Both partners contributed expertise and intellectual input as well as contributing to the writing of a joint paper. Experimental contributions from my group comprised mainly NMR structural analyses of fragments and subcomplexes of protein and protein mutants with DNA, as well as biophysical and biochemical characterisations of these species. During 2015 the collaborating group moved to University of Montreal, so this collaboration is now listed as having ceased and a new collaboration is listed for University of Montreal. The joint paper published in 2015 is listed under the collaboration with University of Montreal.
Collaborator Contribution We are collaborating on the DNA-recognition and activation of the enzyme human PARP-1. Both partners will contribute expertise and intellectual input as well as contributing to the writing of joint papers. Experimental contributions from my partners comprise further biophysical and biochemical characterisations of fragments and subcomplexes of protein and protein mutants with DNA, as well as in vivo characterisations of these species.
Impact None so far
Start Year 2014
 
Description Rhodes group 
Organisation Medical Research Council (MRC)
Department MRC Laboratory of Molecular Biology (LMB)
Country United Kingdom 
Sector Public 
PI Contribution Our collaborators initiated the project and carried out biological experiments, we determined structures.
Collaborator Contribution Intellectual input and materials (samples for structural characterisation)
Impact Joint publications
Start Year 2006
 
Description Stewart group 
Organisation Medical Research Council (MRC)
Department MRC Laboratory of Molecular Biology (LMB)
Country United Kingdom 
Sector Public 
PI Contribution Our collaborators initiated the project and carried out biological experiments, we determined structures and characterised ligand binding by NMR spectroscopy.
Collaborator Contribution Intellectual input and materials (samples for structural characterisation)
Impact Joint publications
Start Year 2006
 
Description Walker group 
Organisation Medical Research Council (MRC)
Department MRC Mitochondrial Biology Unit
Country United Kingdom 
Sector Public 
PI Contribution Our collaborators initiated the project and carried out biological experiments, we determined structures and charaterised ligand binding by NMR spectroscopy.
Collaborator Contribution Intellectual input
Impact joint publications
Start Year 2006
 
Description West group 
Organisation Cancer Research UK
Department Cancer Research UK London Research Institute (LRI)
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Our collaborators initiated the project and carried out biological experiments, we determined structures and charaterised ligand binding by NMR spectroscopy and other biophysical techniques.
Collaborator Contribution Intellectual input
Impact Joint publication
Start Year 2007
 
Description Cambridge Independent interview 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact I was interviewed by the Cambridge Independent newspaper in connection with a story they published on the collaboration between LMB and AstraZenecca concerning NMR spectroscopy. This story was about both the sharing of laboratory space (whereby one of AZ's NMR spectrometers has been accommodated within the LMB's dedicated building for magnetic resonance spectroscopy), and also my research project on the activation mechanism of human PARP-1, which is in part joint-funded under an LMB-AZ Blue skies award.
Year(s) Of Engagement Activity 2018
URL http://www.cambridgeindependent.co.uk/business/science/magnetic-attraction-of-astrazeneca-to-mrc-lab...
 
Description Participation in LMB Open Day 2017 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact I gave a short talk on the principles and applications of NMR spectroscopy to groups of visitors to the LMB Open Day who had selected the Structural Studies lab tour.
Year(s) Of Engagement Activity 2017
 
Description School Visit 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact 26 AS-level students from all over the UK visited the LMB and took part in a day of practical work at 1st year degree level. This helped the students put their coursework into perspective and gave insight into the workings of a real laboratory, with the aim of inspiring students to choose a degree, or indeed a career, in biological sciences.
Year(s) Of Engagement Activity 2014,2015