The mechanism and roles of nucleoside triphosphatases in cell regulation and motility

Lead Research Organisation: MRC National Inst for Medical Research

Abstract

Cells move objects such as proteins, organelles or DNA by a variety of methods. Mostly the methods share some degree of underlying mechanism by which motility proteins undergo a series of cyclical interactions with other macromolecules. The interactions involve considerable changes in shape of the proteins that in turn produce the motion. The energy for movement is provided by enzymic hydrolysis of the small, high energy molecule, ATP. The project is aimed at understanding the mechanism of such motility systems. Two systems are being studied in particular. The archetypical motility system is the organized array of protein filaments in muscle that produce tension and contraction. In contrast, helicases are proteins involved in most DNA processing. They are responsible for separating double-stranded DNA into single strands, that can then undergo many of the sequence-dependent functions or, for example, repair. The project in particular uses physical techniques to follow the biochemical processes in real time and compare with the motility.|Signalling pathways within cells also requires a series of cyclical interactions, particularly between proteins and one class of molecules involved in signalling pathways, small G proteins also depend on hydrolysis of a closely related high energy molecule, GTP. This hydrolysis also drives protein-protein interactions and this relationship is being elucidated.

Technical Summary

Motility within cells involves precise, dynamic interactions between proteins, normally driven by nucleoside triphosphate hydrolysis. We aim to understand the molecular basis for the coupling between this hydrolysis and movement, with particular emphasis on the actomyosin system of muscle and movement of proteins along DNA (energy transduction). Small G proteins are involved in signalling within cells and their action also depends on precise interaction with other proteins in order to transmit signals (signal transduction), coupled to nucleoside triphosphate hydrolysis. As part of the project to understand such coupling, novel biosensors are being developed to probe ligand (inorganic phosphate and nucleoside diphosphate) release during the coupled nucleoside triphosphate hydrolysis reactions. A variety of biochemical approaches are used with particular emphasis on understanding the chemical mechanisms involved in these transducing systems and the dynamics of the reactions. Isotope labelling and exchange, fluorescence probes are used to determine the kinetics of individual reaction steps, at different levels of organization and activation. Protein engineering is being used for mutational structure-function studies and to develop new protein-based sensors. Processes are followed in real time, particularly using fluorescence signals. Novel fluorescent protein sensors for inorganic phosphate and for nucleoside diphosphate have been developed in this laboratory. They are being used to investigate several systems where these products are released, with particular emphasis on motility involving ATP hydrolysis. They are being applied in muscle fibres to probe the relationship between the biochemical cycle of ATP hydrolysis on myosin and the mechanical properties of tension generation and muscle contraction. Studies are revealing how the tension generation is controlled at particular points of the ATP hydrolysis cycle.|Helicases are responsible for separating duplex DNA strands and as such are involved in many DNA functions. Most, if not all, require ATP hydrolysis in order to move along DNA. The relationship between DNA movement and ATP hydrosis is being elucidated, particularly making use of specific fluorescence signals introduced to ATP, protein or DNA. Thus a fluorescent label located along DNA can provide a signal for when the helicase passes that point and thereby provide a timing assessment for movement. The phosphate sensor can provide a measurement of the amount and rate of ATP hydrolysis over exactly the same time period. Phosphate and ADP sensors are potentially important tools both clinically (both sensors) and environmentally (phosphate). Use of such sensors is widespread in biochemical research. Some of the sensors have been patented and are available commercially.

Publications

10 25 50
 
Description MRCT Development Gap Fellowship
Amount £100,732 (GBP)
Organisation MRC-Technology 
Sector Private
Country United Kingdom
Start 01/2012 
End 01/2014
 
Description MRCT Development Gap Funding
Amount £85,084 (GBP)
Organisation MRC-Technology 
Sector Private
Country United Kingdom
Start  
 
Description MRCT Development Gap Funding
Amount £74,746 (GBP)
Funding ID A853/0164 
Organisation MRC-Technology 
Sector Private
Country United Kingdom
Start 08/2013 
End 04/2015
 
Title ADP biosensor 
Description Reagentless biosensor developed, based on fluorescent proteins that respond to the target molecule. 
Type Of Material Technology assay or reagent 
Year Produced 2006 
Provided To Others? Yes  
Impact ADP biosensor available commercially. 
 
Title ADP biosensor 
Description Reagentless biosensor for ADP measurement by fluorescence 
Type Of Material Technology assay or reagent 
Year Produced 2010 
Provided To Others? Yes  
Impact Licenced to biotech companies / pharma 
 
Title ATP biosensor 
Description Fluorescent reagentless biosensor for ATP 
Type Of Material Technology assay or reagent 
Year Produced 2016 
Provided To Others? Yes  
Impact Novel methhod to measure ATP in real time 
 
Title New phosphate biosensor (rhodamine) 
Description Reagentless biosensor for phosphate with rhodamine fluorescence read-out 
Type Of Material Technology assay or reagent 
Year Produced 2010 
Provided To Others? Yes  
Impact Licenced to companies for potential as a laboratory reagent 
 
Title Phosphate assay 
Description New colorimetric, coupled-enzyme assay for inorganic phosphate. 
Type Of Material Technology assay or reagent 
Year Produced 2006 
Provided To Others? Yes  
Impact Now commercially available from four companies 
 
Title Phosphate biosensor 
Description Reagentless biosensor developed, based on fluorescent proteins that respond to the target molecule. 
Type Of Material Technology assay or reagent 
Year Produced 2007 
Provided To Others? Yes  
Impact A phosphate sensor available commercially. 
 
Title Wide concentration range phosphate biosensor 
Description Development of a novel form of the phosphate biosensor to enable measurements over a wide concentration range 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact Thgis enable measurements over a wide concentration range of inorganic phosphate in real tim e 
 
Title ssDNA biosensor 
Description Reagentless biosensor for fluorescent measurement of ssDNA 
Type Of Material Technology assay or reagent 
Year Produced 2009 
Provided To Others? Yes  
Impact Used to assay helicase activity. New version now available with 20X fluorescence change 
 
Description DNA helicases 
Organisation University of Bristol
Department School of Biochemistry Bristol
Country United Kingdom 
Sector Academic/University 
PI Contribution Reagents, methodology, development of biosensor
Collaborator Contribution Reagents, kinetic measurements
Impact 18599625, patent application, 21071401, 20350930
 
Description Muscle and myosin 
Organisation Imperial College London
Country United Kingdom 
Sector Academic/University 
PI Contribution Development of assay techniques, interpretation of data
Collaborator Contribution Wider reagents, techniques
Impact 16540476, 19383472, 17496049, 20923650, paper in press
Start Year 2006
 
Description Myosin kinetics 
Organisation Eastern Virginia Medical School
Country United States 
Sector Academic/University 
PI Contribution Development of assay techniques (Pi sensor, coumarin-labeled nucleotides), kinetic measurements
Collaborator Contribution Wider techniques and protein preparationsWider techniques and protein preparations
Impact 19008235, 18668042, 17965414, 17059220
 
Description Myosin kinetics 
Organisation National Institutes of Health (NIH)
Country United States 
Sector Public 
PI Contribution Development of assay techniques (Pi sensor, coumarin-labeled nucleotides), kinetic measurements
Collaborator Contribution Wider techniques and protein preparationsWider techniques and protein preparations
Impact 19008235, 18668042, 17965414, 17059220
 
Description Plasmid replication 
Organisation University of Leeds
Department School of Biochemistry and Microbiology Leeds
Country United Kingdom 
Sector Academic/University 
PI Contribution Reagents, methodology, mehanistic measurements
Collaborator Contribution Reagents, methodology
Impact 19473041
Start Year 2007
 
Description Single molecule studies on ParM 
Organisation University of Illinois
Department Department of Physics
Country United States 
Sector Academic/University 
PI Contribution Preparation of reagents. Design of experiments and analysis
Collaborator Contribution DProvided data to address future eevelopment of project
Impact Paper ipublished
Start Year 2011
 
Title ADP biosensor 
Description Reagentless biosensor for fluorescent assay of ADP 
IP Reference WO2010032001 
Protection Patent application published
Year Protection Granted 2010
Licensed Yes
Impact Assay for kinases
 
Title ATP biosensor 
Description Fluorescent reagentless biosensor for real time assays of ATP 
IP Reference GB1505662.5 
Protection Patent application published
Year Protection Granted 2015
Licensed No
Impact Method to measure ATP form,ation
 
Title Biosensor for detection and visualisation of single-stranded DNA 
Description Reagentless biosensor for assaying ssDNA 
IP Reference WO2008152379 
Protection Patent application published
Year Protection Granted 2008
Licensed No
Impact Used to assay helicases
 
Title IMPROVED INORGANIC PHOSPHATE ASSAYS 
Description Reagentless biosensor for fluorescent determination of Pi 
IP Reference WO2007026155 
Protection Patent granted
Year Protection Granted 2007
Licensed Yes
Impact Patents applied for in UK and granted in US, Australia and Europe in 2000. Current licenses.
 
Title Improved biosensor for single-stranded DNA 
Description Improved method to assay ssDNA formation in real time 
IP Reference P10276GB 
Protection Patent application published
Year Protection Granted 2015
Licensed No
Impact None yet
 
Title Wide range phosphate biosensor 
Description Method to measure a wide range of inorganic phosphate concentrations 
IP Reference GB1505256.6 
Protection Patent application published
Year Protection Granted 2015
Licensed No
Impact None yet
 
Description Nuffield Science Bursary Scheme 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Schools
Results and Impact Lab hosted a summer student in this scheme. Student then took part in a poster day.

No subsequent impact
Year(s) Of Engagement Activity 2007,2008,2009,2010