MRC National Institute for Medical Research, London
Science for Health
Martin Webb group project:
Techniques and expertise
An important feature of a research group is the type of core techniques used. Within this research team there is expertise in a number of techniques.
Fluorescence spectroscopy
A core method of the laboratory with a number of different types of labeling strategy to provide sensitive probes of biochemical function, outlined in other web pages of this research team.
Kinetic measurements
A core approach is to follow reaction kinetics in real time, to explore the biochemical processes underlying the biological system of interest. Because the time scales of processes of interest are often on the millisecond to second timescale, this requires the use of rapid reaction techniques. We use two rapid mixing techniques, 'stopped flow fluorescence' and 'quenched flow'. In the former, two reactants are mixed rapidly and a fluorescence signal is followed with time.
Stopped flow apparatus
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In quenched flow measurements, the two reactants are mixed and the reaction is allowed to proceed for a particular time. The reaction is then quenched (usually in acid to denature proteins), for subsequent analysis of the extent, for example by hplc.
We also use laser flash photolysis of caged compounds to initiate a reaction so that it can be followed on a millisecond timescale.
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For example, caged ATP is a precursor of ATP that is biochemically inert and so can be present in a reaction mix that is otherwise complete. On photolysis by a laser flash, ATP is released and can then interact with the protein of interest
Isotope exchange methods
using stable oxygen isotopes to follow the fate of particular oxygen atoms during ATPase reactions. In doing so this gives information about the kinetics of various steps during these reactions. Analysis is by mass spectroscopy.
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The [18O3]ATP typically used for these investigations.
Nucleotide chemistry
Used to synthesise novel fluorescent nucleotides. Examples also include the [18O3]ATP and caged ATP shown above.
Analytical ultracentrifugation
To investigate complex formation between proteins and/or DNA by measuring sedimentation.
Basic methods
Along with many biochemical research teams, there is a mixture of basic techniques needed to prepare our target proteins: mutagenesis, expression especially in bacteria, chromatography including hplc, along with standard analytical techniques such as gel electrophoresis and absorbance spectroscopy.
Collaborative techniques
The research team collaborates within the Division and wider NIMR to make use of others’ expertise, for example to use single molecule techniques (Justin Molloy).
Selected publications
- Toseland, CP; Martinez-Senac, MM; Slatter, AF and Webb, MR (2009)
The ATPase cycle of PcrA helicase and its coupling to translocation on DNA.
Journal of Molecular Biology 392, 1020-1032 PubMed abstract - Phillips, R. A., Hunter, J. L., Eccleston, J. F., and Webb, M. R. (2003)
The mechanism of Ras GTPase activation by neurofibromin.
Biochemistry 42, 3956-3965 - Dillingham, M. S., Wigley, D. B., and Webb, M. R. (2000)
Demonstration of unidirectional single-stranded DNA translocation by PcrA helicase: measurement of step size and translocation speed.
Biochemistry 39, 205-212 - Webb, M. R., and Corrie, J. E. T. (2001)
Fluorescent coumarin-labeled nucleotides to measure ADP release from actomyosin.
Biophysical Journal 81, 1562-1569
Webb group
Recent publications
Research projects
- DNA helicases: mechanism of coupling ATP hydrolysis to DNA translocation
- Myosins: the relationship between ATP hydrolysis and mechanical events
- Development of fluorescent biosensors and other probes
- The role of small G proteins in cell signalling
Techniques and expertise
Webb biography
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