Manchester Centre for Biophysics and Catalysis (MCBC)

(Time resolved) Spectroscopy

The Centre has major interests in the application of both static and time-resolved spectroscopic methods for studying catalytic and dynamical processes in biological macromolecules. Such techniques allow us to obtain important structural and mechanistic information about the molecule or reaction of interest.

To support our ‘high-end’ spectroscopy equipment we also have a number of peripheral instruments, including standard spectrophotometers and fluorimeters, a number of which are maintained in anaerobic environments. (see Anaerobic facilities).

[-] Advanced fluorescence techniques

Fluorescence spectroscopy is a powerful research tool that we use in MCBC to probe a wide range of molecular processes, including the interactions of fluorophores with the solvent, conformational changes, binding interactions, rotational diffusion of biomolecules, distances between sites on biomolecules and also the rate of chemical reactions.

As well as our conventional fluorescence techniques we also use a variety of more sophisticated fluorescence methods to obtain dynamical information about the molecules of interest. These include fluorescence lifetime measurements, fluorescence resonance energy transfer, fluorescence anisotropy and time-resolved fluorescence. In addition, we also use fluorescence spectroscopy for our single molecule studies (see single molecule approaches).

[-] Circular dichroism spectroscopy

CD spectrometer

Circular Dichroism (CD) provides important information about the secondary structure of biological macromolecules and is a key technique for studying the folding/unfolding of proteins. CD spectra are obtained when the optically active molecules absorb left and right hand circular polarised light slightly differently. CD spectroscopy is a quick method that allows us to probe the secondary structure of proteins and DNA in solution over a range of different conditions.

[-] Electrochemical approaches

Electrochemical approaches

We are able to probe the redox properties of biological molecules by using different electrochemical approaches. Our potentiometry apparatus provides a technique to measure the potentials of various molecules and allows us to determine electron transfer pathways in complex redox systems. In addition, we are able to couple surface plasmon resonance methods with elecrodes in order to probe for structural changes associated with electrochemical changes in the biomolecule.

[-] FTIR spectroscopy

FTIR data

We use FTIR vibrational spectroscopy to probe the structure and dynamics of biological molecules. It uses the infra-red region of the electromagnetic spectrum to provide a ‘fingerprint’ of a molecule with absorption peaks that correspond to the frequencies of the vibrations between the bonds of the molecule. We have recently coupled our FTIR instrument to a stopped-flow device to facilitate time-resolved measurements of this spectroscopic technique (see stopped-flow spectroscopy). In addition, we have the capability to perform infrared imaging from solid surfaces using focal plane array detectors.

[-] Raman spectroscopy

We use a range of Raman spectroscopies (conventional Raman, UV resonance Raman, Raman optical activity and surface enhanced Raman scattering) to study the structure and behaviour of biological molecules. These Raman methods are powerful vibrational tools for studying the conformations, and conformational changes, of proteins, nucleic acids and viruses in solution (water is not a problem with Raman spectroscopy). Inherent advantages of these methods are their sensitivity to biomolecular structure (tertiary fold, secondary structure motifs, specific residues), their wide applicability (solutions, solids, films, living cells, tissue samples, no molecular size limits, no crystals required), and portability as well as microscopic analysis.

[-] Stopped flow spectroscopy

Stopped-flow spectroscopy is one of the most frequently used rapid kinetics techniques enabling reactions to be followed on a millisecond timescale by using a variety of spectroscopic methods. In addition to our conventional stopped-flow instruments we also have a number of highly specialised stopped-flows:

  • High Pressure stopped-flow instrument: allows stopped-flow measurements to be made at pressures up to 2000 bar.
  • Cryogenic stopped-flow instrument: to facilitate measurements at temperatures down to -90°C.
  • Magnetic Field Effect stopped-flow instrument: modified to enable magnetic field effect experiments on a range of enzyme reaction systems.
  • FTIR stopped-flow instrument: to follow the kinetics of spectral changes in the infra-red region that are associated with vibrational changes in the reactants/enzyme.

[-] Temperature jump

Temperature jump spectroscopy provides the opportunity to follow reactions on the microsecond timescale. An equilibrated reaction solution is rapidly heated within a few microseconds by 10-12°C using a short pulse of current at high voltage. A new equilibrium is reached at the higher temperature which can then be followed spectroscopically.

Related technologies: