The British Biophysical Society

Biophysics Instrumentation and Methods, and its Interface with Biology and Chemistry

John Helliwell

 

Biophysics is an interdisciplinary subject with a dynamic interplay between biology, chemistry and physics. Instrumentation and methods are key components for elucidating structure and function at the molecular level.

X-rays, electrons and neutrons are very important for the study of biological macromolecules by diffraction methods using crystals, fibres or solution states of matter, and which allow their structures to be probed in detail.

Electrons can also be readily focused, making direct imaging feasible, reaching to the atomic level (~5Å) in the electron microscope. X-rays are less readily focused but nevertheless can be to some extent thus allowing the current development of X-ray microscope techniques, at ~100Å resolution, on wet biological specimens.

X-ray crystallography remains the paramount method as the most detailed structure determination technique. Its capability has been transformed for biological macromolecules by harnessing the synchrotron X-radiation (SR) emitted from electron (or positron) particle accelerators or storage rings. SR is intense and tunable opening up new methods for the study of structure relevant to chemical reactivity or other biological processes. Several categories of application include the following. Firstly, MAD (multi-wavelength anomalous dispersion) opens up more certain and quicker structure determination as well as better element discrimination to finely probe the role of key metals in special cases. Secondly with SR it is possible to reach new levels of structural detail, ie ultra high resolution, which can thereby reveal fine differences in chemical bonding or hydrogen atoms in the protein. Thirdly, it is now possible to undertake even time-resolved diffraction covering picosecond through nanosecond, microsecond, millisecond to kilosecond time domains of structural processes to study biological functions directly, such as enzyme mechanism and photosensitive cycles.

Crystallisation is possibly the most difficult aspect of X-ray crystallography. The biophysical chemistry of protein crystallisation is under intense study. The fluid physics conditions of microgravity, for example, allows sedimentation and convection free protein crystal growth, which can therefore be diffusion controlled, thus allowing the weak interplay of intermolecular forces involved in crystallising proteins to develop under quiescent conditions. This is a relatively new area where choice of crystallisation geometry, and criteria to define good, stable conditions of microgravity in a given space flight mission are being established. Moreover a battery of techniques for crystallisation monitoring (e.g. interferometry, CCD video), crystal growth (atomic force microscopy) and crystal characterisation techniques (e.g. mosaicity, topography) are now allowing optimisation of protocols for crystallisation and crystal growth. This has important implications for the growth of crystals that have been found hitherto to be difficult to grow. Also it is of considerable interest to produce bigger protein crystals for neutron diffraction studies of hydrogen deuterium exchange, in particular cases.

X-rays damage crystals and ultimately limit the diffraction data but through the freezing of protein crystals, greatly extended crystal lifetime has been obtained. Cryocrystallography is also used for freeze trapping of structural intermediates. The time of such a structural state has to be established in the crystalline state. This is achieved by room temperature microspectrophotometry, if a chromophore is available, and/or by time-slicing diffraction data acquisition. Cryocrystallography thus complements time-resolved diffraction techniques.

Dynamical aspects of structures are very important. These are experimentally accessible through spectroscopic techniques such as NMR, as well as theoretically through molecular dynamics (MD) calculations. NMR also is used extensively now for structure determination of biological macromolecules and without the need for a single crystal! NMR has a current molecular weight ceiling of ~20 kDa and a coordinate precision of ~1Å. Thus NMR and X-ray crystallography complement each other well.

This is just a glimpse of biophysics. The BBS home page contains various further examples of the application of these biophysical and chemical methods in the study of biological macromolecules. Moreover the respective links to the home pages of the Committee members' laboratories more extensively show the range of fundamental research work that is going on in Britain today, as well as the more applied aspects such as molecular sensors/biosensors or drug design, although the details of these may be restricted for commercial reasons!

Reading List

J.R. Helliwell
"Macromolecular Crystallography with Synchrotron Radiation"
Cambridge University Press (1992)

J.R. Helliwell and P M Rentzepis
"Time-Resolved Diffraction" Oxford University Press 1997

J.R. Helliwell and M. Helliwell
"X-ray crystallography in structural chemistry and molecular biology" (1996) Feature Article for Chem. Comm., No. 14, 1595-1602.

N.E. Chayen, T.J. Boggon, A. Cassetta, A. Deacon, T. Gleichmann, J. Habash, S.J. Harrop, J.R. Helliwell, Y.P. Nieh, M.R. Peterson, J. Raftery, E.H. Snell, A. Hadener, A.C. Niemann, D.P. Siddons, V. Stojanoff, A.W. Thompson, T. Ursby and M Wulff
"Trends and challenges in experimental macromolecular crystallography" (1996) Quarterly Reviews in Biophysics, 29:3, 227-278.

John Helliwell is Professor of Structural Chemistry at the University of Manchester and is a Joint Appointee with CCLRC.