the area under such a curve represents the entropy change for the transition. From this information the
value of Gcan be evaluated at any temperature.
Differential scanning calorimetry data can also provide important information concerning the co-operativity
of a thermal transition. This can be achieved by comparison of the magnitudes of the model-dependent
van’t Hoff enthalpy (obtained by shape analysis of the calorimetric data) and the calorimetric enthalpy. If
HvHHcaland there are no chemical or instrumental kinetic limitations, such that Tmis independent of
both scan rate and concentration, then the transition proceeds in a two-state manner and meaningful thermo-
dynamic data can be obtained by examination of the temperature dependence of an equilibrium property. If
HvHHcal, then the transition must involve a significant number of intermediate states. Conversely, if
HvHHcal, then aggregation is implicated. The HvH/Hcalratio provides quantitative insight into the
nature of the transition. Specifically, it provides a measure of the fraction of the structure that melts as a
single thermodynamic entity (i.e.it defines the size of the co-operative unit). This is a unique advantage of
DSC in the study of biological molecules and when combined with ITC, thermodynamic studies over a
wide temperature range and under a variety of solution conditions can be carried out. These techniques
offer a powerful tool to probe the energetics of macromolecule stability and ligand association reactions.
11.5 Microscop y
11.5.1 Electron Microscop y
Electron microscopy (EM) is an invaluable tool for gaining structural information on systems that are too
large for the atomic resolution methods of X-ray diffraction or NMR, and yet too small for optical micro-
scopy.34,35Electron microscopy uses beams of electrons with wavelengths between 0.001 and 0.01nm. The
resolving power of EM can be below 2nm but is largely restricted by factors such as radiation damage and
the process of sample preparation.
The lenses in an electron microscope consist of shaped electric or magnetic fields. Electrons from a heated
tungsten filament are accelerated across a voltage difference of up to 100kV and the sample under study is
examined in a vacuum. In transmission EMthe electron beam is passed through the sample, then through a
suitable lens system and is detected on a fluorescent plate or photographic film. In scanning EM, the electron
beam is focused down to a point and scanned across the specimen. Secondary electrons are produced when
electrons interact with the target specimen and these secondary electrons are detected and used to build up
a raster image of the sample.
Both transmission and scanning EM require samples to be dehydrated and often coated by deposition
of a film of either carbon or a metal such as tungsten. For nucleic acid analysis, one of the oldest means of
sample preparation involves the combination of the nucleic acid with a thin film of denatured protein floating
on water. The nucleic acid and protein film can be lifted off the surface onto a grid and then, after drying,
coated with a fine layer of palladium or uranium. The process of coating, or shadow casting, involves deposit-
ing the metal from a heated filament that is oriented at an acute angle to the supporting grid. The effect of
the metal deposition is to highlight regions that are raised above the grid. In order to achieve good contrast,
either the samples can be shadowed from several angles or can be rotated during the coating procedure.
The protein film has two benefits: one is that it supports and protects the DNA during preparation; the other
is it causes thickening of the DNA strands. Nucleic acids will also bind directly to carbon supports that have
been physically and chemically pre-treated in a suitable fashion. They can then be dried and shadowed in
the same manner as in film deposition.
The technique of cryogenic EM(cryo EM)^36 uses samples that have been flash frozen into a thin film of
vitreous water. The sample is prepared as an aqueous solution and applied to a carbon grid with holes of
about 3m. Blotting of the grid followed by quick freezing in ethane at near its freezing temperature produces
vitrified samples which can be directly imaged by EM.
While the contrast in cryo EM is relatively poor, it has the great advantage that a biological specimen is
preserved in its solution conformation without many of the possible artefacts that can result from the
446 Chapter 11