Section 19.4.3.2. Variation of internal contrast

A second form of contrast variation can be achieved by replacing the hydrogen atoms in biological molecules with deuterium. If, in addition, it is possible to deuterate selected regions of a particle, internal contrasts can be modified to gain information about substructures. In some cases, the opportunity arises because significant biochemical differences are present, as between the RNA and protein portions of a ribosome, the DNA and protein of a nucleosome, or the lipid and protein of a lipoprotein particle. While the intrinsic contrast may be sufficient to provide key information [as, for example, the early finding that DNA is on the outside of nucleosomes (Bradbury et al., 1976; Uberbacher et al., 1982)], it can be accentuated by incorporation of deuterated biochemical precursors or reconstitution from separately labelled components.

Internal contrast can be created in single molecules by differential incorporation of biosynthetic precursors or by chemical synthesis. Differential incorporation was first used to test models of bacteriorhodopsin, using the incorporation of deuterated amino acids supplied to a culture of halophilic archae (Engelman & Zaccai, 1980), and internal labelling was used to document conformations of cholesteryl esters that had been chemically labelled in key positions (see below). Reconstitution from purified components has been used to place deuterated proteins in ribosomes and in other complexes, again with the aim of creating internal contrast to enhance the information obtained in a neutron experiment.

In general, the creation of internal contrast can be viewed as a strategy for enriching the low information content of a solution-scattering experiment by building additional information into the sample. By design, it is known what has been labelled, so the scattering given by contrasting elements provides information about the relationships of the labelled parts to each other or to the particle as a whole. A particularly informative (but difficult) strategy is to use internal contrast to measure distances between locations in a molecule or complex. Such measurements are discussed below.