Peripatetic proteins

A common perception of X-ray crystallography is that it is the method of choice for determining the ‘‘static’’ structures of proteins and macromolecular complexes, but that it provides little information about dynamics. Crystallographers themselves would argue that this characterization is unjustified. In the first place, crystallographic ‘‘B-factors’’ can provide an atom-byatom measure of the displacements experienced by each atom within the crystal. Second, proteins in different crystallographic environments, or in the presence of ligands, or for other reasons, can display striking changes in structure, which are often relevant to function. That B-factors are not just an artifact of the crystal environment was shown in early experiments by David Phillips’ group. They compared the B-factors of hen and human lysozymes in unrelated crystal forms. Although the packing of the lysozyme molecules was different, and involved different residues on the surface of the protein, the B-factors in the respective crystal structures remained largely the same. In other words, the parts of lysozyme that were most mobile in one crystal were also most mobile in the other. It was reasonable to assume that these regions also had higher mobility in solution. In this issue of Protein Science, Alber and coworkers discuss crystallographic evidence of a different type for the dynamic behavior of individual protein side chains. Specifically, they present evidence from well-refined crystal structures suggesting that 18% or so of the side chains have multiple conformations. There are anecdotal reports from early crystal structure determinations of cases where individual amino acid side chains are peripatetic in the sense that they wander between two or more distinct rotamer conformations. This behavior has become more apparent as higher-resolution protein structures have become available. Alber and coworkers have taken this analysis to the next level by developing the program Ringer to systematically search for weak electron density that might suggest alternative side-chain configurations. Take, as an example, a methionine residue. If the side chain is rotated about the CaACb bond (i.e., about v), the Cc atom will trace out a circle (or a ‘‘ring’’). If the methionine side chain is ‘‘well ordered,’’ there will be one location on the circle (e.g., with v 60 ) at which the electron density has an obvious maximum perhaps three standard deviations or so above the mean density. What Lang et al. show, however, is that there may also be a weaker electron density peak at another rotamer position, i.e., v 180 or v 300 . To the extent that this weaker peak is statistically significant, it suggests that the methionine side chain has two distinct conformations. The question is ‘‘What is statistically significant? ’’ To address this question, Lang et al. carry out a control in which they apply the Ringer search procedure to alanine residues. Because alanine has no Cc atom, the behavior of the electron density at putative Cc sites should correspond to ‘‘noise. ’’ On this basis, they conclude that an electron density threshold of 0.3r can be used to identify alternative side-chain conformations. The value of 0.3r is a guide rather than a hard threshold. *Correspondence to: Brian W. Matthews, Institute of Molecular Biology, 1229 University of Oregon, Eugene, OR 97403-1229. E-mail: brian@uoregon.edu