The Conformational Preference of Chemical Crosslinkers Determines the Crosslinking Probability of Reactive Protein Residues.

Chemical cross-linking mass spectrometry (XLMS) is an emerging technique in structural biology. Providing the cross-linked peptides are identified by mass spectrometry with high confidence, a distance restraint can be applied between the two reactive protein residues, with the upper bound corresponding to the maximal span of the cross-linker. However, as the upper bound is typically over 20 angstrom, cross-link distance restraints are unrestrictive and provide a marginal improvement in protein structural refinement. Here we analyze the experimental cross-links for lysine or acidic residues and show that the distribution of C beta-C beta' distances can be described with two overlapping Gaussian species. In addition to the pairwise occurrence probability of the reactive protein residues, we show that the distribution profile of the cross link distances is determined by the intrinsic conformational propensity of the cross-linker. The cross-linker prefers either a compact or extended conformation and, once attached to a reactive protein residue, predominantly an extended conformation. Consequently, the long-distance Gaussian species occurs at a much higher probability than the short-distance species in the observed cross-links. Together, the probabilistic distribution of the cross-link distance allows the construction of a more restrictive restraint for structural modeling and better use of the XLMS data.