Enhancing ubiquitin crystallization through surface-entropy reduction

Abstract

Ubiquitin has many attributes suitable for a crystallization chaperone, including high stability and ease of expression. However, ubiquitin contains a high surface density of lysine residues and the doctrine of surface-entropy reduction suggests that these lysines will resist participating in packing interactions and thereby impede crystallization. To assess the contributions of these residues to crystallization behavior, each of the seven lysines of ubiquitin was mutated to serine and the corresponding single-site mutant proteins were expressed and purified. The behavior of these seven mutants was then compared with that of the wild-type protein in a 384-condition crystallization screen. The likelihood of obtaining crystals varied by two orders of magnitude within this set of eight proteins. Some mutants crystallized much more readily than the wild type, while others crystallized less readily. X-ray crystal structures were determined for three readily crystallized variants: K11S, K33S and the K11S/K63S double mutant. These structures revealed that the mutant serine residues can directly promote crystallization by participating in favorable packing interactions; the mutations can also exert permissive effects, wherein crystallization appears to be driven by removal of the lysine rather than by addition of a serine. Presumably, such permissive effects reflect the elimination of steric and electrostatic barriers to crystallization.

Systematic analysis of residual density suggests that a major limitation in well‐refined X‐ray structures of proteins is the omission of ordered solvent

“…Yet, each of log[ P ( x > 0)], log[ P ( x < 0)], log[Φ( x > 0)], and log[Φ( x < 0)] bears a striking similarity across all the three models (Fig. ), even though they were obtained independently in different space groups . This similarity suggests that a common physical basis may exist for why they are incomplete.…”

“…Three highest‐resolution models for human ubiquitin models in the PDB are 4XOF at 1.15 Å, and 4PIJ and 4PIH both at 1.50 Å (Table ) . Ubiquitin is a small protein of 76‐amino acid residues.…”

“…This model is one of the most complete protein models in the PDB examined so far (Table I), and has the smallest R-factor gap with R Ratio of 1.22 (see Methods). [4][5][6][7][8][9][10][11][12][13][14][15] Evidence will be provided below that its corresponding residual ED map is the closest to true featurelessness with all the residual peaks contributed mainly by random noise present in the intensity data.…”

“…17 A few are selected here for this analysis ( Table I). [4][5][6][7][8][9][10][11][12][13][14][15] It is shown that the 5BV2 model 4 appears to be the only one whose intensity differences between the model and data are truly limited by random noise present in the diffraction data (see below).…”

“…3 and 4). [7][8][9][10] By this criterion, all of these models should be considered to be incomplete.…”