Salvage of Failed Protein Targets by Reductive Alkylation

Abstract

The growth of diffraction-quality single crystals is of primary importance in protein X-ray crystallography. Chemical modification of proteins can alter their surface properties and crystallization behavior. The Midwest Center for Structural Genomics (MCSG) has previously reported how reductive methylation of lysine residues in proteins can improve crystallization of unique proteins that initially failed to produce diffraction-quality crystals. Recently, this approach has been expanded to include ethylation and isopropylation in the MCSG protein crystallization pipeline. Applying standard methods, 180 unique proteins were alkylated and screened using standard crystallization procedures. Crystal structures of 12 new proteins were determined, including the first ethylated and the first isopropylated protein structures. In a few cases, the structures of native and methylated or ethylated states were obtained and the impact of reductive alkylation of lysine residues was assessed. Reductive methylation tends to be more efficient and produces the most alkylated protein structures. Structures of methylated proteins typically have higher resolution limits. A number of well-ordered alkylated lysine residues have been identified, which make both intermolecular and intramolecular contacts. The previous report is updated and complemented with the following new data; a description of a detailed alkylation protocol with results, structural features, and roles of alkylated lysine residues in protein crystals. These contribute to improved crystallization properties of some proteins.

The His-tag as a decoy modulating preferred orientation in cryoEM

“…interfaces , Tan et al, 2014, Klebl et al, 2020, and this is fully consistent with our data. For polar particles, an advantage of our method is that it provides a quick and convenient characterization of asymmetry between two surfaces, and it also allows for retrospective analysis of cryoEM SPR data.…”

“…pegylation, or using reductive methylation of lysine residues (Means & Feeney, 1990, Rypniewski et al, 1993, to name just a few. Reductive methylation was used to change the pattern of hydrophobicity on the surface of proteins to promote their crystallization (Tan et al, 2014, Kim et al, 2008. One can expect that reductive methylation will have a similar impact on interactions with supports used in cryoEM SPR.…”

The “Sticky Patch” Model of Crystallization and Modification of Proteins for Enhanced Crystallizability

“…Detailed protocols have been published [165,166]; only the free amino groups (ε-amino groups of lysines, and the N-terminal amino group) are modified. Formaldehyde is the methylating agent with dimethylamine-trifluoroborane acting as the reducing agent.…”

“…As is invariably the case in crystallization screens, assessment of success rates is not trivial. Nevertheless, HT Structural Genomics centers reported 10 – 30% success rates with selections of proteins recalcitrant to crystallization in unmodified form [167,168,166]. Why does this strategy enhance crystallization?…”

“…It should be noted that methylation constitutes just one variation of reductive alkylation, which may involve introduction of larger groups, such as ethyl and isopropyl [166]. However, examples of the latter are rare and have not been reported to be very successful.…”