Large-scale evaluation of protein reductive methylation for improving protein crystallization

Abstract

To the editor:Obtaining diffraction-quality crystals is a major bottleneck in protein X-ray crystallography. For example, the current success rate for protein structure solution at the Midwest Center for Structural Genomics (starting from purified protein) is ~10%. Protein crystallization is influenced by many factors, and many methods have been developed to enhance crystallization. In particular, reductive methylation of proteins has been successfully applied to obtain high-quality crystals 1-4 . Several studies 3,5,6 have indicated that methylating the solvent-exposed ε-amino group of lysines changes protein properties (pI, solubility and hydropathy) 7,8 , which may promote crystallization via improving crystal packing. Reductive methylation of proteins is a simple, generic method; it is fast, specific and requires few steps under relatively mild buffer and chemical conditions and can be executed for several proteins in parallel. Native and methylated proteins have very similar structures, and, in most cases, methylated proteins maintain their biochemical function 2,5,9 . Some proteins can only be crystallized after methylation 3,10 , and crystals of modified proteins often diffract to higher resolution 3,9 . The efficacy of the method has been previously tested on 10 proteins, with a 30% success rate 3 .Here we investigated the application of reductive methylation on a large scale. We applied a previously described reductive methylation protocol 2,11 (Supplementary Methods online) to 370 sequence-diverse proteins selected from protein families that had no structural homologs with >30% sequence identity. We expressed 370 recombinant proteins and purified them using standard methods 12 and screened them using standard crystal screening methods (Supplementary Methods). Of the 370 proteins, 269 proteins had not previously yielded crystals suitable for structure determination (crystals were too small, poorly ordered, twinned, highly mosaic or multiple), 85 proteins had previously failed to crystallize and 16 proteins were a reference set (not previously screened for crystallization; Table 1 and Supplementary Tables 1 and 2 online). After reductive methylation, we obtained diffraction-quality crystals for 40 of the 370 proteins, and so far we solved 26 crystal structures ( NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptWe also determined the structures of 4 proteins in their native as well as their methylated states (Supplementary Methods). By comparing these structures, we obtained insight into how methylation affects protein crystallization. We observed a decrease in the isotropic B factor (Fig. 1), which is likely a result of more ordered crystal packing and which leads to better diffraction limits. Indeed, the resolution of the methylated structures (average, 2.07 Å) was better than that of their native counterparts (average, 3.05 Å; Supplementary Table 1). The methylated lysines were engaged in various intra-and intermolecular interactions with protein and solvent (carboxylates and ...

Nanovolume optimization of protein crystal growth using the microcapillary protein crystallization system

“…3a-3f; for crystal optimization data, see Table 2). This salvage rate compares favorably to published data from reductive methylation (Kim et al, 2008) and limited proteolysis (Dong et al, 2007). In two cases, high-quality diffraction was also generated from crystals grown from subsequent sittingdrop vapor-diffusion experiments (in one case, X-ray diffraction from the MPCS crystal was of slightly higher resolution and in one case diffraction from the MPCS crystal was of slightly lower resolution).…”

Biotinylation of lysine method identifies acetylated histone H3 lysine 79 in Saccharomyces cerevisiae as a substrate for Sir2

“…Lysine is subject to other reversible modifications such as methylation and ubiquitination, and identifying the substrates for demethylases and deubiquitinating enzymes is similarly of great utility. Our method could be modified to study demethylase substrates by chemically methylating substrates instead of acetylating them (61). Perhaps more significantly, the method could, in principle, be adapted to screen for substrates of deubiquitinating enzymes using endogenously ubiquitinated proteins, with chemical acetylation or methylation to block all unmodified lysines while retaining native ubiquitination sites.…”

Structure-Guided Expansion of the Substrate Range of Methylmalonyl Coenzyme A Synthetase (MatB) of Rhodopseudomonas palustris

“…In order to obtain an ATP-bound structure of RpMatB, it was necessary to methylate the lysines within RpMatB (51). This is an established method to change the nature of the surface-exposed residues of a protein, which play a large role in the formation of the crystal lattice (18,45), without changing the intrinsic structure of the protein, and it has been successfully utilized to crystallize numerous recalcitrant proteins (31,35,39,52,57). Additionally, the RpMatB K488A mutant was utilized, as this protein binds ATP ϳ10-fold more tightly than wild-type RpMatB (Table 6), and mutation of the active-site lysine to alanine prevented predicted complications due to methylated K488 within the active site.…”