Systematic replacement of lysine with glutamine and alanine inEscherichia colimalate synthase G: effect on crystallization

Abstract

Two proposals recommend substitution of surface lysine residues as a means to improve the quality of protein crystals. In proposal I, substitution of lysine by alanine has been suggested to improve crystallization by reducing the entropic cost of ordering flexible side chains at crystal contacts. In proposal II, substitution of lysine by residues more commonly found in crystal contacts, such as glutamine, has been proposed to improve crystallization. 15 lysine residues on the surface of Escherichia coli malate synthase G, distributed over a variety of secondary structures, were individually mutated to both alanine and glutamine. For 28 variants, detailed studies of the effect on enzymatic activity and crystallization were conducted. This has permitted direct comparison of the relative effects of the two types of mutations. While none of the variants produced crystals suitable for X-ray structural determination, small crystals were obtained in a wide variety of conditions, in support of the general approach. Glutamine substitutions were found to be more effective than alanine in producing crystals, in support of proposal II. Secondary structure at the site of mutation does not appear to play a major role in determining the rate of success.

Atomic resolution structures of Escherichia coli and Bacillus anthracis malate synthase A: Comparison with isoform G and implications for structure‐based drug discovery

“…Sequence comparisons suggest that the larger molecular size of MSG isoforms can be attributed to the presence of one or more insertions (Molina et al 1994;Howard et al 2000;Smith et al 2003), while the conserved segments of the amino acid sequence show only ;18% identity. Crystal structures of MSG from E. coli (Howard et al 2000;Anstrom et al 2003) and from mTB (Smith et al 2003;Anstrom and Remington 2006) have been determined, but unfortunately the resolution of the diffraction data is modest, which limits structure-based drug discovery efforts (Anstrom et al 2005).…”

Erkennt die Signale

“…Crystal contact engineering involving substitution of lysine by arginine, a residue more frequently observed to form crystal contacts, has been already described in the literature 36, 37. This method was demonstrated to be a successful alternative to the SER method: instead of decreasing the size of the entropic barrier, a residue is replaced with another with similar conformation entropy but an increased probability to form noncovalent interactions.…”

Nuclear Localization Sequences in Cytomegalovirus Capsid Assembly Proteins (UL80 Proteins) Are Required for Virus Production: Inactivating NLS1, NLS2, or Both Affects Replication to Strikingly Different Extents

“…It was found that nuclear translocation was blocked only when both NLS were disabled. To verify the functionality of these NLS in the HCMV pAP, and to determine whether Gln could be used instead of Ala as a more conservative replacement for the NLS Arg and Lys residues (2,8,26,36), WT and the NLS1 Ϫ and NLS2…”

“…The NLS1 target sequence in UL80 was knocked out using a recombination cassette, composed of the streptomycin-sensitive RpsL gene linked to the neomycin gene, in plasmid pRpsL-neo (K002; Gene Bridges, Dresden, Germany) and the NLS1 flanking sequences. The RpsL-neomycin cassette was replaced in a second recombination by using a mutagenic double-strand oligonucleotide that changed only the NLS1 coding sequence (i.e., from AAACGCCGTAAG1539 to CAACAGCAGCAA1539), substituting glutamine (Gln) for lysine (Lys) and arginine (Arg) in the original NLS1 amino acid sequence KRRK513 (2,8,26,36). The resulting mutant bacmid is called bHCMV-UL80NLS1 Ϫ .…”