Systematic replacement of lysine with glutamine and alanine in Escherichia coli malate synthase G: effect on crystallization.
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.
Anstrom
David M
DM
Institute of Molecular Biology, University of Oregon, USA.
Colip
Leslie
L
Moshofsky
Brian
B
Hatcher
Eric
E
Remington
S James
SJ
D000409:Alanine / Q000737:chemistry*; D003460:Crystallization; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology*; D005973:Glutamine / Q000737:chemistry*; D008239:Lysine / Q000737:chemistry*; D008292:Malate Synthase / Q000737:chemistry*; D009154:Mutation; D011487:Protein Conformation; D017433:Protein Structure, Secondary
D005973:Glutamine; D008292:Malate Synthase; D008239:Lysine; D000409:Alanine