The impact of Glu-->Ala and Glu-->Asp mutations on the crystallization properties of RhoGDI: the structure of RhoGDI at 1.3 A resolution.
It is hypothesized that surface residues with high conformational entropy, specifically lysines and glutamates, impede protein crystallization. In a previous study using a model system of Rho-specific guanine nucleotide dissociation inhibitor (RhoGDI), it was shown that mutating Lys residues to Ala results in enhanced crystallizability, particularly when clusters of lysines are targeted. It was also shown that one of these mutants formed crystals that yielded diffraction to 2.0 A, a significant improvement on the wild-type protein crystals. In the current paper, an analysis of the impact of surface mutations replacing Glu residues with Ala or Asp on the stability and crystallization properties of RhoGDI is presented. The Glu-->Ala (Asp) mutants are generally more likely to produce crystals of the protein than the wild-type and in one case the resulting crystals yielded a diffraction pattern to 1.2 A resolution. This occurs in spite of the fact that mutating surface Glu residues almost invariably affects the protein's stability, as illustrated by the reduced deltaG between folded and unfolded forms measured by isothermal equilibrium denaturation. The present study strongly supports the notion that rational surface mutagenesis can be an effective tool in overcoming problems stemming from the protein's recalcitrance to crystallization and may also yield dramatic improvements in crystal quality.
Mateja
Agnieszka
A
Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia 22908, USA.
Devedjiev
Yancho
Y
Krowarsch
Daniel
D
Longenecker
Kenton
K
Dauter
Zbigniew
Z
Otlewski
Jacek
J
Derewenda
Zygmunt S
ZS
D000409:Alanine / Q000737:chemistry*; D001224:Aspartic Acid / Q000737:chemistry*; D003460:Crystallization; D018360:Crystallography, X-Ray; D018698:Glutamic Acid / Q000737:chemistry*; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors
D020730:Guanine Nucleotide Dissociation Inhibitors; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; D001224:Aspartic Acid; D018698:Glutamic Acid; D000409:Alanine