Take a sneak peek at the new NIST.gov and let us know what you think!
(Please note: some content may not be complete on the beta site.).

View the beta site
NIST logo

Publication Citation: Protein crystal engineering of YpAC-IV using a strategy of excess charge reduction

NIST Authors in Bold

Author(s): David T. Gallagher; N N. Smith; Sung Kim; Howard Robinson; Prasad T. Reddy;
Title: Protein crystal engineering of YpAC-IV using a strategy of excess charge reduction
Published: August 05, 2009
Abstract: The class IV adenylyl cyclase from Yersinia pestis has been engineered to enable crystallization at neutral pH for mechanism studies. The wild-type enzyme crystallized only at pH below 5. Based on the unliganded wild-type structure 2FJT at 1.9 Angstrom resolution, two different approaches were tested with the goal of producing a higher-pH crystal needed for substrate complexation. In one approach, Asp 19, which forms a growth-limiting dicarboxylate contact in wild-type triclinic crystals, was modified to Ala and Asn in hopes of relieving the acid-dependence of that crystal form. In the other approach, wild-type residues Met 18, Glu 25, and Asp 55 were (individually) changed to lysine to reduce the protein s excess negative charge in hopes of enabling growth of new, higher-pH forms. These 3 sites were selected based on their high solvent exposure and lack of intraprotein interactions. The D19A and D19N mutants had reduced solubility and did not crystallize. The other 3 mutants all crystallized, producing several new forms at neutral pH. One of these forms, with the D55K mutant, enabled a substrate-analog complex at 1.8 Angstrom resolution. This structure shows why the new crystal form required the mutation in order to grow at neutral pH. This approach could be useful in other cases where low-pI proteins are too highly charged to crystallize at neutral pH.
Citation: Journal of Crystal Growth
Volume: 9
Issue: 8
Pages: pp. 3570 - 3574
Keywords: crystal contact,crystal growth,cyclic AMP,isoelectric point,x-ray diffraction
Research Areas: Life Sciences Research, Bioscience & Health