Skip to main content
U.S. flag

An official website of the United States government

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you’ve safely connected to the .gov website. Share sensitive information only on official, secure websites.

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

Published

Author(s)

David T. Gallagher, N N. Smith, Sung Kim, Howard Robinson, Prasad T. Reddy

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

Keywords

crystal contact, crystal growth, cyclic AMP, isoelectric point, x-ray diffraction

Citation

Gallagher, D. , Smith, N. , Kim, S. , Robinson, H. and Reddy, P. (2009), Protein crystal engineering of YpAC-IV using a strategy of excess charge reduction, Journal of Crystal Growth (Accessed April 19, 2024)
Created August 5, 2009, Updated February 19, 2017