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Disulfide Bond Formation and Evolution of Protein-Folding Catalysts
Summary: James Bardwell studies protein-folding catalysts and chaperones—the critical molecular machines that help proteins shape up. Bardwell focuses on disulfide bonds, which act like bolts or stiffening struts for folding proteins.
Proteins are synthesized as linear chains of amino acids. To be biologically active, proteins must rapidly fold into their unique three-dimensional structures. They are assisted in this process by folding catalysts and chaperones.
The formation of disulfide bonds is vital for the proper folding of most secreted proteins, including many of medical importance. Without their disulfides, these proteins are disordered and nonfunctional. We found that the formation of disulfides is catalyzed in the cell. DsbA directly donates disulfides to secreted proteins and in doing so catalyzes their folding. DsbA, therefore, is essential for disulfide bond formation in endogenous Escherichia coli proteins, many virulence factors, and in eukaryotic proteins expressed in E. coli. Although DsbA was discovered only relatively recently, we and others have used the power of E. coli as a model organism to make rapid progress in the understanding of DsbA's function. We now know in mechanistic detail how this folding catalyst functions. DsbA is reoxidized by DsbB; DsbB, in turn, uses the oxidizing power of quinones to generate disulfides de novo. This novel catalytic activity is the major source of disulfide bonds in vivo.
Our in vitro reconstitution of the disulfide bond catalytic system established where the oxidative power for protein folding originates and how this vital step in the catalysis of protein folding is linked to cellular metabolism (see figure). In the endoplasmic reticulum of eukaryotes, oxidative protein folding has been found to involve a set of catalysts and disulfide cascade reactions, similar to those we have found in E. coli. Many of these discoveries are directly applicable to all branches of life.
Currently, we are using a multifaceted genetic, biophysical, and structural approach to determine how disulfide bonds are generated de novo. We will use disulfide bond formation as a tool to investigate and to optimize the protein-folding pathways of eukaryotic proteins in E. coli. We will use directed evolution to engineer novel ways of catalyzing the formation of disulfide bonds in vivo. Using a powerful genetic selection for the oxidation of disulfide bonds, we succeeded in replacing the entire disulfide catalytic pathway with a single protein. This system provides an ideal platform to investigate the evolution of enzymes and the coevolution of folding catalysts with their substrates.
We have also used a laboratory evolution approach to determine functionally important structural differences between a number of distantly related disulfide oxidoreductases. The in vitro folding pathways of some proteins, such as bovine pancreatic trypsin inhibitor (BPTI), are well studied. In contrast, very little is known about the in vivo folding pathway of any protein. We have been able to directly select variants of BPTI that fold more effectively in vivo. Our results imply that important aspects of the in vitro folding pathway of BPTI are shared with its in vivo pathway.
Our work will help illuminate the mechanism of disulfide bond formation, a basic process vital for protein folding, and the mechanism of evolution, a process basic to all of biology.
Last updated: July 2, 2007
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