The Role of Cysteines and Disulfide Bonds in the Protein Folding of P22 Tailspike
 

Brenda L. Danek, Sujata K. Bhatia¹, and Anne S. Robinson

University of Delaware
Department of Chemical Engineering
150 Academy Street
Newark, DE 19716

¹Currently at:
University of Pennsylvania
Philadelphia, PA 19104

danek@che.udel.edu
(302) 831-6697

The flourishing biotechnology industry has led to increased interest in many biological molecules, including proteins.  In order for proteins to function correctly they must properly fold.  Several diseases, including sickle cell anemia, cystic fibrosis, and Alzheimer’s disease, have been linked to misfolded proteins.  Additionally, biotechnology companies produce recombinant proteins in foreign hosts, where non-native cellular environments often alter protein assembly and lead to aggregation and decreased yields.  In order to make advances in any of the above areas, it is necessary that researchers obtain a thorough understanding of the pathway, mechanism and principles in which large proteins fold and assemble.   

A model system for studying the folding and assembly of complex proteins is the tailspike protein of the P22 bacteriophage.  In its native state, P22 tailspike is a homotrimer with three monomer units intertwined and held together through non-covalent molecular interactions. There are eight cysteine residues per monomer chain, and all twenty-four residues are buried within the core of the protein.  While the native trimer structure of tailspike lacks any disulfide bonds, experimental evidence has indicated that some folding intermediates are disulfide bonded.  We have been examining the effect of this disulfide bond formation on the kinetics and partitioning between folding and aggregation. The nature of the disulfide linkage is not known, but mutational analysis has indicated that the cysteines at 496, 613 and 635 are critical residues for protein folding.  Single mutants, each containing one point mutation at each of the three residues of interest, were expressed and their folding constants characterized in vivo.  These mutants folded five to ten times slower than wild type tailspike, indicating that these residues are important in protein folding.  Rates for in vitro folding of the single mutants have also been found to be around one tenth to one fifth as fast as wild type tailspike.  Studies comparing wild type and single mutant stability to guanidine chloride treatment have shown that wild type and C613S trimer are similar in stability once folded.  Using this information, we can gain insight into the role of the redox environment of in vivo or in vitro refolding from inclusion bodies.