Poster
Presentation 13:
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.
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