P22 tailspike:

Background

Folding:

On-pathway

Off-pathway

Funding

Background:

Folding and aggregation are important competing pathways in association of polypeptide chains that are still poorly understood. Breakdowns of both processes lead to numerous diseases, including cystic fibrosis, Alzheimer’s, Creutzfeldt-Jacob and Parkinson’s disease. Our lab has been using the trimeric P22 tailspike protein to understand both the process of folding and the determinants of aggregation. P22 tailspike is a member of the family of elongated b-helix viral adhesion proteins, such as T4 ligase, etc (Mitraki, Miller et al. 2002). The P22 tailspike system offers the advantages of an established folding and aggregation pathway, the ability to study the association of a multimeric protein in electrophoresis, numerous folding mutations that affect different regions of the folding pathway, and a high degree of b-sheet structure, similar to many disease related proteins, such as the amyloid-forming proteins (Wetzel 2002).

Structure of the P22 tailspike protein with the four major domains highlighted (Steinbacher, Seckler et al. 1994; Steinbacher, Miller et al. 1997).

Folding:

Shown below is a schematic of the current model of the in vitro folding and aggregation pathways. A monomer, dimer and a disulfide-bonded protrimer have been identified as folding intermediates in the folding pathway of tailspike. The initial step in folding is the folding of the unfolded polypeptide chain to form a stable monomer species (Fuchs, Seiderer et al. 1991). Partially folded monomer chains add to one another to form dimer, which has also been shown to be disulfide-bonded (Sather and King 1994; Robinson and King 1997; Haase-Pettingell, Betts et al. 2000; Danek and Robinson 2003). A final monomer chain is added to the dimer to form an immature trimer (protrimer) species. The protrimer species undergoes structural rearrangements to generate the mature tailspike trimer. The disulfide-bonded dimer and protrimer are of particular interest as there are no disulfide bonds in the final, native structure.

Schematic of the folding pathway of the P22 tailspike protein.

On-pathway:

Our studies to understand on-pathway folding has focused on 1) understanding the role of the C-terminus in protrimer assembly and maturation and 2) understanding the role of the transient disulfide bond in folding.

Understanding the role of the C-terminus in folding:

The C-terminus of the P22 tailspike was recognized as important to assembly of the tailspike protein after the structure of the protein was determined in 1994 (Steinbacher, Seckler et al. 1994). Our lab has shown that deletion of the C-terminus or disruption of the hydrophobic core of the C-terminus by mutagenesis inhibits trimerization. In addition, ANS, which normally is used as a fluorescent probe for exposed hydrophobic patches, can inhibit assembly of the protrimer. These results have shown that the hydrophobic core of the C-terminus plays a critical role in protrimer assembly (Gage and Robinson 2003).

Our lab has also shown that protrimer maturation involves interaction of R549, R563 and D572. Mutation of arginine 549 to glutamine and arginine 563 to lysine affects the kinetics of the protrimer to trimer transition, resulting in an accumulation of protrimer species in vivo and monomer, dimer and protrimer in vitro. Purified R549Q and R563K trimers also have reduced stability relative to native tailspike trimer and are more sensitive to pH during refolding. We have proposed that R563 interacts with D572 to stabilize a loop in the monomer and positions D572 to interact with R549 during protrimer maturation (Gage, Zak et al.).

C_a-trace of the P22 tailspike protein with the R549, R563 and D572 residues highlighted in ball and stick representation.

Role of the transient disulfide bond:

The three cysteine residues toward the carboxyl terminus, at 496, 613 and 635, have been shown to exhibit sulfur reactivity, indicating that these are the most likely candidates to be involved in transient disulfide bonding during the folding reaction ((Sather and King 1994; Robinson and King 1997). Single serine mutants at each of these residues were expressed and purified from E. coli. In vitro characterization of these mutants, in trimer form, demonstrate native-like circular dichroism spectra, and similar stability to wild type tailspike when treated with guanidine chloride. However, significant differences were observed between the single mutant and wild type folding reactions under both low protein concentrations, which favor productive folding, and high concentrations, which lead toward aggregation. The single mutants fold two to five times slower than wild type under productive folding conditions, but this difference is even greater at aggregating conditions, where the appearance of trimer is more than fifty times slower than wild type (Danek and Robinson 2003; Danek and Robinson 2004).

Off-pathway:

Our studies on off-pathway aggregation are focused on 1) using hydrostatic pressure to recover folded protein from aggregates and 2) using the b-helix domain of the protein to study the relationship between the kinetics and thermodynamics of aggregation.

Hydrostatic pressure:

In order to address the aggregation problems in production and to understand the molecular determinants that drive aggregation in vivo and in vitro, we are investigating the inhibition and reversal of protein aggregation of P22 tailspike by application of hydrostatic pressure. The results we obtain here will serve as a foundation for studying other important protein systems, of both industrial and medical relevance.

High pressure apparatus.

We have shown that application of hydrostatic pressure to tailspike aggregates results dissociation of about 60% of tailspike aggregates formed at 37° C. Aggregates dissociate primarily into folding-competent monomeric and dimeric intermediates, of which ~80% form trimer. Interestingly, 40% of the aggregates are unperturbed by pressure and repeated cycles of pressure do not disrupt this pool of aggregates, indicating that there are differences in the morphology of the aggregates (Lefebvre and Robinson 2003; Lefebvre, Gage et al. 2004).

Pressure has also proven to be a useful tool in studying the phenotypes of two different tsf mutants, G244R and E196K. The E196K mutant refolds extremely poorly in vitro at 20° C, but shows nearly native-like recovery of trimer after hydrostatic pressure treatment. This suggests that the E196K affects the formation of the folding-competent monomer, since pressure-treatment dissociates the aggregates to primarily dimers. In contrast, the G244R mutant, which refolds with near wild-type yields in vitro at 20° C shows very poor recovery of trimer after pressure treatment of aggregates, suggesting that the G244R mutation acts after formation of the folding-competent, at the dimer stage. Since pressure-treated G224R protein must still pass through this step, there is no increased yield by pressure-treatment. Hydrostatic pressure has helped to distinguish between these two different misfolding mechanisms (Lefebvre, Comolli et al. 2004).

Kinetics and thermodynamics of aggregation:

The b-helix domain of the tailspike protein serves as a good model for the proposed protofibril structure (Miller, Schuler et al. 1998). Our lab has purified this domain of the protein in order to characterize its aggregation properties. This domain aggregates readily at temperatures slightly above physiological (37°C) and forms fibril-like aggregates. Using a combination of size-exclusion chromatography, circular dichroism, electron microscopy, mass-spectrometry and calorimetry, we are characterizing the relationship between the kinetics and thermodynamics of aggregation to develop a model of the steps involved in aggregation of this domain. Parts of this project are in collaboration with Dr. Chris Roberts.

Electron micrograph of aggregates of the b-helix domain of the P22 tailspike protein.

Funding:

NIH P20 RR15588

NSF Career 99 84312

NIH RO1 GM060543

References:

Danek, B. L. and A. S. Robinson (2003). "Nonnative interactions between cysteines direct productive assembly of P22 tailspike protein." Biophys J 85(5): 3237-47.

Danek, B. L. and A. S. Robinson (2004). "P22 tailspike trimer assembly is governed by interchain redox associations." Biochim Biophys Acta 1700(1): 105-16.

Fuchs, A., C. Seiderer, et al. (1991). "In vitro folding pathway of phage P22 tailspike protein." Biochemistry 30: 6598-6604.

Gage, M. J. and A. S. Robinson (2003). "C-terminal hydrophobic interactions play a critical role in oligomeric assembly of the P22 tailspike trimer." Protein Sci 12(12): 2732-47.

Gage, M. J., J. L. Zak, et al. "It All Comes Down to Positioning: Three Amino Acids that are Critical to Formation and Stability of the P22 Tailspike Trimer." in preparation.

Haase-Pettingell, C., S. D. Betts, et al. (2000). "Role for cysteine residues in the in vivo folding and assembly of the phage P22 tailspike." Prot. Sci. 10: 397-410.

Lefebvre, B. G., N. K. Comolli, et al. (2004). "Pressure dissociation studies provide insight into oligomerization competence of temperature-sensitive folding mutants of P22 tailspike." Protein Sci 13(6): 1538-46.

Lefebvre, B. G., M. J. Gage, et al. (2004). "Maximizing Recovery of Native Protein from Aggregates by Optimizing Pressure Treatment." Biotechnology Progress.

Lefebvre, B. G. and A. S. Robinson (2003). "Pressure treatment of tailspike aggregates rapidly produces on-pathway folding intermediates." Biotechnol Bioeng 82(5): 595-604.

Miller, S., B. Schuler, et al. (1998). "A Reversibly Unfolding Fragment of P22 Tailspike Protein with Native Structure: The Isolated b-Helix Domain." Biochemistry 37: 9160-9168.

Mitraki, A., S. Miller, et al. (2002). "Review: conformation and folding of novel beta-structural elements in viral fiber proteins: the triple beta-spiral and triple beta-helix." J Struct Biol 137(1-2): 236-47.

Robinson, A. S. and J. King (1997). "Disulphide-bonded intermediate on the folding and assembly pathway of a non-disulphide bonded protein." Nature Struct Biol 4(6): 450-455.

Sather, S. and J. King (1994). "Intracellular Trapping of a Cytoplasmic Folding Intermediate of the Phage P22 Tailspike Using Iodoacetamide." Journal of Biological Chemistry 269(41): 25268-25276.

Steinbacher, S., S. Miller, et al. (1997). "Phage P22 Tailspike Protein: Crystal Structure of the Head-binding Domain at 2.3 Å, Fully Refined Structure of the Endorhamnosidase at 1.56 Å Resolution, and the Molecular Basis of O-Antigen Recongnition and Cleavage." Journal of Molecular Biology 267: 865-880.

Steinbacher, S., R. Seckler, et al. (1994). "Crystal Structrue of P22 Tailspike Protein: Interdigitated Subunits in a Thermostable Trimer." Science 265: 383-385.

Wetzel, R. (2002). "Ideas of order for amyloid fibril structure." Structure (Camb) 10(8): 1031-6.