Structural Investigation into Chemically Synthesized Peptides using Modern Biophysical Techniques

Abstract

This thesis explores the total chemical synthesis and characterization of three target proteins, EspC, ORFV002 and SHONα, to probe their biophysical and biochemical properties with the goal of producing racemic X-ray crystal structures. EspC, a novel Mycobacterium tuberculosis (Mtb)-secreted antigen (103 amino acids, 10.8 kDa), displays immunodominance similar to CFP-10 and ESAT-6, the two specific immunodiagnostic T-cell antigens of Mtb. Following the effective synthesis of native, linear L-EspC using Boc solid phase peptide synthesis (SPPS) and native chemical ligation (NCL), the protein was screened by differential scanning fluorimetry yielding a useful folding buffer in which the protein was successfully folded into its α-helical native structure as evidenced by circular dichroism and size-exclusion chromatography (SEC). All polypeptide building blocks could be prepared in tens of milligram amounts and underwent near quantitative single-step ligation to yield the full EspC sequence, which did not require any post-NCL modifications. The analysis of the protein by SEC, circular dichroism (CD) and SEC-MALS (size-exclusion chromatography-multiangle light scattering) identified the formation of oligomeric species, the largest of which was estimated at containing 7-12 EspC monomers. This oligomer appeared thermally unstable in some experiments, potentially disassociating into a number of smaller oligomeric species at a temperature as low as 30 ºC. Using either L-EspC or a racemic mixture of L-/D-EspC, various trials of crystallization were attempted at 18 ºC, a temperature at which the large oligomer of EspC appeared stable. ORFV002, an orf virus-encoded protein (117 amino acids, 11.8 kDa), is involved in modulation of the nuclear function of nuclear factor kappa B (NF-κB), the master regulator of human gene transcription that plays a central role in the integration of stress-inducing stimuli and innate immune responses in the epidermis. Native, linear ORFV002 was successfully synthesized using the techniques of Boc SPPS and NCL – all three polypeptide building blocks could be prepared in tens of milligram amounts and underwent near quantitative ligation reactions. A final desulfurization step afforded linear ORFV002 in an overall yield of 7.1 %. Following folding buffer screening, it was hypothesized that the protein may be intrinsically unfolded. This hypothesis was supported by the ORFV002 protein undergoing induced-folding upon addition of osmolytes such as trimethylamine N-oxide (TMAO) and 2,2,2-trifluoroethanol (TFE). Upon successful recombinant expression and purification of NF-κB p6519-320, the natural binding partner of ORFV002, pull-down assays demonstrated that the unfolded, synthetic ORFV002 binds the expressed His6-NF-κB p6519-320 and presumably forms a folded structure. Further characterization of the binding event, for example, by isothermal titration calorimetry (ITC) or co-crystallization experiments was not possible, due to intrinsic instability associated with the purified His6-NF-κB p6519-320. SHONα is the major secreted protein isoform of a human protein translated from the secreted hominoid-specific oncogene (SHON). This novel hominoid-specific oncogene appears to play an important role in the oncogenicity of breast cancer. Native SHONα (93 amino acids, 9.6 kDa) and its non-disulfide forming alanine-analogue were successfully synthesized in a multi-milligram scale using protein chemical synthesis methodologies. The native SHONα was successfully folded into its native structure and the foldedness was confirmed by circular dichroism and differential scanning fluorimetry studies. The folded, native SHONα and its alanine-analogue were tested for their activity in estrogen receptor positive (ER+) breast cancer cell lines, MCF-7 and T47D, upon exogenous treatment. The folded, native SHONα stimulated AKT/PKB, ERK1/2 and GSK3α/β intracellular growth stimulating pathways in both of the cell lines tested, while the alanine-analogue of SHONα did not seem to similarly stimulate the growth signalling pathways.