| Macromolecular analysis by electrospray (ESI) mass spectrometry and Matrix Assisted Laser Desorption Ionization (MALDI) mass spectrometry has been utilized for characterization of many macromolecules. Presented here are: (i) organometallic oligomer detection and characterization for potential solar energy applications (ii) and Interferon Stimulated Gene (ISG)15 structural stability in solution and effect on ISG15 interaction with UBE1L due to changes in structure.; Synthesized rhenium oligomers are photosensitive and may be destabilized in the presence of varying solvents, ionizing agents, and salt adducts. MALDI-TOF-MS data was compared with ESI-MS data for a series of oligomers with varying complexity; i.e., varying solvents, bridging ligands, matrices, and counterions with implications for synthesizing larger oligomers with similar or better photochemical behavior.; ISG15 is up-regulated in response to interferon tau during early pregnancy in pigs, cows, sheep, humans, and mice. Structurally, ISG15 resembles two ubiquitin domains (30% identity) separated by a hinge region. Recombinant (r) bovISG15 is not stable in solution; a hinge region cysteine was hypothesized to contribute to the instability of rbovISG15. Within 24 hours of dialysis (PBS, pH 7.4, 22°C), rbovISG15 formed other complexes (despite reducing SDS-PAGE) which were resolved using western blot (antibody against rISG15). Chemical modification of cysteine prevented rbovISG15 complexes over time. Also, a site-directed mutant, Cys80Ser rbovISG15 was isomeric (MALDI-TOF-MS and western blot) and more stable (CD spectroscopy) than rbov ISG15 (P < 0.05, 48 hours). Structural stability of ovine (ov) rISG15 was examined since ovISG15 has three more amino acids present in the hinge region than the rbovISG15. Cys80Ser rovISG15 was more stable than rovISG15 over time in solution (CD spectroscopy) and was able to specifically bind to its initiating enzyme, E1 after 48 hours of dialysis (western blot). Structural models were generated for bovISG15, ovISG15, and Cys78Ser ovISG15 based on the structure of ubiquitin, which revealed that the cysteine and spacing within the hinge region of ISG15 may be critical to structural stability and interaction with E1.; In conclusion, mass spectrometry of the rhenium oligomer series produced useful characterization including a trimetallic compound. Structural stability of ISG15 and the ability to bind E1 were dependent on hinge region intramolecular actions. |
