Variable-temperature Fourier transform infrared (VT-FTIR) spectroscopy in conjunction with 2D correlation analysis was employed to study the unfolding of aspartate transcarbamylase (ATCase) and its individual subunits. The regulatory subunit (RSU) was uniformly labeled with 13C/15N and then reconstituted with the unlabeled catalytic subunit (CSU) to form the holoenzyme. The activity of the holoenzyme was shown to be unaffected by the isotopic labeling of the RSU. The VT-FTIR investigation of the isolated CSU and the CSU in the holoenzyme revealed that the CSU is more thermally stable when bound to the RSU (i.e., in the holoenzyme). The RSU also showed more thermal stability when bound to the CSU. The sequences of events leading to the unfolding of the isolated CSU and RSU and the CSU in the holoenzyme were deduced by 2D correlation analysis of the VT-FTIR spectra. The results for the isolated CSU demonstrated that beta-sheets unfold first, followed by a-helices and then turns, and finally aggregates form. The sequence of unfolding of the RSU showed an increase of turns followed by a loss of intramolecular beta sheets, then a loss of alpha-helices and the formation of aggregates. The CSU in the holoenzyme exhibited a slightly different unfolding pathway and was observed to unfold subsequent to the unfolding of the RSU, consistent with the two thermal transitions observed by differential scanning calorimetry.

Fourier transform infrared (FTIR) spectroscopy is a powerful tool for probing protein structure-function relationships. With the use of isotope editing, it can also be employed to elucidate protein-nucleic acid interactions. This technique was used to study the sequence of heat-induced unfolding of the uniformly labeled 13C regulatory subunit (RSU) of E. coli aspartate transcarbamylase (ATCase) with its inhibitor CTP. The absorption of CTP in the amide I' region limits our ability to detect protein conformational changes upon binding of CTP. Therefore, by labeling the protein with 13C shifts the amide I' band ~ 40 cm -1 and clearly separates the protein bands from those of CTP. Variable-temperature (VT) FTIR spectroscopy was then employed to monitor the thermal unfolding of the labeled RSU in the presence and absence of CTP. / In addition, isotope editing was further explored to probe domain-domain interactions of the two domains of RSU using intein technology. Intein technology provides a novel means by which isotope editing can be performed to extract information on protein inter-domain and inter-subunit interactions by spectroscopic analysis but has not yet been exploited in Fourier transform infrared (FTIR) spectroscopy. The objective of this project is to present for the first time the feasibility of segmental labeling through intein-mediated protein ligation (IPL) for the purpose of studying conformational changes by FTIR spectroscopy, using ATCase as a model enzyme. In the first phase of this project, the RSU of ATCase, which houses a Zn-binding domain and a nucleotide binding domain, was reconstructed from its isolated domains using commercially available intein-base expression vectors. As steps towards obtaining an isotope labeled RSU, we have fused each domain to separate inteins. Following affinity purification, the intein tags were chemically cleaved and the reactive ends of the two RSU domains were ligated together to form a peptide. Although ligation was successful, improved yields are required for the FTIR spectroscopic studies.

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