Tryptophan hydroxylase (TPH) is a ubiquitous enzyme in the human nervous system. TPH plays an important role in the biosynthesis of serotonin, a multifunctional neurotransmitter, regulating, among others, moods, the circadian rhythm, and pancreatic and liver function. Major depression is among the disorders associated with incorrect serotonin levels in the brain. The regulation of TPH activity is currently poorly understood, due to the unavailability of a full-length high-resolution experimental structure of the protein.
This thesis deals with the characterization of one of the human isoforms of TPH using multiple biophysical characterization techniques. Small-angle X-ray scattering (SAXS) and multi-angle light scattering (MALS) are employed to find the molecular parameters and shape of the TPH macromolecule in solution. One of the achievements of the study is the first experimental model of the protein in its native oligomeric state and encompassing all its functional domains. This structure provides new information on the conformation of the protein in the presence of L-phenylalanine - its suspected regulator and one of the proteinogenic amino acids. The behavior of the protein in the presence of L-phenylalanine is further explored using hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS). This study identifies the locations of the L-phenylalanine binding sites, the interface between two of the protein’s domains, as well as the interface between two TPH molecules in its dimeric form.
The results presented in this work provide new structural insights into the regulatory domain of the studied TPH isoform and shed new light on its functionality. Properly applied, these results can aid in future drug discovery studies aimed at the development of new antidepressants, as well as other drugs regulating the serotonergic system.