A quantitative FRET approach to characterize protein-protein interactions in living cells

Butz, Elisabeth (2017): A quantitative FRET approach to characterize protein-protein interactions in living cells. Dissertation, LMU München: Fakultät für Chemie und Pharmazie

Abstract

The ability of proteins to specifically interact with each other is a key feature in the regulation of biological processes. Knowledge about interaction partners and characterization of protein-protein interactions contribute to the understanding of proper protein function and cell physiology. In particular, Förster resonance energy transfer (FRET) is a suitable method to analyze interactions between proteins in living cells. During the progress of this thesis, a quantitative FRET approach was established that aims to evaluate binding curves for interaction partners. Moreover, the quantitative FRET approach was applied to study biological questions, including the investigation of putative interaction partners of the endolysosomal ion channel two-pore channel 2 (TPC2), the Kv7.2 potassium channel and the photoreceptor-specific transmembrane protein peripherin 2. The FRET approach described in manuscript I computes calibrated FRET efficiencies from fluorescent measurements using three-filter cubes and correlates the FRET efficiencies to the concentration of donor and acceptor molecules to determine binding curves, which bear information about maximal FRET efficiencies and relative binding constants for individual FRET pairings. Calibration factors that represent the optical properties of the imaging setup and the fluorophores are crucial for quantitative measurements. A detailed description how to assess these factors is provided. The quantitative FRET approach is very robust as both donor-centric (E-FRET) and acceptor-centric (SE-FRET) efficiencies are obtained simultaneously from multiple cells. The method was further applied to investigate protein-protein interactions of membrane proteins. First of all, in manuscript II, an epilepsy-causing mutation in the Kv7.2 potassium channel was shown to be implicated in a reduced calmodulin binding affinity to the channel, which affects channel regulation. A second study identified SNARE proteins, such as syntaxin 7 and syntaxin 6, as novel interaction partners of the intracellular ion channel TPC2 (manuscript III), revealing TPC2 as a putative member of the late endosome-lysosome fusion machinery. In manuscript IV, the impact of polymorphic variants of TPC2 on channel dimerization and mTOR binding was investigated. Furthermore, in a study covered by manuscripts V and VI, rhodopsin as well as S- and M-opsins were identified as novel interaction partners of the retinal protein peripherin 2 in rods and cones, respectively. The binding domain underlying the interaction between peripherin 2 and rhodopsin, could be assigned to the fourth transmembrane domain of peripherin 2. Moreover, it could be demonstrated that disease-associated mutations in peripherin 2 attenuated this particular binding, suggesting differential pathophysiological consequences of disrupted interactions in rods and cones. In manuscript VIII, peripherin 2 and its homolog Rom-1 were shown to have opposing effects on rod outer segment targeting of disease-linked peripherin 2 mutants by evaluating their binding affinities. Peripherin 2 is a scaffold protein exclusively expressed in outer segments of rods and cones. As photoreceptors are polarized cells, FRET measurements were not only performed on transfected HEK293 cells but also on acutely isolated outer segments of virally transduced murine photoreceptors (manuscript VII). The results gained in this thesis demonstrate that protein interactions play a crucial role in the regulation of proper protein function. Loss of binding partners or a reduced binding affinity to particular proteins may result in pathophysiological conditions. A deeper knowledge about molecular interactions will contribute to the understanding of cellular mechanisms, etiology of diseases and may further evaluate putative targets of pharmacological interest.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: Förster Resonanz Energie Transfer
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 540 Chemie
• Fakultäten: Fakultät für Chemie und Pharmazie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 20. November 2017
• 1. Bericht­erstatter:in: Wahl-Schott, Christian