Dipolar Spectroscopy Inside the Cell : Methods and Applications for the Study Of Protein Conformations

Abstract

Proteins are essential macromolecules that are involved in virtually all cellular processes, and their functional diversity is closely tied to their structure. Far from being static, proteins are dynamic entities that exist as ensembles of interconverting conformations, which can be strongly influenced by their environment. Gaining insight into these structural dynamics within the native cellular context is key to understanding protein function. Consequently, a central goal of modern structural biology is to investigate protein structure and dynamics under physiologically relevant conditions. This thesis addresses this fundamentally challenging task by applying SDSL-EPR spectroscopy, specifically pulsed dipolar spectroscopy, to study protein conformational ensembles inside cells. First, it was shown that DEER spectroscopy can detect even subtle conformational changes within the C2 domain of CaLB1, highlighting its sensitivity for probing protein conformations. The methodological requirements for in-cell EPR spectroscopy were then addressed. Due to the reducing nature of the intracellular environment, the use of stable, reduction-resistant spin labels is essential. Such labels were tested and compared, including the novel cysteine-targeting 4-vinyl-Gd(III)PymiMTA spin label, which proved to be highly suitable for protein studies in a cellular context. Two approaches for in-cell EPR spectroscopy were explored: (1) the delivery of exogenously spin-labeled proteins into HeLa cells, and (2) in-cell spin labeling in E. coli via incorporation of ncAAs and click chemistry. For the first approach, electroporation and thermal stimulation were identified as the most effective methods for intracellular delivery. For the second approach, four novel azido-functionalized Gd(III)-based spin labels were tested. While successful labeling was achieved in vitro and in cell lysates, in-cell labeling was unsuccessful, likely due to limited membrane permeability of the spin labels. Finally, DEER spectroscopy was applied to investigate the conformational ensembles of the disease-relevant proteins αS and Akt1, both in vitro and in HeLa cells. M-Gd(III)DOTA-labeled αS was efficiently delivered into the cytoplasm of HeLa cells by electroporation. DEER spectroscopy revealed that αS adopts both disordered and α-helical conformations inside cells, suggesting potential binding to endogenous membranes. However, it could not be excluded that the observed changes in inter-spin distance distributions arose from intracellular αS compaction. Akt1, labeled with 4-vinyl-Gd(III)PymiMTA, was delivered into HeLa cells by thermal stimulation. Results obtained from in-cell DEER measurements indicate that Akt1 adopts a different conformational ensemble in cells compared to in vitro conditions. Altogether, this thesis presents a comprehensive methodological framework and demonstrates the feasibility of using SDSL-EPR spectroscopy to study protein conformations in the native cellular environment, contributing to the advancement of in-cell structural biology.