Most biological processes require the production and degradation of proteins, a task that weighs heavily on the cell. Mutations that compromise the conformational stability of proteins place both specific and general burdens on cellular protein homeostasis (proteostasis) in ways that contribute to numerous diseases. Efforts to elucidate the chain of molecular events responsible for diseases of protein folding address one of the foremost challenges in biomedical science. However, relatively little is known about the processes by which mutations prompt the misfolding of α-helical membrane proteins, which rely on an intricate network of cellular machinery to acquire and maintain their functional structures within cellular membranes. In this review, we summarize the current understanding of the physical principles that guide membrane protein biogenesis and folding in the context of mammalian cells. Additionally, we explore how pathogenic mutations that influence biogenesis may differ from those that disrupt folding and assembly, as well as how this may relate to disease mechanisms and therapeutic intervention. These perspectives indicate an imperative for the use of information from structural, cellular, and biochemical studies of membrane proteins in the design of novel therapeutics and in personalized medicine.

Myriad biological processes proceed through states that defy characterization by conventional atomic-resolution structural biological methods. The invisibility of these ‘dark’ states can arise from their transient nature, low equilibrium population, large molecular weight, and/or heterogeneity. Although they are invisible, these dark states underlie a range of processes, acting as encounter complexes between proteins and as intermediates in protein folding and aggregation. New methods have made these states accessible to high-resolution analysis by nuclear magnetic resonance (NMR) spectroscopy, as long as the dark state is in dynamic equilibrium with an NMR-visible species. These methods – paramagnetic NMR, relaxation dispersion, saturation transfer, lifetime line broadening, and hydrogen exchange – allow the exploration of otherwise invisible states in exchange with a visible species over a range of timescales, each taking advantage of some unique property of the dark state to amplify its effect on a particular NMR observable. In this review, we introduce these methods and explore two specific techniques – paramagnetic relaxation enhancement and dark state exchange saturation transfer – in greater detail.