Where do we go from here? Membrane protein research beyond the structure-function horizon (original) (raw)

2018, Biochimica Et Biophysica Acta - Biomembranes

Where do we go from here? Membrane protein research beyond the structure-function horizon Biology has evolved and thrives in an aqueous environment. The water-insolubility of membrane proteins is one of the biggest reasons why membrane protein research has been among the most challenging aspects of protein biology, with every study being plagued by uncertainties regarding the physiological relevance of its findings. But thanks to savvy technical innovations (see below), researchers are closer than ever to getting a firm handle on the biology of several membrane proteins, even to the point where bench-to-bedside research is becoming a consideration. This Special Issue is dedicated to such advances and is our humble attempt at highlighting some key areas. For a while now, the workflow of membrane protein research has proceeded as follows: (i) A select protein of interest is cloned out from its native genome, and tested in a battery of functional biochemical assays, either in a convenient heterologous host or in purified form. (ii) Mutations are made across the protein to assess their various effects, and predictions are made about its structure and mechanism(s). In the past few decades, this cycle was iterated several times over with many discoveries of novel classes of membrane proteins being made, based on topology mappings and functional nuances. The advances in biophysical tools in the form of high-resolution X-ray and cryo-electron microscopy (cryoEM) structures have allowed the addition of architecture to be ascribed to protein function. However, for the longest time, obtaining the structure of a researcher's favorite protein (especially if it happened to be a membrane protein) was the culmination of a lifetime's worth of workif it was ever attained. Membrane protein research certainly has changed, and although the first high-resolution structure of a membrane protein by cryoEM was only published four years ago [1], the resulting avalanche of high-resolution cryoEM membrane protein structures certainly profits from the fact that their structure-function relationships had been heavily investigated for decades. It is not a stretch, then, to expect to see protein structures for all major membrane protein families, and potentially all medically relevant targets in the next couple of years. But where do we go from there? What is left to do and what can structures fall short of revealing? In the first paper in this Special Issue, Seeger muses on how it is time for us to reflect on what we, as a field, can do in "times of countless structures" [2]. And encouragingly, he reminds us that structural biology will not become obsolete any time soon. Instead, a new species of structural biologist is emerging-that of an integrated scientist, who works with experts on the functional, computational, and biophysical facets of membrane proteins to arrive at a holistic understanding for each protein of interest. Advances in biophysical tools have, in close collaboration with high resolution structures and functional data, provided means to detect interatomic distance changes in membrane-embedded proteins, to assess their dynamics and now even allow to predict their behavior to a certain degree through molecular dynamic simulations of proteins in modelled native-like environments. But no matter how advanced our techniques become, ultimately, they still rely on a careful sample preparation. And while it may by now seem reasonably straight forward to pull a membrane protein out of its lipid environment to then reconstitute it back into a lipid bilayer, there has also been a huge demand for optimizing these approaches and obtain more native-like reconstitution conditions. The introduction of nanodiscs clearly revolutionized the field (e.g. [3-5]) as they allow to study membrane proteins in near-native conditions. Another novel technology for the reconstitution of membrane proteins for functional studies using styrene-maleic acid copolymer discs is discussed in Pollock et al. [6]. Here, a major advantage is that the protein can be purified in its native membrane, thus maintaining lipid composition and presumably, at least for a short amount of time, asymmetry. Together, combining advances in protein preparation with biophysical approaches do a fantastic job validating and accelerating functional experimentation much further than would otherwise have been possible with the individual techniques alone. As spectroscopic techniques become more advanced, they begin to shed light on intricate details of protein dynamics as described by Spadaccini et al., using solid-state NMR (nuclear magnetic resonance) in combination with DNP (dynamic nuclear polarization) [7], Zhogbi et al., using LRET (luminescence resonance electron transfer) [8] and Bordignon and Bleicken with EPR (electron paramagnetic resonance) [9]. EPR spectroscopy has long been recognized as a formidable tool to study membrane protein dynamics, and in the present review, the authors also give an overview over recent hardware, pulse sequence and spin label improvements. EPR studies are often combined with NMR studies [10,11], as global and local conformational changes can be studied in a complementary fashion. DNP-enhanced NMR additionally allows spectra to be recorded in a fraction of the previously required time, thus making lowly populated conformations or difficult to obtain samples accessible to inherently insensitive NMR studies. Spadaccini et al. show how substrate interactions with MsbA lead to specific changes in helix 4 and 6 of this ABC transporter [7]. Likewise, Zhogbi et al. use this ABC transporter to demonstrate the advantages of LRET in the study of membrane proteins, such as the long lifetime of excited states for the lanthanide donor and the line spectra emissions [8].