Stabilizing membrane proteins through protein engineering
Introduction
Integral membrane proteins (IMPs) are encoded by over 30% of all human genes [1] and are involved in critical cellular processes including cellular communication and sensing, molecular transport across lipid bilayers, biosynthesis and cell adhesion. Because many IMPs are located on the surface of cells, it is not surprising that they constitute the major class of drug targets, with 39 of the top 50 selling U.S. prescription drugs in 2010 acting on IMPs (http://www.drugs.com/top200.html). IMPs have specifically evolved to function in the hydrophobic environment of lipid bilayers, which they need to be removed from with detergents for biochemical and structural analysis (Figure 1).
Detergents are amphiphilic molecules containing a wide range of polar groups and, usually, a linear alkyl group, allowing them to form micelles in solution (Figure 1). IMPs solubilized in detergents become enveloped in detergent micelles, which partially mimic the membrane bilayer. A plethora of detergents exist for IMP purification: generally, milder detergents (long-chain detergents) are preferred, and they are uncharged and form large micelles that more closely mimic cell membranes (e.g., n-dodecyl-β-d-maltopyranoside (DDM)), while harsher detergents have shorter alkyl chains (e.g. n-octyl-β-d-glucopyranoside (OG)), and thus form smaller micelles that expose more of the IMP to the hydrophilic solvent (Figure 1). In IMPs without extensive extracellular domains, such as most G protein-coupled receptors (GPCRs), mild long-chain detergents tend to hinder the formation of protein–protein contacts and thus crystal formation, making the use of the harsh short-chain detergents a requirement for crystallization from detergents. Detergent molecules may also get access to internal cavities of the IMP, depending on the molecular structure of the detergent. All of these factors cause the environment within a detergent micelle to be different from the cell membrane, and thus IMP solubilization is typically denaturing, which has resulted in a large intellectual gap between our knowledge on the molecular aspects of IMP function in comparison to that of soluble proteins [2].
Much effort has been dedicated to methods that minimize the denaturation of IMPs during solubilization [3, 4, 5]. However, the inherent adaptability of IMPs can be harnessed to circumvent this problem in a different way. Because of their localization in a lipid environment, there are fewer protein fold classes for IMPs compared to soluble proteins [6]. The resultant protein families are, however, among the most evolutionary successful genes and have adapted to fulfill many biological functions [7, 8, 9, 10]. Thus, rather than modify the conditions to suit the protein — which may not be sufficient to reach the goal —, several methods have now been established that allow the modification of the IMP to be more resistant to detergent solubilization. Here we will review and compare the protein engineering methods that have been applied to IMPs to find sequence modifications that confer increased IMP stability without disrupting biological functionality.
Section snippets
Stabilized IMPs through mutations
Pioneering work in the membrane protein stabilization field was carried out in the laboratory of James Bowie. Unlike many soluble proteins, the prototypical bacterial IMP diacylglycerol kinase (DGK), later found to be a domain-swapped trimer with three transmembrane helices per subunit [11], was found to be very tolerant to mutations throughout its sequence [12]. A set of DGK mutants was then generated containing cysteine substitutions into 20 residues in the second transmembrane domain, two of
Alanine scanning mutagenesis
Bowie and coworkers were also the first to perform alanine scanning mutagenesis on a 7-transmembrane helix containing IMP, bacteriorhodopsin, to identify stabilizing mutations by monitoring the unfolding of the mutants upon exposure to SDS [17]. The main finding of this work was that a high frequency of alanine mutations, all within the second transmembrane helix of bacteriorhodopsin, resulted in receptors that were more stable when solubilized. This concept was then expanded upon and applied
Directed evolution for high expression and stability
Directed evolution, which has been used for years to stabilize soluble proteins, allows for screening of a much larger number of mutants than conventional alanine scanning. It uses an iteration of diversification and selection and can optionally be used to focus on specific protein residues or regions. Accordingly, the iterative progression can cover a larger sequence space than contained in a single library, or covered by alanine scanning. Also, neutral mutations can occur, which can be the
Cellular High Throughput Encapsulation, Solubilization and Screening (CHESS)
Bacterial display for high expression is able to easily select IMP GPCR variants with stability in longer chain detergents, however stability in harsher short-chain detergents required additional steps of receptor screening [48•]. The ability to perform directed evolution experiments where the stability of an IMP in detergent could be selected for directly would remove the need for such additional screening. Technically, this cannot be done using unmodified bacterial cells, because the
Computational design of stabilized IMPs
While directed evolution is less laborious and allows the sampling of many more mutations compared to alanine scanning, there is still concern that mutational coverage may not be complete, at least at the stage of combining mutations. The ultimate way of stabilizing an IMP would be through knowledge-based design. However, we still know very little about how IMPs fold and remain stable in the membrane, nor do we understand the different environment in synthetic detergent micelles in atomic
Conclusions
Over the past few years IMP stabilization has grown to become an established field with proven utility in the biochemical and structural study of IMPs, particularly GPCRs. Significant innovation has provided the field with several possible methods for stabilizing an IMP of interest, each with advantages and disadvantages (Table 1). It is important to note that the functionality of stabilized IMP mutants needs to be tested thoroughly to assess the influence of mutations on function. This is
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
• of special interest
•• of outstanding interest
References (54)
Stabilizing membrane proteins
Curr Opin Struct Biol
(2001)Detergents for the stabilization and crystallization of membrane proteins
Methods
(2007)- et al.
Detergent selection for enhanced extraction of membrane proteins
Protein Expr Purif
(2012) - et al.
Helical membrane proteins: diversity of functions in the context of simple architecture
Curr Opin Struct Biol
(2001) - et al.
Learning from the past: evolution of GPCR functions
Trends Pharmacol Sci
(2007) - et al.
Changing single side-chains can greatly enhance the resistance of a membrane protein to irreversible inactivation
J Mol Biol
(1999) - et al.
Building a thermostable membrane protein
J Biol Chem
(2000) - et al.
Crystal structure of a thermally stable rhodopsin mutant
J Mol Biol
(2007) - et al.
Side-chain contributions to membrane protein structure and stability
J Mol Biol
(2004) - et al.
Thermostabilization of the neurotensin receptor NTS1
J Mol Biol
(2009)