Structural studies of large integral membrane proteins in reverse micelles by solution nuclear magnetic resonance

The structural characterization of integral membrane proteins represents one of the many challenges of the post-genomic era. While membrane proteins comprise approximately 50% of current and potential drug targets, their structural characterization lags far behind that of soluble proteins. Nuclear magnetic resonance (NMR) offers tremendous potential for the investigation of membrane proteins in aqueous environments with respect to structural characterization, relaxation properties, and the details of small ligand interactions. However, the size limitations of solution NMR due to the slow tumbling problem have restricted comprehensive structural characterization of membrane protein NMR structures to the relatively small β-barrel proteins or helical proteins of simple topology. Here we detail an approach for the encapsulation of integral membrane proteins in reverse micelles, allowing for their study in low viscosity solvents and thus limiting the slow tumbling issue. This approach obviates the traditional compromises in sample preparation for large proteins in NMR. Using a 54 kDa construct of the homotetrameric potassium channel KcsA, we present a hybrid surfactant screen to optimize NMR conditions and describe utilization of 3D NMR pulse sequences and backbone assignment strategies normally restricted to proteins of much smaller size. We are able to confirm the helical structure of KcsA’s transmembrane domains in reverse micelles, as well as proper quaternary arrangement of the monomers and preservation of potassium coordination in the selectivity filter. Additionally we show that the solvation properties of the channel in reverse micelles are analogous to a membrane protein solubilized by a traditional aqueous micelle. Relaxation studies of the channel are also presented

A Suppression Strategy for Antibiotic Discovery

AbstractHigh-throughput phenotype screening and target identification have been combined in an effort to isolate antimicrobial, small-molecule therapeutics [1]. This approach, developed by Brown and colleagues and reported in this issue, is a major technological advance for antimicrobial drug discovery

Reverse Micelles in Integral Membrane Protein Structural Biology by Solution NMR Spectroscopy

SummaryIntegral membrane proteins remain a significant challenge to structural studies by solution NMR spectroscopy. This is due not only to spectral complexity, but also because the effects of slow molecular reorientation are exacerbated by the need to solubilize the protein in aqueous detergent micelles. These assemblies can be quite large and require deuteration for optimal use of the TROSY effect. In principle, another approach is to employ reverse micelle encapsulation to solubilize the protein in a low-viscosity solvent in which the rapid tumbling of the resulting particle allows for use of standard triple-resonance methods. The preparation of such samples of membrane proteins is difficult. Using a 54 kDa construct of the homotetrameric potassium channel KcsA, we demonstrate a strategy that employs a hybrid surfactant to transfer the protein to the reverse micelle system

Mechanistic studies of the biogenesis and folding of outer membrane proteins in vitro and in vivo: what have we learned to date?

Research into the mechanisms by which proteins fold into their native structures has been on-going since the work of Anfinsen in the 1960s. Since that time, the folding mechanisms of small, water-soluble proteins have been well characterised. By contrast, progress in understanding the biogenesis and folding mechanisms of integral membrane proteins has lagged significantly because of the need to create a membrane mimetic environment for folding studies in vitro and the difficulties in finding suitable conditions in which reversible folding can be achieved. Improved knowledge of the factors that promote membrane protein folding and disfavour aggregation now allows studies of folding into lipid bilayers in vitro to be performed. Consequently, mechanistic details and structural information about membrane protein folding are now emerging at an ever increasing pace. Using the panoply of methods developed for studies of the folding of water-soluble proteins. This review summarises current knowledge of the mechanisms of outer membrane protein biogenesis and folding into lipid bilayers in vivo and in vitro and discusses the experimental techniques utilised to gain this information. The emerging knowledge is beginning to allow comparisons to be made between the folding of membrane proteins with current understanding of the mechanisms of folding of water-soluble proteins