Rohini R. Rana

Maltose-neopentyl glycol (MNG) amphiphiles for solubilization, stabilization and crystallization of membrane proteins

The understanding of integral membrane protein (IMP) structure and function is hampered by the difficulty of handling these proteins. Aqueous solubilization, necessary for many types of biophysical analysis, generally requires a detergent to shield the large lipophilic surfaces of native IMPs. Many proteins remain difficult to study owing to a lack of suitable detergents. We introduce a class of amphiphiles, each built around a central quaternary carbon atom derived from neopentyl glycol, with hydrophilic groups derived from maltose. Representatives of this maltose–neopentyl glycol (MNG) amphiphile family show favorable behavior relative to conventional detergents, as manifested in multiple membrane protein systems, leading to enhanced structural stability and successful crystallization. MNG amphiphiles are promising tools for membrane protein science because of the ease with which they may be prepared and the facility with which their structures may be varied.

Tandem Facial Amphiphiles for Membrane Protein Stabilization

We describe a new type of synthetic amphiphile that is intended to support biochemical characterization of intrinsic membrane proteins. Members of this new family displayed favorable behavior with four of five membrane proteins tested, and these amphiphiles formed relatively small micelles.

A New Class of Amphiphiles Bearing Rigid Hydrophobic Groups for Solubilization and Stabilization of Membrane Proteins

Integral membrane proteins (IMPs) are crucial cellular components, mediating the transfer of material and signals between the environment and the cytoplasm, or between different cellular compartments. Structural and functional analysis of IMPs is important; more than half of current pharmaceutical agents target proteins in this class. [1] IMP characterization is often challenging, and sometimes impossible, because of difficulties associated with handling these macromolecules.[2] IMPs in the native state display large hydrophobic surfaces, which are not compatible with an aqueous environment; therefore, detergents are required to extract IMPs from the lipid bilayer and to maintain the native state of the protein in solution.[3] Nonionic detergents, such as dodecyl-β-D-maltoside (DDM) and octyl-β-D-glucoside (OG), are generally preferred for these applications. Despite the comparatively mild nature of DDM, OG and related detergents, many membrane proteins denature and/or aggregate upon solubilization with these agents.[4]

Large-scale functional expression of WT and truncated human adenosine A2A receptor in Pichia pastoris bioreactor cultures

BackgroundThe large-scale production of G-protein coupled receptors (GPCRs) for functional and structural studies remains a challenge. Recent successes have been made in the expression of a range of GPCRs using Pichia pastoris as an expression host. P. pastoris has a number of advantages over other expression systems including ability to post-translationally modify expressed proteins, relative low cost for production and ability to grow to very high cell densities. Several previous studies have described the expression of GPCRs in P. pastoris using shaker flasks, which allow culturing of small volumes (500 ml) with moderate cell densities (OD600 ~15). The use of bioreactors, which allow straightforward culturing of large volumes, together with optimal control of growth parameters including pH and dissolved oxygen to maximise cell densities and expression of the target receptors, are an attractive alternative. The aim of this study was to compare the levels of expression of the human Adenosine 2A receptor (A2AR) in P. pastoris under control of a methanol-inducible promoter in both flask and bioreactor cultures.ResultsBioreactor cultures yielded an approximately five times increase in cell density (OD600 ~75) compared to flask cultures prior to induction and a doubling in functional expression level per mg of membrane protein, representing a significant optimisation. Furthermore, analysis of a C-terminally truncated A2AR, terminating at residue V334 yielded the highest levels (200 pmol/mg) so far reported for expression of this receptor in P. pastoris. This truncated form of the receptor was also revealed to be resistant to C-terminal degradation in contrast to the WT A2AR, and therefore more suitable for further functional and structural studies.ConclusionLarge-scale expression of the A2AR in P. pastoris bioreactor cultures results in significant increases in functional expression compared to traditional flask cultures.

Bacterial TIR-containing proteins and host innate immune system evasion

The innate immune system provides the first line of host defence against invading pathogens. Key to upregulation of the innate immune response are Toll-like receptors (TLRs), which recognize pathogen-associated molecular patterns (PAMPs) and trigger a signaling pathway culminating in the production of inflammatory mediators. Central to this TLR signaling pathway are heterotypic protein–protein interactions mediated through Toll/interleukin-1 receptor (TIR) domains found in both the cytoplasmic regions of TLRs and adaptor proteins. Pathogenic bacteria have developed a range of ingenuous strategies to evade the host immune mechanisms. Recent work has identified a potentially novel evasion mechanism involving bacterial TIR domain proteins. Such domains have been identified in a wide range of pathogenic bacteria, and there is evidence to suggest that they interfere directly with the TLR signaling pathway and thus inhibit the activation of NF-κB. The individual TIR domains from the pathogenic bacteria Salmonella enterica serovar Enteritidis, Brucella sp, uropathogenic E. coli and Yersinia pestis have been analyzed in detail. The individual bacterial TIR domains from these pathogenic bacteria seem to differ in their modes of action and their roles in virulence. Here, we review the current state of knowledge on the possible roles and mechanisms of action of the bacterial TIR domains.

Novel tripod amphiphiles for membrane protein analysis.

Integral membrane proteins play central roles in controlling the flow of information and molecules across membranes. Our understanding of membrane protein structures and functions, however, is seriously limited, mainly due to difficulties in handling and analysing these proteins in aqueous solution. The use of a detergent or other amphipathic agents is required to overcome the intrinsic incompatibility between the large lipophilic surfaces displayed by the membrane proteins in their native forms and the polar solvent molecules. Here, we introduce new tripod amphiphiles displaying favourable behaviours toward several membrane protein systems, leading to an enhanced protein solubilisation and stabilisation compared to both conventional detergents and previously described tripod amphiphiles.

A Toll/interleukin (IL)-1 receptor domain protein from Yersinia pestis interacts with mammalian IL-1/Toll-like receptor pathways but does not play a central role in the virulence of Y. pestis in a mouse model of bubonic plague

The Toll/interleukin (IL)-1 receptor (TIR) domain is an essential component of eukaryotic innate immune signalling pathways. Interaction between TIR domains present in Toll-like receptors and associated adaptors initiates and propagates an immune signalling cascade. Proteins containing TIR domains have also been discovered in bacteria. Studies have subsequently shown that these proteins are able to modulate mammalian immune signalling pathways dependent on TIR interactions and that this may represent an evasion strategy for bacterial pathogens. Here, we investigate a TIR domain protein from the highly virulent bacterium Yersinia pestis, the causative agent of plague. When overexpressed in vitro this protein is able to downregulate IL-1β- and LPS-dependent signalling to NFκB and to interact with the TIR adaptor protein MyD88. This interaction is dependent on a single proline residue. However, a Y. pestis knockout mutant lacking the TIR domain protein was not attenuated in virulence in a mouse model of bubonic plague. Minor alterations in the host cytokine response to the mutant were indicated, suggesting a potential subtle role in pathogenesis. The Y. pestis mutant also showed increased auto-aggregation and reduced survival in high-salinity conditions, phenotypes which may contribute to pathogenesis or survival.

New Tools for Membrane Protein Research

The last five years have seen a dramatic increase in the number of membrane protein structures. The vast majority of these 191 unique structures are of membrane proteins from prokaryotic sources. Whilst these have provided unprecedented insight into the mechanism of action of these important molecules our understanding of many clinically important eukaryotic membrane proteins remains limited by a lack of high resolution structural data. It is clear that novel approaches are required to facilitate the structural characterization of eukaryotic membrane proteins. Here we review some of the techniques developed recently which are having a major impact on the way in which structural studies of eukaryotic membrane proteins are being approached. Several different high throughput approaches have been designed to identify membrane proteins most suitable for structural studies. One approach is to screen large numbers of related or non-related membrane proteins using GFP fusion proteins. An alternative involves generating large numbers of mutants of a single protein with a view to obtaining a fully functional but highly stable membrane protein. These, and other novel techniques that aim to facilitate the production of protein likely to yield well-diffracting crystals are described.

Glucose-Neopentyl Glycol (GNG) Amphiphiles for Membrane Protein Solubilization, Stabilization and Crystallization

The development of a new class of surfactants for membrane protein manipulation, “GNG amphiphiles”, is reported. These amphiphiles display promising behavior for membrane proteins, as demonstrated recently by the high resolution structure of a sodium-pumping pyrophosphatase reported by Kellosalo et al.