Solution NMR study of integral membrane proteins.
ABSTRACT Signals between a cell and its environment are often transmitted through membrane proteins; therefore, many membrane proteins, including G protein-coupled receptors (GPCRs) and ion channels, are important drug targets. Structural information about membrane proteins remains limited owing to challenges in protein expression, purification and the selection of membrane-mimicking systems that will retain protein structure and function. This review describes recent advances in solution NMR applied to the structural study of integral membrane proteins. The examples herein demonstrate that solution NMR spectroscopy will play a unique role not only in structural analysis, but also drug discovery of membrane proteins.
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ABSTRACT: Fragments of large membrane proteins have the potential to facilitate structural analysis by NMR, but their folding state remains a concern. Here we determined the quality of folding upon heterologous expression for a series of N- or C-terminally truncated fragments of the human Y4 G-protein coupled receptor, amounting to six different complementation pairs. As the individual fragments lack a specific function that could be used to ascertain proper folding, we instead assessed folding on a basic level by studying their membrane topology and by comparing it to well-established structural models of GPCRs. The topology of the fragments was determined using a reporter assay based on C-terminal green fluorescent protein- or alkaline phosphatase-fusions. N-terminal fusions to Lep or Mistic were used if a periplasmic orientation of the N-terminus of the fragments was expected based on predictions. Fragments fused to Mistic expressed at comparably high levels, whereas Lep fusions were produced to a much lower extent. Though none of the fragments exclusively adopted one orientation, often the correct topology predominated. In addition, systematic analysis of the fragment series suggested that the C-terminal half of the Y4 receptor is more important for adopting the correct topology than the N-terminal part. Using the detergent dodecylphosphocholine, selected fragments were solubilized from the membrane and proved sufficiently stable to allow purification. Finally, as a first step toward reconstituting a functional receptor from two fragments, we observed a physical interaction between complementing fragments pairs upon co-expression.Biochimica et Biophysica Acta 07/2012; 1818(12):3055-63. · 4.66 Impact Factor
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ABSTRACT: Flavivirus non-structural protein 2A (NS2A) is a component of viral replication complex, functions in virion assembly, and antagonizes host immune response. Although flavivirus NS2A is known to associate with endoplasmic reticulum (ER) membrane, the detailed topology of this protein has not been determined. Here we report the first topology model of flavivirus NS2A on ER membrane. Using dengue virus (DENV) NS2A as a model, we show that (i) the N-terminal 68 amino acids are located in ER lumen with one segment (amino acids 30-52) that interacts with ER membrane without traversing the lipid bilayer; (ii) amino acids 69 to 209 form five trans-membrane segments, each of which integrally spans the ER membrane; (iii) the C-terminal tail (amino acids 210-218) is located in cytosol. NMR structural analysis showed that the first membrane-spanning segment (amino acids 69-93) consists of two helices separated by a helix "breaker". The helix "breaker" is formed by amino acid P85 and one positively charged residue R84. Functional analysis using replicon and genome-length RNAs of DENV-2 indicates that P85 is not important for viral replication. However, when R84 was substituted with E, the R84E mutation attenuated both viral RNA synthesis and virus production. Remarkably, when R84 was changed to A, the R84A mutation did not affect viral RNA synthesis, but blocked intracellular formation of infectious virions. Collectively, the mutagenesis results demonstrate that NS2A functions in both DENV RNA synthesis and virion assembly/maturation. The topology model of DENV NS2A provides a good starting point to study how flavivirus NS2A modulates viral replication and evasion of host immune response.Journal of Virology 02/2013; · 5.08 Impact Factor
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ABSTRACT: The G protein-coupled β(2)-adrenoreceptor (β(2)AR) signals through the heterotrimeric G proteins G(s) and G(i) and β-arrestin. As such, the energy landscape of β(2)AR-excited state conformers is expected to be complex. Upon tagging Cys-265 of β(2)AR with a trifluoromethyl probe, (19)F NMR was used to assess conformations and possible equilibria between states. Here, we report key differences in β(2)AR conformational dynamics associated with the detergents used to stabilize the receptor. In dodecyl maltoside (DDM) micelles, the spectra are well represented by a single Lorentzian line that shifts progressively downfield with activation by appropriate ligand. The results are consistent with interconversion between two or more states on a time scale faster than the greatest difference in ligand-dependent chemical shift (i.e. >100 Hz). Given that high detergent off-rates of DDM monomers may facilitate conformational exchange between functional states of β(2)AR, we utilized the recently developed maltose-neopentyl glycol (MNG-3) diacyl detergent. In MNG-3 micelles, spectra indicated at least three distinct states, the relative populations of which depended on ligand, whereas no ligand-dependent shifts were observed, consistent with the slow exchange limit. Thus, detergent has a profound effect on the equilibrium kinetics between functional states. MNG-3, which has a critical micelle concentration in the nanomolar regime, exhibits an off-rate that is 4 orders of magnitude lower than that of DDM. High detergent off-rates are more likely to facilitate conformational exchange between distinct functional states associated with the G protein-coupled receptor.Journal of Biological Chemistry 08/2012; 287(43):36305-11. · 4.65 Impact Factor