Expression, Purification, Circular Dichroism, and NMR Analysis of Triple Transmembrane Domain Containing Fragments of a GPCR

G protein-coupled receptors (GPCRs) are the largest class of signaling molecules in eukaryotes and are important pharmacological targets. Structural characterization of GPCRs is of paramount importance to the discovery of more efficient drugs; however, these studies are hindered by the inherent hydrophobicity, flexibility, and large size of these signaling proteins. Since their flexibility makes crystallization difficult, stabilizing mutations or substitutions are required to facilitate crystal-packing contacts. The size of the receptor/membrane mimetic complex required for solution-state nuclear magnetic resonance (NMR) analysis is too large to enable efficient isotropic tumbling. For these reasons, high-resolution structural information is available for only thirteen of the ∼1000 GPCRs identified to date.;Insights into conformational preferences and the three-dimensional (3D) structure of domains of these receptors can be obtained using polypeptide fragments of these proteins. This approach is relevant because functional GPCRs can be reconstituted from fragments. Our lab has been using the yeast &agr;-factor receptor as a paradigm for methods development for GPCR structural characterization. We have studied isolated fragments of this GPCR containing 1 and 2 transmembrane domains (TMs). For my dissertation project, I set out to characterize 3TM containing fragments of Ste2p. The goals of my project were 1) to determine whether 3TM containing fragments form more definied tertiary structures than a 2TM containing counterpart and 2) to determine whether chemical shifts from smaller fragments could be used to assign larger fragments.;Two 3TM-containing fragments of Ste2p were recombinantly expressed, purified by reverse phase high performance liquid chromatography (RP-HPLC), and subjected to extensive biophysical analysis by circular dichroism (CD) and NMR spectroscopy. A 131-residue fragment containing the first 3TMs of Ste2p, TM1-TM3 (G31-R161) and a 151-residue fragment containing the first, second, and seventh TMs, TM127 (G31-T114,T274-L340) were cloned and expressed as TrpDeltaLE fusion proteins in Escherichia coli. The expressed proteins were subjected to CNBr cleavage to remove the fusion tag and TM1-TM3 and TM127 were purified by reverse-phase HPLC. The cleavage products were isolated in yields of up to 20 mg per liter of culture in a variety of isotopic-labeled forms. The secondary structure of TM1-TM3 and TM127 was determined to be helical in a number of membrane mimetic environments, including 2,2,2-trifluoroethanol (TFE):water and detergent micelles by CD spectroscopy. Preliminary HSQC analysis in 50% TFE:water and detergent micelles revealed that these fragments were suitable for structural analysis by NMR spectroscopy. Complete backbone and side chain assignments and a detailed localization of the secondary structural elements of TM1-TM3 in 50% TFE:water have been achieved. Under these conditions, an NMR structure was determined to have low convergence, and no tertiary contacts were observed. Attempts are currently being made to ascertain tertiary contacts by paramagnetic relaxation enhancement (PRE). NMR structural characterization of both TM1-TM3 and TM127 in detergent micelles is being conducted at the University of Zurich.;To determine the transferability of chemical shifts for small to large fragments of Ste2p, two smaller constructs were analyzed. A 1TM containing fragment of Ste2p, TM1 [Ste2p(G31-T78)] was cloned into a direct expression vector and expressed with an N-terminal histidine tag in E.coli. The fragment was purified by RP-HPLC. This construct and a previously expressed TM7CT40 construct [Ste2p(S267-S339)](1) were subject to extensive heteronuclear NMR analysis in lysopalmitoylphosphatidylglycerol:dodecylphosphocholine (LPPG:DPC) micelles for backbone and side chain assignment. Backbone and side chain assignments for the TM1 fragment have been completed. Comparison of amide chemical shifts obtained for this fragment to those obtained for the TM1-TM2, TM1-TM3, and TM127 fragments of Ste2p suggests that chemical shifts are transferable for all regions except the flexible GXXXG region. A calculated NMR structure for TM1 reveals a large kink at this region. Comparison of the amide TM7 chemical shifts to those of the TM127 fragment reveals that all obtained chemical shifts are similar for this region. Detailed computational analysis is currently being performed at the Frankfurt Institute of Advanced Studies of Goethe University in order to assess complete chemical shift transferability for these fragments.