Numerous examples of phage display applied to soluble proteins demonstrate the power of the technique for protein engineering, affinity reagent discovery and structure-function studies. Recent reports have expanded phage display to include membrane proteins. The scope and limitations of membrane protein display remain undefined. Therefore, we report data from the phage display of representative types of membrane-associated proteins including plasma, nuclear, peripheral, single and multipass. The peripheral membrane protein neuromodulin displays robustly with packaging by conventional M13-KO7 helper phage. The monotopic membrane protein, Nogo-66 can also display on the phage surface, if packaged by the modified M13-KO7+ helper phage. The modified phage coat of KO7+ can better mimic the zwitterionic character of the plasma membrane. Four examples of putatively α-helical, integral membrane proteins failed to express as fusions to an anchoring phage coat protein, and therefore did not display on the phage surface. However, the β-barrel membrane proteins ShuA and MOMP, which pass through the membrane 22- and 16-times respectively, can display surprisingly well on the surfaces of both conventional and KO7+ phage. The results provide a guide for protein engineering and large-scale mutagenesis enabled by the phage display of membrane proteins.
Membrane proteins comprise a third of the human genome, yet present challenging targets for reverse chemical genetics. For example, though implicated in numerous diseases including multiple myeloma, the membrane protein caveolin-1 appears to offer a poor target for the discovery of synthetic ligands due to its largely unknown structure and insolubility. To break this impasse and identify new classes of caveolae controlling lead compounds, we applied phage-based, reverse chemical genetics for the discovery of caveolin-1 ligands derived from the anti-HIV therapeutic T20. Substitution of homologous residues into the T20 sequence used a process analogous to medicinal chemistry for the affinity maturation to bind caveolin. The resultant caveolin-1 ligands bound with >1000-fold higher affinity than wild-type T20. Two types of ELISAs and isothermal titration calorimetry (ITC) measurements demonstrated high affinity binding to caveolin by the T20 variants with Kd values in the 150 nM range. Microscopy experiments with the highest affinity caveolin ligands confirmed colocalization of the ligands with endogenous caveolin in NIH 3T3 cells. The results establish the foundation for targeting caveolin and caveolae formation in living cells.
Human reticulon 4 (RTN-4) has been identified as the neurite outgrowth inhibitor (Nogo). This protein contains a span of 66 amino acids (Nogo-66) flanked by two membrane helices at the C-terminus. We previously determined the NMR structure of Nogo-66 in a native-like environment and defined the regions of Nogo-66 expected to be membrane embedded. We hypothesize that aromatic groups and a negative charge hyperconserved among RTNs (Glu26) drive the remarkably strong association of Nogo-66 with a phosphocholine surface. Glu26 is an isolated charge with no counterion provided by nearby protein groups. We modeled the docking of dodecylphosphocholine (DPC) with Nogo-66 and found that a lipid choline group could form a stable salt bridge with Glu26 and serve as a membrane anchor point. To test the role of the Glu26 anion in binding choline, we mutated this residue to alanine and assessed the structural consequences, association with lipid and affinity for the Nogo receptor. In an aqueous environment, Nogo-66 Glu26Ala is more helical than WT and binds the Nogo receptor with higher affinity. Thus, we can conclude that in the absence of a neutralizing positive charge provided by lipid, the glutamate anion is destabilizing to the Nogo-66 fold. Although the Nogo-66 Glu26Ala free energy of transfer from water into lipid is similar to that of WT, NMR data reveal a dramatic loss of tertiary structure for the mutant in DPC micelles. These data show that Glu26 has a key role in defining the structure of Nogo-66 on a phosphocholine surface.
The twin-arginine translocase (Tat) system is used by many bacteria and plants to move folded proteins across the cytoplasmic membrane. Tat substrates contain a signature S/TRRxFLK twin-arginine motif in their N-terminal sequence. In most bacteria, the translocon consists of the TatABC subunits where TatA is the postulated pore subunit through homo-oligomerization with other TatA protomers, whereas TatBC is the substrate-receptor complex. The predicted structure of TatA includes a transmembrane helix, an amphipathic helix and a potentially unstructured C-terminal region. Biochemical and structural investigations were targeted at a peptide which represents the amphipathic region consisting of residues 22 to 44 of TatA (TatAH2). The dual topology of the region corresponding to TatAH2 in TatA was previously shown to be dependent on the membrane potential (Chan et al. 2007 Biochemistry 46: 7396-404), and thus warranted further investigations on its role for protein translocation. NMR and CD spectroscopy of TatAH2 show that it adopts helical structure in a membrane mimetic environment, in comparison to the random coil structure in aqueous solution. Microcalorimetry studies also show that it interacts with DPPG lipid vesicles to affect the phase transition temperatures. The solution NMR structure of TatAH2 shows conformation flexibility of the peptide around the acidic Asp31 at the center of the helix, a residue potentially important for the function of the TatA pore. The C-terminal half (residue 32 onwards) is a-helical, whereas the N-terminal half (23 to 30) has helical-like structure, suggesting that TatAH2 does not form a 'typical'a-helix.
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