The function of membrane proteins is inextricably linked to the proper packing and assembly of their independently helical transmembrane (TM) segments. Here we examined whether an externally added TM peptide analogue could specifically inhibit the function of the membrane protein from which it is derived by competing for native TM helix packing sites, thereby producing a non-functional peptide-protein complex. This hypothesis was tested using Lys-tagged peptides synthesized with sequences corresponding to the three TM segments of the homotrimeric Escherichia coli diacylglycerol kinase (DGK). The peptide corresponding to wild-type DGK TM-2 inhibited the protein's enzymatic activity in a dose-dependent manner through formation of an inactive pseudo-complex, whereas peptides derived from TM-1 and TM-3 were benign toward DGK structure/function. Also, substitution of a conserved residue (Glu-69) within the TM-2 peptide abolished these effects, demonstrating the strict sequence requirements for TM-2-mediated association. This strategy, coupled with the practical advantages of the water solubility of Lys-tagged TM peptides, may constitute an attractive approach for the design of therapeutic membrane protein modulators even in the absence of a high resolution structure.
"While one would prefer to perform structural investigations on intact CFTR, the facts that (i) the protein cannot be readily expressed in preparative amounts; and (ii) the local structural effects of point mutations in the 1480-residue protein may fall below detection by spectroscopic analysis, have greatly limited biophysical studies on the full-length molecule. As well, TM segments have been shown to behave as independent folding domains even when excised from the protein, and to retain the native contacts that they exhibit within the intact protein   . These realities have encouraged us and others to develop the use of defined domain constructs of CFTR that can be manipulated by established biophysical techniques, and can be used to characterize the molecular events that link CF disease to CFphenotypic mutations. "
[Show abstract][Hide abstract] ABSTRACT: Mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) cause CF disease by altering the biosynthesis, maturation, folding and ion conductance of this protein. Our laboratory has focused on expression and structural analysis of the CFTR transmembrane (TM) domains using two-TM segments (i.e., helix-loop-helix constructs) which we term 'helical hairpins'; these represent the minimal model of tertiary contacts between two helices in a membrane. Previous studies on a library of TM3/4 hairpins of the first CFTR TM domain suggested that introduction of non-native polar residues into TM4 can compromise CFTR function through side chain-side chain H-bonding interactions with native Q207 in TM3 [Choi, M. Y., Cardarelli, L., Therien, A. G., and Deber, C. M. Non-native interhelical hydrogen bonds in the cystic fibrosis transmembrane conductance regulator domain modulated by polar mutations, Biochemistry 43 (2004) 8077-8083]. In the present work, we combine gel shift assays with a series of NMR experiments for comparative structural characterization of the wild type TM3/4 hairpin and its mutants V232D, I231D, Q207N/V232E. Over 95% of the backbone resonances of a 15N,13C-labelled V232D-TM3/4 construct in the membrane-mimetic environment of perfluorooctanoate (PFO) micelles were successfully assigned, and the presence and boundaries of helical segments within TM3 and TM4 were defined under these conditions. Comparative analysis of 15N and 1H chemical shift variations among HSQC spectra of WT-, V232D-, I231D- and Q207N/V232E-TM3/4 indicated that hairpin conformations vary with the position of a polar mutation (i.e., V232D and I231D vs. WT), but remain similar when hairpins with identically-positioned polar partners are compared (i.e., V232D vs. Q207N-V232E). The overall findings suggest that a polar mutation in a TM helix can potentially distort native interfacial packing determinants in membrane proteins such as CFTR, with consequences that may lead to disease.
"The present study investigates the abilities of a model peptide corresponding to the predicted TM sequence of DGKε to interact with membrane components, using circular dichroism, tryptophan fluorescence, and FRET; to inhibit DGK activity ; and to interact with cholesterol in membrane bilayers. The results provide insights into the functional importance of this segment in intact DGKε. "
[Show abstract][Hide abstract] ABSTRACT: Diacylglycerol kinase epsilon (DGKepsilon) is unique among mammalian DGK isoforms in having a segment of hydrophobic amino acids as a putative membrane anchor. To model the conformation, and stoichiometry of this segment in membrane-mimetic environments, we have prepared a peptide corresponding to this hydrophobic segment of DGKepsilon of sequence KKKKLILWTLCSVLLPVFITFWKKKKK-NH(2). Flanking Lys residues mimic the natural setting of this peptide in DGKepsilon, while facilitating peptide synthesis and characterization. Circular dichroism and fluorescence spectroscopic analysis demonstrated that the peptide has increased helical content and significant blue shifts in the presence of anionic--but not zwitterionic--bilayer membranes. When labeled with fluorophores that can undergo fluorescence resonance energy transfer, the peptide was found to dimerize--a result also observed from migration rates on SDS-PAGE gels under both reducing and non-reducing disulfide bridge conditions. The peptide was shown to preferentially interact with cholesterol in lipid films comprised of homogeneous mixtures of cholesterol and phosphatidylcholine, yet the presence of cholesterol in hydrated vesicle bilayers decreases its helical content. The peptide was also able to inhibit the activity of DGKepsilon protein in vitro. Our overall findings suggest that the peptide ultimately cannot leave the bulk water for attachment/insertion into the outer leaflet of an erythrocyte-like bilayer, yet its core sequence is sufficiently hydrophobic to insert into membrane core regions when membrane attachment is promoted by electrostatic attraction to anionic lipid head groups of the inner leaflet of an erythrocyte-like bilayer.
[Show abstract][Hide abstract] ABSTRACT: Studies that focus on packing interactions between transmembrane (TM) helices in membrane proteins would greatly benefit from the ability to investigate their association and packing interactions in multi-spanning TM domains. However, the production, purification, and characterization of such units have been impeded by their high intrinsic hydrophobicity. We describe the polar tagging approach to biophysical analysis of TM segment peptides, where incorporation of polar residues of suitable type and number at one or both peptide N- and C-termini can serve to counterbalance the apolar nature of a native TM segment, and render it aqueous-soluble. Using the native TM sequences of the human erythrocyte protein glycophorin A (GpA) and bacteriophage M13 major coat protein (MCP), properties of tags such as Lys, His, Asp, sarcosine, and Pro-Gly are evaluated, and general procedures for tagging a given TM segment are presented. Gel-shift assays on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) establish that various tagged GpA TM segments spontaneously insert into micellar membranes, and exhibit native TM dimeric states. Sedimentation equilibrium analytical centrifugation is used to confirm that Lys-tagged GpA peptides retain the native dimer state. Two-dimensional nuclear magnetic resonance (NMR) spectroscopy studies on Lys-tagged TM MCP peptides selectively enriched with N-15 illustrate the usefulness of this system for evaluating monomer-dimer equilibria in micelle environments. The overall results suggest that polar-tagging of hydrophobic (TM) peptides approach constitutes a valuable tool for the study of protein-protein interactions in membranes.
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