Spontaneous, pH-Dependent Membrane Insertion of a Transbilayer α-Helix

Hunt, J.F.; Rath, Parshuram; Rothschild, Kenneth J.; Engelman, Donald M.

doi:10.1021/bi970147b

Cited by 247 publications

(369 citation statements)

References 95 publications

Supporting

Mentioning

344

Contrasting

Order By: Relevance

“…In contrast to previously studied amphipathic peptides, it does not aggregate, form helical structure on the surface of a membrane, or induce pore formation (6)(7)(8)(9)12). Folding (formation of a transmembrane helix) accompanies insertion of the peptide into the lipid bilayer.…”

Section: Resultsmentioning

confidence: 78%

“…pHLIP contains two tryptophan residues having fluorescence that reports the attachment of the peptide to the membrane surface and the insertion of the peptide across the lipid bilayer as an ␣-helix (6,8,9). We chose 100-nm large unilamellar vesicles (LUV) containing palmitoyloleoylphosphatidylcholine (POPC) lipids with zwitterionic head groups to minimize any electrostatic component of the pHLIP interaction and to study peptide-membrane interactions over a wider range of temperatures than was possible in earlier work with dimyristoylphosphatidylcholine (DMPC) (6).…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Energetics of peptide (pHLIP) binding to and folding across a lipid bilayer membrane

Reshetnyak

Andreev

Segala

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

166

180

View full text Add to dashboard Cite

The pH low-insertion peptide (pHLIP) serves as a model system for peptide insertion and folding across a lipid bilayer. It has three general states: (I) soluble in water or (II) bound to the surface of a lipid bilayer as an unstructured monomer, and (III) inserted across the bilayer as a monomeric ␣-helix. We used fluorescence spectroscopy and isothermal titration calorimetry to study the interactions of pHLIP with a palmitoyloleoylphosphatidylcholine (POPC) lipid bilayer and to calculate the transition energies between states. We found that the Gibbs free energy of binding to a POPC surface at low pHLIP concentration (state I-state II transition) at 37°C is approximately ؊7 kcal/mol near neutral pH and that the free energy of insertion and folding across a lipid bilayer at low pH (state II-state III transition) is nearly ؊2 kcal/mol. We discuss a number of related thermodynamic parameters from our measurements. Besides its fundamental interest as a model system for the study of membrane protein folding, pHLIP has utility as an agent to target diseased tissues and translocate molecules through the membrane into the cytoplasm of cells in environments with elevated levels of extracellular acidity, as in cancer and inflammation. The results give the amount of energy that might be used to move cargo molecules across a membrane.thermodynamics ͉ drug delivery ͉ imaging ͉ membrane protein T he folding of helical membrane proteins can be conceptualized in terms of distinct steps: (i) peptide insertion and folding to form independently stable helices across a lipid bilayer, (ii) helix association to form a helix bundle intermediate, and (iii) further rearrangements of protein structure and/or binding of prosthetic groups to achieve a functional state (1). Insertion of most membrane proteins is facilitated in vivo by complex molecular machines, such as the translocon, which assist in placing hydrophobic sequences across the bilayer (2). In contrast to more hydrophobic sequences, moderately polar transmembrane domains of Cterminally-anchored proteins can postranslationally translocate themselves into membranes in a translocon-defective yeast strain (3, 4), seeking the free-energy minimum (5). Thus, insertion of a disordered peptide in aqueous solution to form a transbilayer ␣-helix is accompanied by a release of energy, so thermodynamic studies of the interaction of proteins and peptides with a lipid bilayer can provide insights regarding folding. However, such studies are significantly complicated by the poor solubility of membrane peptides, which has made it difficult to separate the contributions of peptide interactions with a lipid bilayer from self-interactions and aggregation.We have been studying a useful system for measurement of the thermodynamics and kinetics of peptide insertion and folding across a lipid bilayer. It is based on the pH low-insertion peptide (pHLIP), which has three major states: (I) soluble in water in an unstructured, monomeric state, (II) bound to the surface of a lipid bilayer in an unstructu...

show abstract

Section: Resultsmentioning

confidence: 78%

Section: Resultsmentioning

confidence: 99%

Energetics of peptide (pHLIP) binding to and folding across a lipid bilayer membrane

Reshetnyak

Andreev

Segala

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

166

180

View full text Add to dashboard Cite

show abstract

“…In addition to Bcl-x L , the translocation domain of diphtheria toxin also reversibly inserts into membranes in a pH-dependent manner. Furthermore, protonation of acidic residues is involved in the reversibility of membrane spanning for the C helix of bacteriorhodopsin, in which two of its six acidic residues are embedded in the bilayer (49). We speculate here that acidic residues are responsible for the observed reversibility because of the innate differences in the membrane partitioning properties of protonated and ionized Glu and Asp, which could readily be exploited as a reversibility switch.…”

Section: Membrane Association Arises From Weakening Electrostatic Repmentioning

confidence: 82%

The N-Terminal Domain of Bcl-x_L Reversibly Binds Membranes in a pH-Dependent Manner

et al. 2006

View full text Add to dashboard Cite

Bcl-x L regulates apoptosis by maintaining the integrity of the mitochondrial outer membrane by adopting both soluble and membrane-associated forms. The membrane-associated conformation does not require a conserved, C-terminal transmembrane domain and appears to be inserted into the bilayer of synthetic membranes as assessed by membrane permeabilization and critical surface pressure measurements. Membrane association is reversible and is regulated by the cooperative binding of approximately two protons to the protein. Two acidic residues, Glu153 and Asp156, that lie in a conserved hairpin of Bcl-x L ΔTM appear to be important in this process on the basis of a 16% increase in the level of membrane association of the double mutant E153Q/D156N. Contrary to that for the wild type, membrane permeabilization for the mutant is not correlated with membrane association. Monolayer surface pressure measurements suggest that this effect is primarily due to less membrane penetration. These results suggest that E153 and D156 are important for the Bclx L ΔTM conformational change and that membrane binding can be distinct from membrane permeabilization. Taken together, these studies support a model in which Bcl-x L activity is controlled by reversible insertion of its N-terminal domain into the mitochondrial outer membrane. Future studies with Bcl-x L mutants such as E153Q/D156N should allow determination of the relative contributions of membrane binding, insertion, and permeabilization to the regulation of apoptosis.Bcl-2 proteins act as checkpoints in the regulation of apoptosis by integrating intracellular signals to control the permeability and possibly the morphology of the mitochon-drion (1-9). For many Bcl-2 proteins, this checkpoint involves a change in localization from the cytosol to the mitochondrion (10-13). For example, Bcl-x L displays mixed localization to the cytosol and to organellar membranes, including the mitochondrion. After an apoptotic stimulus, Bcl-x L appears to localize primarily to the mitochondrial outer membrane where it may bind other apoptotic factors such as Bad (10,11,14) or form an ion channel thought to maintain the integrity of the mitochondrial membrane (11,(15)(16)(17). While mixed localization of Bcl-x L is well-established, the relative importance of cytosolic-and membranous-localized protein to the regulation of apoptosis is not. Intriguingly, the membrane activity of Bcl-x L may contribute more to its anti-apoptotic activity than its ability to sequester pro-apoptotic proteins: a mutant

show abstract

“…[29]). A synthetic peptide corresponding to helix C of bacteriorhodopsin had similar characteristics [69]. By inspection of the sequence (Fig.…”

Section: Discussionmentioning

confidence: 92%

Spectroscopic studies of the interaction of Ca²⁺‐ATPase‐peptides with dodecyl maltoside and its brominated analog

Soulié

Foresta

Møller

et al. 1998

European Journal of Biochemistry

View full text Add to dashboard Cite

The transmembrane sector of sarcoplasmic reticulum Ca 2ϩ -ATPase comprises ten putative transmembrane spans (M1ϪM10) in current topology models. We report here the structure and properties of three synthetic peptides with a single Trp representing the M6 and M7 regions implicated in Ca 2ϩ binding: peptide M6 (amino acid residues 785Ϫ810), peptide M7-L (amino acid residues 808Ϫ847) corresponding to loop 6-7 and the majority of span M7, and peptide M7-S (amino acid residues 818Ϫ847) which contains a shorter version of loop 6-7 than M7-L. After uptake of the peptides in the hydrophobic environment of dodecyl maltoside micelles, the peptides gain a significant amount of secondary structure, as indicated by their CD spectra. However, the A-helical content of M6 is lower than would be expected for a classical transmembrane segment. For M7-L peptide, the L6-7 loop is subject to specific proteolytic cleavage by proteinase K, as in intact Ca 2ϩ -ATPase. The formation of the peptide-detergent complexes was followed from the resulting fluorescence intensity changes, either enhancement using n-dodecyl β- D-maltoside Our results indicate that M7-L and M7-S are completely taken up by the detergent micelles. In contrast, the M6 peptide, which is highly water soluble, is more loosely associated with the detergent, as is also demonstrated by size-exclusion chromatography. The location of Trp in micelles was evaluated from the quenching observed in mixed micelles of n-dodecyl β-D-maltoside/ 7,8-dibromododecyl β-maltoside, using tryptophan octyl ester and solubilized Ca 2ϩ -ATPase as reference compounds. We conclude that W832 in M7 appears to be located near the surface of the micelle, in agreement with its membrane interfacial localization predicted in most Ca 2ϩ -ATPase topology models. In contrast, our data suggest that W794 in M6 has a deeper insertion in the micelle although not to the extent predicted by current models of Ca 2ϩ -ATPase and the rather short A-helix span of M6 may lead to exposure of a significant part of the C-terminal of this peptide to the micelle surface. The results are discussed in relation to the proposed roles of these membrane segments in active transport of Ca 2ϩ ions, in particular, the demonstration that M6 does not behave as a classical transmembrane helix may be correlated with the evidence, from site-directed mutagenesis, that this transmembrane segment should be essential in Ca 2ϩ binding.

show abstract

Spontaneous, pH-Dependent Membrane Insertion of a Transbilayer α-Helix

Cited by 247 publications

References 95 publications

Energetics of peptide (pHLIP) binding to and folding across a lipid bilayer membrane

Energetics of peptide (pHLIP) binding to and folding across a lipid bilayer membrane

The N-Terminal Domain of Bcl-x_L Reversibly Binds Membranes in a pH-Dependent Manner

Spectroscopic studies of the interaction of Ca²⁺‐ATPase‐peptides with dodecyl maltoside and its brominated analog

Contact Info

Product

Resources

About

Spontaneous, pH-Dependent Membrane Insertion of a Transbilayer α-Helix

Cited by 247 publications

References 95 publications

Energetics of peptide (pHLIP) binding to and folding across a lipid bilayer membrane

Energetics of peptide (pHLIP) binding to and folding across a lipid bilayer membrane

The N-Terminal Domain of Bcl-xL Reversibly Binds Membranes in a pH-Dependent Manner

Spectroscopic studies of the interaction of Ca2+‐ATPase‐peptides with dodecyl maltoside and its brominated analog

Contact Info

Product

Resources

About

The N-Terminal Domain of Bcl-x_L Reversibly Binds Membranes in a pH-Dependent Manner

Spectroscopic studies of the interaction of Ca²⁺‐ATPase‐peptides with dodecyl maltoside and its brominated analog