Roles of Carboxyl Groups in the Transmembrane Insertion of PeptidesIntroductionResultsDiscussionMaterials and MethodsPeptide synthesis and assessment of monomeric stateAnalytical ultracentrifugationLiposome preparationFluorescence spectroscopyCircular dichroismOCD measurementsBiotin translocation assayAcknowledgementsReferencesRoles of Carboxyl Groups in the TransmembraneInsertion of PeptidesFrancisco N. Barrera1, Dhammika Weerakkody2, Michael Anderson2,Oleg A. Andreev2, Yana K. Reshetnyak2and Donald M. Engelman1⁎1Department of Molecular Biophysics and Biochemistry, Yale University, PO Box 208114, New Haven,CT 06520, USA2Physics Department, University of Rhode Island, Kingston, RI 02881, USAReceived 6 July 2011;received in revised form3 August 2011;accepted 5 August 2011Available online23 August 2011Edited by J. BowieKeywords:membrane protein folding;pHLIP;pH trigger;carboxyl titration;transmembrane helixWe have used pHLIP® [pH (low) insertion peptide] to study the roles ofcarboxyl groups in transmembrane (TM) peptide insertion. pHLIP binds tothe surface of a lipid bilayer as a disordered peptide at neutral pH; when thepH is lowere d, it inserts across the membrane to form a TM helix. Peptideinsertion is reversed when the pH is raised above the characteristic pKa(6.0).A key event that facilitates membrane insertion is the protonation ofaspartic acid (Asp) and/or glutamic acid (Glu) residues, since theirnegatively charged side chains hinder membrane insertion at neutral pH.In order to gain mechanistic understanding, we studied the membraneinsertion and exit of a series of pHLIP variants where the four Asp residueswere sequentially mutated to nonacidic residues, including histidine (His).Our results show that the presence of His residues does not prevent the pH-dependent peptide membrane insertion at ∼ pH 4 driven by the protonationof carboxyl groups at the inserting end of the peptide. A further pH dropleads to the protonation of His residues in the TM part of the peptide, whichinduces peptide exit from the bilayer. We also find that the number ofionizable residues that undergo a change in protonation during membraneinsertion correlates with the pH-dependent insertion into the lipid bilayerand exit from the lipid bil ayer, and that cooperativity increases with theirnumber. We expect that our understanding will be used to improve thetargeting of acidic diseased tissue by pHLIP.© 2011 Elsevier Ltd. All rights reserved.IntroductionExtracellular acidification is a hallmark of differ-ent pathologies, includ ing cancer, inflammation,ischemic stroke, and atherosclerotic plaques. Acido-sis might be a useful biomarker for diagnosis ortreatment if means can be found to target tissueacidity. We have found that a peptide derived fromhelix C of bacteriorhodopsin,1named pHLIP® [pH(low) insertion peptide], is capable of targetingacidic tissu es and inserting into the cell plasmamembrane.2pHLIP is able to target mouse tumorsin vivo with high specificity,2opening the possibilityof its use for cancer imaging. Additionally, pHLIPhas a promising therapeutic potential, as it is able totranslocate cell-impermeable cargo molecules, suchas organic dyes, peptides, peptide nucleic acids, andtoxins, across the plasma membrane into thecytoplasm of tumor cells.2,3pHLIP itself does nothave obvious acute toxicity in cells3or in mice.2pHLIP is monomeric at low concentrations, with amostly unstructured conformation in neutral and*Corresponding author. E-mail address:[email protected] used: TM, transmembrane; wt, wild type;POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine;OCD, oriented circular dichroism; PEG, polyethyleneglycol; pH, extracellular pH.doi:10.1016/j.jmb.2011.08.010 J. Mol. Biol. (2011) 413, 359–371Contents lists available at www.sciencedirect.comJournal of Molecular Biologyjournal homepage: http://ees.elsevier.com.jmb0022-2836/$ - see front matter © 2011 Elsevier Ltd. All rights reserved.basic solutions (state I). If lipid vesicles or mem-branes are present at neutral pH, pHLIP binds totheir external surface with an energy of 6–7 kcal/mol (state II).4In the membrane-attached state,pHLIP remains largely unstructured.1However, ifthe solution pH is lowered, pHLIP inserts to form atransmembrane (TM) α-helix (state III). The inser-tion is fully reversible and unidirectional, with theC-terminus being translocated acrossthe membrane.3The pKaof peptide insertion into lipid bilayers is 6.0,and the energy difference between the attached stateand the inserted state is 1.8 kcal/mol at 37 °C.4The pH LIP sequence is relatively rich in acidicresidues (Table 1). At neutral pH, the combinednegative charges of these residues, together with thecarboxy terminus, constitute a large energeticbarrier to pHLIP insertion across the membrane.The estimated energetic cost of the transfer of asingle aspartic acid residue from water to thehydrophobic core of the membrane is unfavorableby 3.6 kcal/mol for the unprotonated (negativelycharged) state, but only by 0.4 kcal/mol for theprotonated (noncharged) state.5Simultaneouslymoving four charged Asp residues, one Glu residue,and the carboxy terminus into the membrane wouldcost 21.6 kcal/mol, assuming 3.6 kcal/mol for eachcarboxyl group, and peptide partitioning into themembrane at equilibr ium would be about 1:1016.Thus, for pHLIP to be able to insert into membranes,protonation of a large fraction of the acidic residuescan be expected, and knowledge of the protonationpattern of the acidic residues of pHLIP is an essentialpart of understanding the molecular mechanism ofthe membrane insertion process fo r any peptidecontaining carboxyl groups. Two classes of carboxylgroups are of interest: those that rem ain buried inthe membrane after pHLIP is inserted into themembrane and those that traverse the hydrophobiccore of the membrane duri ng insertion.6Accord-ingly, we have studied both the pH-driven mem-brane insertion and the exit process for a series ofpeptides where the key aspartic acid residues aresequentially mutated.ResultsPrevious studies in our laboratories revealed thatsequence variations in the TM region of pHLIP candisrupt the delicate balance that preserves its watersolubility. For example, a simultaneous change in thetwo aspart ic acid residues at positions 14 and 25 tothe homologous glutamic acid (Asp14/25Glu)resulted in a loss of p H-dependent membraneinsertion due to aggregation of the peptide inaqueous solution7(we have