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Terminal deoxynucleotidyl transferase (TdT) catalyzes template free incorporation of arbitrary nucleotides onto single-stranded DNA. Due to this unique feature, TdT is widely used in biotechnology and clinical applications. One particularly tantalizing use is the synthesis of long

The physical properties of lipid bilayers, such as curvature and fluidity, can affect the interactions of polypeptides with membranes, influencing biological events. Additionally, given the growing interest in peptide-based therapeutics, understanding the influence of membrane properties on membrane-associated peptides has potential utility. pH low insertion peptides (pHLIPs) are a family of water-soluble peptides that can insert across cell membranes in a pH-dependent manner, enabling the use of pH to follow peptide-lipid interactions. Here we study pHLIP interactions with liposomes varying in size and composition, to determine the influence of several key membrane physical properties. We find that pHLIP binding to bilayer surfaces at neutral pH is governed by the ease of access to the membrane’s hydrophobic core, which can be facilitated by membrane curvature, thickness, and the cholesterol content of the membrane. After surface binding, if the pH is lowered, the kinetics of pHLIP folding to form a helix and subsequent insertion across the membrane depends on the fluidity and energetic dynamics of the membrane. We showed that pHLIP is capable of forming a helix across lipid bilayers of different thicknesses at low pH. However, the kinetics of the slow phase of insertion corresponding to the translocation of C-terminal end of the peptide across lipid bilayer, vary approximately twofold, and correlate with bilayer thickness and fluidity. Although these influences are not large, local curvature variations in membranes of different fluidity could selectively influence surface binding in mixed cell populations.

pH (Low) Insertion Peptides (pHLIP peptides) find application in studies of membrane-associated folding because spontaneous insertion of these peptides is conveniently triggered by varying pH. Here, we employed small-angle X-ray scattering (SAXS) to investigate a wild-type (WT) pHLIP peptide oligomeric state in solution at high concentrations and monitor changes in the liposome structure upon peptide insertion into the bilayer. We established that even at high concentrations (up to 300 µM) the WT pHLIP peptide at pH 8.0 does not form oligomers larger than tetramers (which exhibit concentration-dependent transfer to the monomeric state, as was shown previously). This finding has significance for medical applications when high concentration of the peptide is injected into blood and diluted in blood circulation. The interaction of WT pHLIP peptide with liposomes does not alter the unilamellar vesicle structure upon peptide adsorption by the lipid bilayer at high pH or upon insertion across the bilayer at low pH. At the same time, SAXS data clearly demonstrate the insertion of the peptide into the membrane at low pH, which opens the possibility of investigating the kinetic process of polypeptide insertion and exit from the membrane in real time by time-resolved SAXS.

The dimeric 44-residue E5 protein of bovine papillomavirus is the smallest known naturally occurring oncoprotein. This transmembrane protein binds to the transmembrane domain (TMD) of the platelet-derived growth factor β receptor (PDGFβR), causing dimerization and activation of the receptor. Here, we use Rosetta membrane modeling and all-atom molecular dynamics simulations in a membrane environment to develop a chemically detailed model of the E5 protein/PDGFβR complex. In this model, an active dimer of the PDGFβR TMD is sandwiched between two dimers of the E5 protein. Biochemical experiments showed that the major PDGFβR TMD complex in mouse cells contains two E5 dimers and that binding the PDGFβR TMD to the E5 protein is necessary and sufficient to recruit both E5 dimers into the complex. These results demonstrate how E5 binding induces receptor dimerization and define a molecular mechanism of receptor activation based on specific interactions between TMDs.

We have developed an improved tool for imaging acidic tumors by reporting the insertion of a transmembrane helix: the pHLIP-Fluorescence Insertion REporter (pHLIP-FIRE). In acidic tissues, such as tumors, peptides in the pHLIP family insert as α-helices across cell membranes. The cell-inserting end of the pHLIP-FIRE peptide has a fluorophore–fluorophore or fluorophore–quencher pair. A pair member is released by disulfide cleavage after insertion into the reducing environment inside a cell, resulting in dequenching of the probe. Thus, the fluorescence of the pHLIP-FIRE probe is enhanced upon cell-insertion in the targeted tissues but is suppressed elsewhere due to quenching. Targeting studies in mice bearing breast tumors show strong signaling by pHLIP-FIRE, with a contrast index of ∼17, demonstrating (i) direct imaging of pHLIP insertion and (ii) cargo translocation in vivo. Imaging and targeted cargo delivery should each have clinical applications.

We have constructed 26-amino acid transmembrane proteins that specifically transform cells but consist of only two different amino acids. Most proteins are long polymers of amino acids with 20 or more chemically distinct side-chains. The artificial transmembrane proteins reported here are the simplest known proteins with specific biological activity, consisting solely of an initiating methionine followed by specific sequences of leucines and isoleucines, two hydrophobic amino acids that differ only by the position of a methyl group. We designate these proteins containing leucine (L) and isoleucine (I) as LIL proteins. These proteins functionally interact with the transmembrane domain of the platelet-derived growth factor β-receptor and specifically activate the receptor to transform cells. Complete mutagenesis of these proteins identified individual amino acids required for activity, and a protein consisting solely of leucines, except for a single isoleucine at a particular position, transformed cells. These surprisingly simple proteins define the minimal chemical diversity sufficient to construct proteins with specific biological activity and change our view of what can constitute an active protein in a cellular context.

The pHLIP® -pH (low) insertion peptide- binds to the surface of lipid bilayers as a disordered peptide at neutral pH. However, when the pH is lowered, pHLIP inserts across the membrane to form a transmembrane (TM) helix. Peptide insertion is reversed when the pH is raised above the characteristic pKa, which is 6.0 for POPC liposomes. Due to its unique properties pHLIP serves as a good model system to study the process of folding/insertion and exit/unfolding of a peptide into/from a membrane. We previously found that the process of insertion of pHLIP in POPC phospholipid bilayer occurs in several steps with different intermediates (Andreev et al 2010, PNAS). The number of intermediates correlates with the number of protonatable groups in pHLIP's sequence. For pHLIP, the pH-dependent insertion and folding processes are coupled, and single sigmoidal transition is observed. Interestingly, we have found that for two pHLIP variants (D14A and D25A where each aspartic residue in TM part is replaced by alanine), the pH curve shows two independent sigmoidal transitions, suggesting that the mutations stabilize an intermediate state along the membrane-attached to transmembrane pathway. We implemented circular dichrosism (CD), oriented CD, fluorescence, FRET to elucidate the nature of these intermediates. From our experiments we inferred that the intermediates correspond to a quasi-transmembrane state. We hypothesize that the intermediate emerges from the protonation of acidic residues at the peptide C-terminal end, and complete TM folding happens with the protonation of the aspartic acid residue located in the hydrophobic core of the membrane. These results further our understanding of formation of transmembrane helix during spontaneous process of insertion of a polypeptide in membrane.

The membrane-associated folding/unfolding of pH (low) insertion peptide (pHLIP) provides an opportunity to study how sequence variations influence the kinetics and pathway of peptide insertion into bilayers. Here, we present the results of steady-state and kinetics investigations of several pHLIP variants with different numbers of charged residues, with attached polar cargoes at the peptide's membrane-inserting end, and with three single-Trp variants placed at the beginning, middle, and end of the transmembrane helix. Each pHLIP variant exhibits a pH-dependent interaction with a lipid bilayer. Although the number of protonatable residues at the inserting end does not affect the ultimate formation of helical structure across a membrane, it correlates with the time for peptide insertion, the number of intermediate states on the folding pathway, and the rates of unfolding and exit. The presence of polar cargoes at the peptide's inserting end leads to the appearance of intermediate states on the insertion pathway. Cargo polarity correlates with a decrease of the insertion rate. We conclude that the existence of intermediate states on the folding and unfolding pathways is not mandatory and, in the simple case of a polypeptide with a noncharged and nonpolar inserting end, the folding and unfolding appears as an all-or-none transition. We propose a model for membrane-associated insertion/folding and exit/unfolding and discuss the importance of these observations for the design of new delivery agents for direct translocation of polar therapeutic and diagnostic cargo molecules across cellular membranes.

The early stages of folding a membrane protein have been conceptualized in terms of the formation of independently stable transmembrane helices, followed by their association to form a bundle, and then followed by further insertions, rearrangements, and binding events. A part of this notion is the formation of transmembrane helices, which is catalyzed by the translocon for hydrophobic sequences, but which can also occur spontaneously for moderately polar sequences. We study spontaneous insertion and folding across a lipid bilayer of moderately polar membrane peptide pHLIP - pH Low Insertion Peptide. pHLIP has three major 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 α-helix. The existence of three distinct equilibrium states makes it possible to separate the process of peptide attachment to a lipid bilayer from the process of peptide insertion/folding. The transitions between states could be easily monitored by the changes of tryptophan fluorescence and circular dichroism signals. We performed steady-state and stopped-flow fluorescence and CD measurements to reveal the molecular mechanism of pHLIP insertion and folding within a POPC lipid bilayer and to calculate the activation energy of formation of transmembrane helix. Global mode analysis allowed us to monitor changes of entire tryptophan fluorescence spectrum during the transition from the state II to the state III.

pHLIP-FIRE (Fluorescence Insertion REporter) is a novel diagnostic tool based on pHLIP (pH Low Insertion Peptide), a water soluble membrane peptide that interacts with membranes by forming a transmembrane helix at low pH. The fluorescence of pHLIP-FIRE is activated by separation of mutually quenching or donor-quencher fluorescent groups via the cleavage of a disulfide-linker when the C-terminus moves into the highly reducing environment inside the cell. Targeting studies in mice bearing breast tumors showed strong targeting of pHLIP-FIRE in vivo, giving a contrast index of 20 with respect to background. This contrast is a significant enhancement in comparison with traditional fluorescent pHLIP constructs. Experiments in vitro and in cultured cells show that the time-course of dequenching in pHLIP-FIRE is pH-dependent and quite slow ( ∼ 1 - 2 days). Direct imaging with pHLIP-FIRE gives the first demonstration of pHLIP insertion and cargo translocation in vivo. Our technology shows promise as a tool for cancer diagnosis or image-guided surgery, and the demonstration of cargo delivery into tumors may lead to a new generation of targeted chemotherapeutic delivery.

The discovery that the C helix of Bacteriorhodopsin exhibits spontaneous, pH-dependent insertion to form a helix across lipid bilayers has led to the use of related peptides, pHLIPs (pH (Low) Insertion Peptides), to study peptide insertion across bilayers, to selectively target cargoes to tumors and other acidic tissues in vivo, and to deliver molecules across the plasma membranes of living cells.Because pHLIP is unfolded on the surface of a bilayer and folding is pH-triggered, we are able to begin to observe and understand the molecular events that accompany the insertion and folding of a peptide entering a bilayer. When the pH is dropped, it is found that a helix forms rapidly on the bilayer surface, followed by a slow insertion across it in several kinetically distinct steps. The exit pathway when the pH is suddenly raised is more rapid, and includes partial unfolding of the helix while still in the bilayer. Lipid composition and sequence features affect the insertion pK and pathway intermediates.pHLIPs target acidic tissues in vivo, where the transmembrane insertion stabilizes them in cells, enabling targeting of imaging and therapeutic agents. We have shown targeting of dyes, radioisotopes, nanogold and Peptide Nucleic Acids (PNAs) to tumors in mice, and will report on the state of our efforts to use PNAs to suppress onco-micro RNAs, a possible route to tumor therapy.

We have discovered a way to target acidity in vivo, which is a hallmark of many pathological states. The tumor targeting is based on the pH-dependent transmembrane insertion and folding of the water-soluble membrane peptide, pHLIP - pH (Low) Insertion Peptide. Here we present the result of the sequence variation study of pHLIP, which was carried out with the main goal to improve blood clearance and tumor targeting at low pH. We have investigated more than 10 pHLIP variants with various mutations in transmembrane and C-terminal parts, including peptides with significantly truncated transmembrane part. Currently our library of pHLIP variants, contains peptides inserting into bilayer with pKa ranging from 4.5 to 6.5, which have different affinity to lipid bilayer at neutral and low pHs. Kinetics measurements indicate that all investigated variants containing truncated or no C-terminal flanking sequence demonstrate fast insertion into lipid bilayer. The results of biophysical studies are in excellent agreement with the tumor targeting and blood clearance data obtained in vivo. Our data contribute in understanding of main principles of peptide-lipid interactions.

The pH-(Low) Insertion Peptide (pHLIP) has potential as a tumor-targeting drug carrier. At neutral pH, pHLIP has affinity for the surface of a lipid bilayer, whereas under slightly acidic conditions (e.g. pH ∼ 6) pHLIP inserts into the membrane, forming a transmembrane helix. Since many solid tumors are more acidic than healthy tissues, pHLIP may be used to translocate chemotherapeutic agents selectively into cancer cells. Knowledge of the exact location of pHLIP insertion in cells can guide the rational design of delivery constructs. We envision two scenarios for pHLIP insertion in cells: First, pHLIP may directly insert into the plasmamembrane; alternatively, cells may internalize pHLIP molecules via endocytosis, and subsequently pHLIP may insert into the endosomal membrane. In this study, several fluorescently self-quenched pHLIP constructs were synthesized to visualize to what extend these two scenarios are occurring in cells at pH 7.3 and 6.2. In these self-quenched pHLIP constructs, a rhodamine dye (TAMRA or Alexa Fluor 568) is attached to a C-terminal Lys residue, with the quencher QSY-9 conjugated to an adjacent Cys via a cleavable disulfide linker (or a stable thio-ether bond). Upon insertion, pHLIP would translocate its C-terminus into the cell cytoplasm, where cleavage of the disulfide linkage and release of the quencher QSY9 can take place. In turn, the pHLIP construct would become more fluorescent.

The study of polypeptide insertion into biological membranes can inform our understanding of membrane protein stability and folding, and has potential practical applications. This has been hard to investigate, since peptides hydrophobic enough to form transmembrane helices are likely to be insoluble in aqueous buffers, and soluble peptides are unlikely to insert. pH-Low Insertion Peptides (pHLIPs) provide an opportunity to study insertion since they are soluble monomers that bind to bilayer surfaces at neutral pH, and can be triggered to form monomeric transmembrane helices in acidic conditions.This set of unusual properties allows the study of spontaneous insertion and exit of a transmembrane polypeptide by changing the pH of its microenvironment. Previous studies showed that pHLIP inserts into a POPC liposomes through rapid formation of an interfacial helix (∼ 0.1s), followed by a slow insertion pathway. The time-course of pHLIP insertion can be changed by varying the number of protonatable groups at the inserting end of the peptide, which need to be moved across the bilayer.We have employed various biophysical methods: fluorescence spectroscopy, anisotropy, CD, OCD, and stopped-flow fluorescence, to study the pHLIP insertion into bilayers composed of different monounsaturated lipids (diC(14:1)PC, diC(16:1)PC, diC(18:1)PC, diC(20:1)PC, and diC(22:1)PC). We found that pHLIP can form a TM helix in bilayers of different thickness. The kinetics of pHLIP insertion vary, and correlate with bilayer thickness and fluidity. Further, pHLIP association with bilayer surfaces also depends on the lipid composition. The activation energy of pHLIP insertion increases with the membrane thickness but the process of inserted helix formation does not significantly depend the membrane type. A model for membrane-associated insertion/folding is discussed. This work was supported by grants from National Institute of Health, GM073857, and CA133890.

There are two questions we would like to address: 1) what is the molecular mechanism of a polypeptide insertion into a lipid bilayer and formation of transmembrane helix? 2) Are there any transient changes of a lipid bilayer in process of a polypeptide insertion and folding? As a convenient system we are studying pHLIP (pH (Low) Insertion Peptide) insertion into a membrane and folding, which is modulated by pH. Previously we showed, that insertion of pHLIP occurs in several steps, with rapid (0.1 sec) interfacial helix formation followed by a much slower (100 sec) insertion pathway to form a transmembrane helix. The reverse process of unfolding and peptide exit from the bilayer core, which can be induced by a rapid pH jump from acidic to basic, proceeds much faster than folding/insertion and through different intermediate states. We designed two truncated pHLIP-variants by removing Asp and Glu residues from the C-terminus, which goes across a membrane. Our kinetic studies indicate that truncated versions of pHLIP insert across a membrane much faster than original (WT) sequence and with less number of intermediate states. Thus, the rate of insertion directly depends on probability of protonation of C-terminal Asp and Glu residues, and translocation of the C-terminus across a bilayer. To understand what are the structural intermediates along the folding pathway, we designed three single-tryptophan pHLIP-variants, where a Trp residue is positioned at the beginning, middle and end of the transmembrane part of the peptide. To study changes, which might occur with a lipid bilayer in a process of peptide insertion and folding, we employed SAXS (small-angle x-ray scattering) technique. A new kinetic model of pHLIP insertion/exit will be discussed.

We are studying the molecular events that occur when a peptide inserts across a membrane or exits from it. Using pH jumps to trigger insertion/exit of the pHLIP (pH Low Insertion Peptide) to enable kinetic analysis, we show that insertion occurs in several steps, with rapid (0.1 sec) interfacial helix formation followed by a much slower (100 sec) insertion pathway to form a transmembrane helix. The reverse process of unfolding and peptide exit from the bilayer core, which can be induced by a rapid pH jump from acidic to basic, proceeds much faster than folding/insertion and through different intermediate states. In the exit pathway, the helix-coil transition is initiated while the polypeptide is still inside the membrane. We also designed two pHLIP-variants where Asp and Glu residues were removed from the C-terminus, which inserts across the membrane. The variants preserve the same pH-dependent properties of pHLIP peptide interaction with the membrane, but insertion occurs 10-30 times faster than in the case of the parent pHLIP peptide. A kinetic model of peptide-membrane insertion/folding and exit/unfolding will be discussed. The work was in part supported by grant from the National Institutes of Health, National Cancer Institute RO1 133890 to OAA, DME, YRK.