Francisco N. Barrera

University of Tennessee | UTK · Department of Biochemistry and Cellular and Molecular Biology

The T cell receptor (TCR) is a complex molecular machine that directs the activation of T cells, allowing the immune system to fight pathogens and cancer cells. Despite decades of investigation, the molecular mechanism of TCR activation is still controversial. One of the leading activation hypotheses is the allosteric model. This model posits that...

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase (RTK) commonly targeted for inhibition by anti-cancer therapeutics. Current therapeutics target EGFR's kinase domain or extracellular region. However, these types of inhibitors are not specific for tumors over healthy tissue and therefore cause undesirable side effects. Our l...

Phosphatidylserine (PS) is a key lipid that plays important roles in disease-related biological processes, and therefore, the means to track PS in live cells are invaluable. Herein, we describe the metabolic labeling of PS in Saccharomyces cerevisiae cells using analogues of serine, a PS precursor, derivatized with azide moieties at either the amin...

Candida albicans is a deadly pathogen responsible for millions of mucosal and systemic infections per year. The pathobiology of C. albicans is largely dependent on the damaging and immunostimulatory properties of the peptide candidalysin (CL), a key virulence factor. When CL forms pores in the plasma membrane of epithelial cells, it activates a res...

Candida albicans causes severe invasive candidiasis. C. albicans infection requires the virulence factor candidalysin (CL) which damages target cell membranes. However, the mechanism that CL uses to permeabilize membranes is unclear. We reveal that CL forms membrane pores using a unique mechanism. Unexpectedly, CL readily assembled into polymers in...

In biology, heterosynaptic plasticity maintains homeostasis in synaptic inputs during associative learning and memory, and initiates long-term changes in synaptic strengths that nonspecifically modulate different synapse types. In bioinspired neuromorphic circuits, heterosynaptic plasticity may be used to extend the functionality of two-terminal, b...

The T cell receptor (TCR) is a complex molecular machine that directs the activation of T cells, allowing the immune system to fight pathogens and cancer cells. Despite decades of investigation, the molecular mechanism of TCR activation is still controversial. One of the leading activation hypotheses is the allosteric model. This model posits that...

In 1987, Susi & Byler published a groundbreaking paper for the determination of the secondary structure of proteins. Notably, they determined the characteristic signature of the β-strand in the infrared spectrum. As a result, Fourier-transform infrared spectroscopy became a general method to determine protein structure.

The study of membrane proteins is undergoing a golden era, and we are gaining unprecedented knowledge on how this key group of proteins works. However, we still have only a basic understanding of how the chemical composition and the physical properties of lipid bilayers control the activity of membrane proteins. Single-molecule (SM) fluorescence me...

The EphA2 receptor is a promising drug target for cancer treatment, since EphA2 activation can inhibit metastasis and tumor progression. It has been recently described that the TYPE7 peptide activates EphA2 using a novel mechanism that involves binding to the single transmembrane domain of the receptor. TYPE7 is a conditional transmembrane (TM) lig...

The EphA2 receptor is a promising drug target for cancer treatment, since EphA2 activation can inhibit metastasis and tumor progression. It has been recently described that the TYPE7 peptide activates EphA2 using a novel mechanism that involves binding to the single transmembrane domain of the receptor. TYPE7 is a conditional transmembrane (TM) lig...

Significance Highly specific protein–protein interactions between transmembrane domains play crucial roles in many biological processes, but are difficult to study because they occur within membranes. The E5 protein of bovine papillomavirus is a 44-residue transmembrane protein that transforms cells by binding the transmembrane domain of the PDGF r...

Several diseases, such as cancer, are characterized by acidification of the extracellular environment. Acidosis can be employed as a target to specifically direct therapies to the diseased tissue. We have used first principles to design an acidity-triggered rational membrane (ATRAM) peptide with high solubility in solution that is able to interact...

For drug delivery purposes, the ability to conveniently attach a targeting moiety that will deliver drugs to cells and then enable controlled release of the active molecule after localization is desirable. Towards this end, we designed and synthesized clickable and photocleavable lipid analog 1 to maximize the efficiency of bioconjugation and trigg...

The pHLIP peptide targets acidic diseases such as cancer. The acidity of the environment causes key aspartic acids in pHLIP to protonate, causing the peptide to insert into membranes. Here we investigate how the negative charge of the membrane influences how pHLIP enters and exits the lipid bilayer. We found that electrostatic repulsion affected di...

MicroRNAs (miRNAs) are short non-coding RNAs that, when expressed in different tissue and cell types, suppress the expression of complementary genes. Certain miRNAs, called oncomiRs, play a causal role in the onset and maintenance of cancer when overexpressed. Tumors that depend on these miRNAs are said to display oncomiR addiction. Inhibition of o...

SUMMARY PARAGRAPH MicroRNAs (miRNAs) are short non-coding RNAs expressed in different tissue and cell types that suppress the expression of target genes. As such, miRNAs are critical cogs in numerous biological processes1,2, and dysregulated miRNA expression is correlated with many human diseases. Certain miRNAs, called oncomiRs, play a causal role...

Transmembrane proteins constitute a large fraction of cellular proteins, and specific interactions involving membrane-spanning protein segments play an important role in protein oligomerization, folding, and function. We previously isolated an artificial, dimeric, 44-amino acid transmembrane protein that activates the human erythropoietin receptor...

Hepatitis C virus NS4A is a single pass transmembrane (TM) protein essential for viral replication and particle assembly. The sequence of the NS4A TM domain is highly conserved, suggesting that it may be important for protein-protein interactions. To test this hypothesis we measured the potential dimerization of the NS4A TM domain in a well-charact...

We have used the pHLIP® (pH Low Insertion Peptide) peptide family to study the role of aspartate embedding depth in pH-dependent transmembrane peptide insertion. pHLIP binds to the surface of a lipid bilayer as a largely unstructured monomer at neutral pH. When pH is lowered, pHLIP inserts spontaneously across the membrane as a spanning α-helix. pH...

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 uni...

pH (low) insertion peptide (pHLIP) is a helical peptide capable of inserting itself across a cell membrane at low pHs, but not at normal physiologic pH. It possesses two carboxyl groups on aspartic acid residues in its transmembrane region which are protonated at low pH, thereby increasing the peptide's hydrophobicity and facilitating transmembrane...

The pHLIP peptide has three states: (I) soluble in aqueous buffer, (II) bound to the bilayer surface at neutral pH, and (III) inserted as a transmembrane (TM) helix at acidic pH. The membrane insertion of pHLIP at low pH can be used to target the acidic tissues characteristic of different diseases, such as cancer. We find that the α-helix content o...

The pHLIP peptide is a new therapeutic and imaging agent for cancer treatment. The pHLIP (pH-Low-Insertion Peptide) is soluble in solution but interestingly, it is able to interact with the plasma membrane and insert as a monomeric transmembrane helix (pKa=6.0). The insertion requires an acidic extracellular environment, such as that found in most...

We have used pHLIP® [pH (low) insertion peptide] to study the roles of carboxyl groups in transmembrane (TM) peptide insertion. pHLIP binds to the surface of a lipid bilayer as a disordered peptide at neutral pH; when the pH is lowered, it inserts across the membrane to form a TM helix. Peptide insertion is reversed when the pH is raised above the...

KcsA, a homotetrameric potassium channel from prokaryotes, contains noncovalently bound lipids appearing in the X-ray crystallographic structure of the protein. The binding sites for such high-affinity lipids are referred to as "nonannular" sites, correspond to intersubunit protein domains, and bind preferentially anionic phospholipids. Here we use...

This work describes a genetic approach to isolate small, artificial transmembrane (TM) proteins with biological activity. The bovine papillomavirus E5 protein is a dimeric, 44-amino acid TM protein that transforms cells by specifically binding and activating the platelet-derived growth factor beta receptor (PDGFbetaR). We used the E5 protein as a s...

The pH (low) insertion peptide (pHLIP) is a 36-aa monomeric peptide which is both soluble in water and able to insert as a transmembrane helix in lipid membranes at low pH. Thus, pHLIP has three major states with characteristic secondary structure: it is unstructured in solution (state I) and when bound to the surface of lipid membranes at neutral...

The bovine papillomavirus E5 protein (BPV E5) is a 44-amino-acid homodimeric transmembrane protein that binds directly to the transmembrane domain of the platelet-derived growth factor (PDGF) β receptor and induces ligand-independent receptor activation. Three specific features of BPV E5 are considered important for its ability to activate the PDGF...

RYBP (Ring1A and YY1 binding protein) is a zinc finger protein with an essential role during embryonic development, which binds transcriptional factors, Polycomb products, and mediators of apoptosis, suggesting roles in, apparently, unrelated functions. To investigate mechanisms underlying its association with functionally diverse partners, we set...

The first comprehensive studies on the structure and thermodynamics of membrane proteins have started revealing the exact architecture of these macromolecules and the physical-chemical rules behind their structures. In this review, the stabilities of several families of membrane proteins, obtained by using spectroscopic, calorimetric and single mol...

The effects of the inactivating peptide from the eukaryotic Shaker BK+ channel (the ShB peptide) on the prokaryotic KcsA channel have been studied using patch clamp methods. The data show that the peptide induces rapid, N-type inactivation in KcsA through a process that includes functional uncoupling of channel gating. We have also employed saturat...

The effects of the inactivating peptide from the eukaryotic Shaker BK(+) channel (the ShB peptide) on the prokaryotic KcsA channel have been studied using patch clamp methods. The data show that the peptide induces rapid, N-type inactivation in KcsA through a process that includes functional uncoupling of channel gating. We have also employed satur...

Moderate concentrations of the alcohol 2,2,2-trifluoroethanol (TFE) cause the coupled unfolding and dissociation into subunits of the homotetrameric potassium channel KcsA, in a process that is partially irreversible when the protein is solubilized in plain dodecyl beta-d-maltoside (DDM) micelles [Barrera et al. (2005) Biochemistry 44, 14344-52]. H...

During maturation of type 1 human immunodeficiency virus, a fraction of the capsid protein (CA) molecules in the budding virus particle form a conical capsid. However, the location and role of the remaining CA molecules are unknown. It has been recently reported that the C-terminal domain of CA is able to interact with lipid bilayers, suggesting th...

The Ring1B is a core subunit protein of the PRC1 (polycomb repressive complex 1), which plays key roles in the regulation of the Homeobox gene expression, X-chromosome inactivation, stem cell self-renewal, and tumorigenesis. The C-terminal region of Ring1B interacts with RYBP, a transcriptional repressor in transiently transfected cells, and also w...

The protein CA forms the mature capsid of human immunodeficiency virus. Hexamerization of the N-terminal domain and dimerization of the C-terminal domain, CAC, occur during capsid assembly, and both domains constitute potential targets for anti-HIV inhibitors. CAC homodimerization occurs mainly through its second helix, and is abolished when its so...

This article reports on the interaction of conducting (K+) and blocking (Na+) monovalent metal ions with detergent-solubilized and lipid-reconstituted forms of the K+ channel KcsA. Monitoring of the protein intrinsic fluorescence reveals that the two ions bind competitively to KcsA with distinct affinities (dissociation constants for the KcsA·K+ an...

This article reports on the interaction of conducting (K(+)) and blocking (Na(+)) monovalent metal ions with detergent-solubilized and lipid-reconstituted forms of the K(+) channel KcsA. Monitoring of the protein intrinsic fluorescence reveals that the two ions bind competitively to KcsA with distinct affinities (dissociation constants for the KcsA...

Different patterns of channel activity have been detected by patch clamping excised membrane patches from reconstituted giant liposomes containing purified KcsA, a potassium channel from prokaryotes. The more frequent pattern has a characteristic low channel opening probability and exhibits many other features reported for KcsA reconstituted into p...

Different patterns of channel activity have been detected by patch clamping excised membrane patches from reconstituted giant liposomes containing purified KcsA, a potassium channel from prokaryotes. The more frequent pattern has a characteristic low channel opening probability and exhibits many other features reported for KcsA reconstituted into p...

The capsid protein, CA, of HIV-1 forms a capsid that surrounds the viral genome. However, recent studies have shown that an important proportion of the CA molecule does not form part of this capsid, and its location and function are still unknown. In the present work we show, by using fluorescence, differential scanning calorimetry and Fourier-tran...

Fur (ferric uptake regulator) is a key bacterial protein that regulates iron acquisition and its storage, and modulates the expression of genes involved in the response to different environmental stresses. Although the protein is involved in several regulation mechanisms, and members of the Fur family have been identified in pathogen organisms, the...

2,2,2-Trifluoroethanol (TFE) effectively destabilizes the otherwise highly stable tetrameric structure of the potassium channel KcsA, a predominantly alpha-helical membrane protein [Valiyaveetil, F. I., Zhou, Y., and MacKinnon, R. (2002) Biochemistry 41, 10771-10777]. Here, we report that the effects on the protein structure of increasing concentra...

2,2,2-Trifluoroethanol (TFE) effectively destabilizes the otherwise highly stable tetrameric structure of the potassium channel KcsA, a predominantly alpha-helical membrane protein [Valiyaveetil, F. I., Zhou, Y., and MacKinnon, R. (2002) Biochemistry 41, 10771-10777]. Here, we report that the effects on the protein structure of increasing concentra...

The lack of a membrane environment in membrane protein crystals is considered one of the major limiting factors to fully imply X-ray structural data to explain functional properties of ion channels [Gulbis, J.M. and Doyle, D. (2004) Curr. Opin. Struct. Biol. 14, 440-446]. Here, we provide infrared spectroscopic evidence that the structure and stabi...

The type 1 human immunodeficiency virus presents a conical capsid formed by several hundred units of the capsid protein, CA. Homodimerization of CA occurs via its C-terminal domain, CA-C. This self-association process, which is thought to be pH-dependent, seems to constitute a key step in virus assembly. CA-C isolated in solution is able to dimeriz...

The bacterial phosphoenolpyruvate (PEP): sugar phosphotransferase system (PTS), formed by a cascade of several proteins, couples the translocation and phosphorylation of specific sugars across cell membranes. The structure and thermal stability of the first protein (enzyme I, EI) of the PTS in Streptomyces coelicolor is studied by using far-UV circ...

KcsA is a prokaryotic potassium channel formed by the assembly of four identical subunits around a central aqueous pore. Although the high-resolution X-ray structure of the transmembrane portion of KcsA is known [Doyle, D. A., Morais, C. J., Pfuetzner, R. A., Kuo, A., Gulbis, J. M., Cohen, S. L., Chait, B. T., and MacKinnon, R. (1998) Science 280,...

The type 1 HIV presents a conical capsid formed by approximately 1500 units of the capsid protein, CA. Homodimerization of CA via its C-terminal domain, CA-C, constitutes a key step in virion assembly. CA-C dimerization is largely mediated by reciprocal interactions between residues of its second alpha-helix. Here, we show that an N-terminal-acetyl...

The alpha splice variant of p73 (p73alpha), a homologue of the tumor suppressor p53, has close to its C terminus a sterile alpha motif (SAM), SAMp73, that is thought to be involved in protein-protein interactions. Here, we report the lipid binding properties of this domain. Binding was assayed against zwitterionic (phosphatidylcholine) and anionic...

Glutamine synthetase (GS) is the key enzyme responsible for the primary assimilation of ammonium in all living organisms, and it catalyses the synthesis of glutamine from glutamic acid, ATP, and ammonium. One of the recently discovered mechanisms of GS regulation involves protein-protein interactions with a small 65-residue-long protein named IF7....

The sterile alpha motif (SAM) domain is a protein module of approximately 65 to 70 amino acids found in many diverse proteins whose functions range from signal transduction to transcriptional repression. The alpha splice variant of p73 (p73 alpha), a homologue of the tumor suppressor p53, has close to its C-terminus a SAM motif. Here, we report the...