Large Hydrophobic Moment Research Articles

Abstract 564: Structure-Function Studies of ApoA-I Mimetic Peptides for ABCA1-Dependent Cholesterol Efflux and HDL Formation

To examine the mechanism of action of ApoA-I mimetics, we designed 4 peptides with a variable number of E, L, K and A residues and determined their ability to form HDL-like particle and promote ABCA1-dependent cholesterol efflux. They were named based on a unique physical property: N=ELK-neutral (EKLKELLEKLLEKLKELL), H=ELK-hydrophobic (EKLLELLKKLLELLKELL), P=ELK-positive (EKLKALLEKLKAKLKELL), Neg=ELK-negative (EELKEKLEELKEKLEEKL). CD-spectroscopy showed that H and P were mostly helical in an aqueous buffer (52% and 22%, respectively), and in a TFE-containing solvent, mimicking a lipid environment, all peptides showed greater than 40% helicity. In DMPC vesicle solubilization assay, we observed following order: P>N>H>>Neg. By non-denaturing gel electrophoresis, N, H, P formed approximately 8, 8, and 18 nm size lipid particles, respectively when combined with DMPC. However, Neg did not form any detectable particle. A mixture of natural lipids (PC:SM:LysoPC:PE:PS:PI; mol% 55:12:5:10:8:10), intended to simulate ABCA1 lipid membrane microdomain, yielded similar results . Using BHK-ABCA1 transfected cells, cholesterol efflux studies showed following results: H>N>>P, whereas Neg peptide was inactive. All-atom MD simulation of N carried out on a 12–nm disc containing POPC:cholest (200:20), 26 peptides, and initially placed in belt-like configuration as head-to-tail dimers, the starting structure was maintained for at least 3 μ-sec, indicated a strong preference for this configuration. However, when N was initially oriented in a picket-fence configuration, it distorted to a belt-like pattern within 1 μ-sec. For Neg, both starting configurations were much less stable with dimers losing their connectivity and monomers migrating to the top and bottom of the disc, leaving large hydrophobic patches of acyl chains exposed. Cross-linking studies were consistent with the ability of H and N to form dimers to stabilize the HDL structure, and thereby, were also consistent with simulation. Together, we show that a net neutral charge, a relatively large hydrophobic moment (0.78), and a broad hydrophobic face (180 degrees) are optimum features for an apoA-I mimetic peptide to promote cholesterol efflux and to stabilize a nascent discoidal HDL structure.

Characterizing alpha helical properties of Ebola viral proteins as potential targets for inhibition of alpha-helix mediated protein-protein interactions.

Ebola, considered till recently as a rare and endemic disease, has dramatically transformed into a potentially global humanitarian crisis. The genome of Ebola, a member of the Filoviridae family, encodes seven proteins. Based on the recently implemented software (PAGAL) for analyzing the hydrophobicity and amphipathicity properties of alpha helices (AH) in proteins, we characterize the helices in the Ebola proteome. We demonstrate that AHs with characteristically unique features are involved in critical interactions with the host proteins. For example, the Ebola virus membrane fusion subunit, GP2, from the envelope glycoprotein ectodomain has an AH with a large hydrophobic moment. The neutralizing antibody (KZ52) derived from a human survivor of the 1995 Kikwit outbreak recognizes a protein epitope on this AH, emphasizing the critical nature of this secondary structure in the virulence of the Ebola virus. Our method ensures a comprehensive list of such `hotspots'. These helices probably are or can be the target of molecules designed to inhibit AH mediated protein-protein interactions. Further, by comparing the AHs in proteins of the related Marburg viruses, we are able to elicit subtle changes in the proteins that might render them ineffective to previously successful drugs. Such differences are difficult to identify by a simple sequence or structural alignment. Thus, analyzing AHs in the small Ebola proteome can aid rational design aimed at countering the `largest Ebola epidemic, affecting multiple countries in West Africa' ( http://www.cdc.gov/vhf/ebola/outbreaks/2014-west-africa/index.html).

Characterizing alpha helical properties of Ebola viral proteins as potential targets for inhibition of alpha-helix mediated protein-protein interactions

Ebola, considered till recently as a rare and endemic disease, has dramatically transformed into a potentially global humanitarian crisis. The genome of Ebola, a member of the Filoviridae family, encodes seven proteins. Based on the recently implemented software (PAGAL) for analyzing the hydrophobicity and amphipathicity properties of alpha helices (AH) in proteins, we characterize the helices in the Ebola proteome. We demonstrate that AHs with characteristically unique features are involved in critical interactions with the host proteins. For example, the Ebola virus membrane fusion subunit, GP2, from the envelope glycoprotein ectodomain has an AH with a large hydrophobic moment. The neutralizing antibody (KZ52) derived from a human survivor of the 1995 Kikwit outbreak recognizes a protein epitope on this AH, emphasizing the critical nature of this secondary structure in the virulence of the Ebola virus. Our method ensures a comprehensive list of such `hotspots'. These helices probably are or can be the target of molecules designed to inhibit AH mediated protein-protein interactions. Further, by comparing the AHs in proteins of the related Marburg viruses, we are able to elicit subtle changes in the proteins that might render them ineffective to previously successful drugs. Such differences are difficult to identify by a simple sequence or structural alignment. Thus, analyzing AHs in the small Ebola proteome can aid rational design aimed at countering the `largest Ebola epidemic, affecting multiple countries in West Africa' (http://www.cdc.gov/vhf/ebola/outbreaks/2014-west-africa/index.html).

Characterizing alpha helical properties of Ebola viral proteins as potential targets for inhibition of alpha-helix mediated protein-protein interactions

Ebola, considered till recently as a rare and endemic disease, has dramatically transformed into a potentially global humanitarian crisis. The genome of Ebola, a member of the Filoviridae family, encodes seven proteins. Based on the recently implemented software (PAGAL) for analyzing the hydrophobicity and amphipathicity properties of alpha helices (AH) in proteins, we characterize the helices in the Ebola proteome. We demonstrate that AHs with characteristically unique features are involved in critical interactions with the host proteins. For example, the Ebola virus membrane fusion subunit, GP2, from the envelope glycoprotein ectodomain has an AH with a large hydrophobic moment. The neutralizing antibody (KZ52) derived from a human survivor of the 1995 Kikwit outbreak recognizes a protein epitope on this AH, emphasizing the critical nature of this secondary structure in the virulence of the Ebola virus. Our method ensures a comprehensive list of such `hotspots'. These helices probably are or can be the target of molecules designed to inhibit AH mediated protein-protein interactions. Further, by comparing the AHs in proteins of the related Marburg viruses, we are able to elicit subtle changes in the proteins that might render them ineffective to previously successful drugs. Such differences are difficult to identify by a simple sequence or structural alignment. Thus, analyzing AHs in the small Ebola proteome can aid rational design aimed at countering the `largest Ebola epidemic, affecting multiple countries in West Africa' ( http://www.cdc.gov/vhf/ebola/outbreaks/2014-west-africa/index.html).

Characterizing alpha helical properties of Ebola viral proteins as potential targets for inhibition of alpha-helix mediated protein-protein interactions

Ebola, considered till recently as a rare and endemic disease, has dramatically transformed into a potentially global humanitarian crisis. The genome of Ebola, a member of the Filoviridae family, encodes seven proteins. Based on the recently implemented software (PAGAL) for analyzing the hydrophobicity and amphipathicity properties of alpha helices (AH) in proteins, we characterize the helices in the Ebola proteome. We demonstrate that AHs with characteristically unique features are involved in critical interactions with the host proteins. For example, the Ebola virus membrane fusion subunit, GP2, from the envelope glycoprotein ectodomain has an AH with a large hydrophobic moment. The ability of this AH to bind to other host proteins is disrupted by a neutralizing antibody derived from a human survivor of the 1995 Kikwit outbreak, emphasizing the critical nature of this secondary structure in the virulence of the Ebola virus. Our method ensures a comprehensive list of such `hotspots'. These helices probably are or can be the target of molecules designed to inhibit AH mediated protein-protein interactions. Further, by comparing the AHs in proteins of the related Marburg viruses, we are able to elicit subtle changes in the proteins that might render them ineffective to previously successful drugs. Such differences are difficult to identify by a simple sequence or structural alignment. Thus, analyzing AHs in the small Ebola proteome can aid rational design aimed at countering the `largest Ebola epidemic, affecting multiple countries in West Africa' (http://www.cdc.gov/vhf/ebola/outbreaks/2014-west-africa/index.html).

Biophysical characterization and membrane interaction of the most membranotropic region of the HIV-1 gp41 endodomain

The membrane fusion protein of HIV-1 is the envelope transmembrane gp41 glycoprotein, which is the responsible of the membrane fusion between the virus and the target cell. Gp41 has an unusual cytoplasmic tail, the endodomain, containing highly helicoidal segments with large hydrophobic moments, the so called lentivirus lytic peptides or LLPs. According to our previous work, one of the most membranotropic regions along the whole gp41 glycoprotein was located in the LLP3 region of the gp41. In order to get new insights into the viral membrane fusion mechanism, a peptide pertaining to the LLP3 domain has been studied by infrared, fluorescence and calorimetry regarding its structure, its ability to induce membrane rupture and aggregation, as well as its affinity towards specific phospholipids. Our results demonstrate that this peptide interacts with phospholipid-containing model membranes, affects the phase-behavior of membrane phospholipids and induces leakage and aggregation of liposomes. The membrane-perturbing properties of LLP3, together with the possibility that the Kennedy sequence could be part of an external loop, open the possibility that these domains might function in modulating viral membrane fusion or budding, synergistically with other membranotropic regions of the gp41 glycoprotein.

Charge distributions and amphipathicity of receptor-binding alpha-helices

Contacts between ligands and cell-surface receptors result in cellular activation. Denning principles which govern these important interactions are of interest and might facilitate pharmacologic intervention. We examined receptor-binding α-helical segments of polypeptide hormones and globular proteins for distinguishing amino acid content and distributions. There was a slight excess of basic residues in both sets of α-helices compared with a panel of control helices. Helical concentrations of charged residues were quantitated using the hydrophobic moment algorithm, adapted to obtain the vector sum of side chain charges. By this analysis we detected increased concentrations of the set of basic residues (arginine, lysine and histidine) on one side of the receptor-binding α-helices of the polypeptide hormones, and to a lesser extent the protein ligands. Comparable data were obtained for “lytic” venom peptides and calmodulin-regulated kinase segments. There was an even greater correlation between receptor-associating α-helical segments and large hydrophobic moments. Receptor-binding helical segments of polypeptide hormones, and to a lesser extent those of protein ligands, often are basic and amphipathic.

The most highly amphiphilic α‐helices include two amino acid segments in human immunodeficiency virus glycoprotein 41

A search for highly amphiphilic alpha-helices has been made in a data base of protein sequences, using the helical hydrophobic moment as a criterion of amphiphilicity. The protein segments of largest hydrophobic moment have been analyzed. For the segments whose structures are known, they are in fact alpha-helices. Two of the segments having very large hydrophobic moments are from the smaller C-terminal portion of the human immunodeficiency virus (HIV) envelope glycoprotein gp41. Also, among segments having large hydrophobic moments, but not among the most extreme, are lytic peptides such as melittin. Melittin seeks surfaces between polar and apolar phases, including the membrane-water interface. It is conceivable that the gp41 segments of extreme hydrophobic moment may participate in one of the membrane-related functions of the HIV virus.

Parameter relation rows: a query system for protein structure function relationships

In the course of molecular modeling or mutant prediction one often wants quick answers to questions such as: 'Are there any residues in a beta-strand that point into an internal cavity, and are highly mutable?' 'Are there large polar residues in a helix that make a contact with a hydrophobic residue in a sheet, and don't make the maximal number of hydrogen bonds?' or 'Which hydrophobic residues are in a helix with a large hydrophobic moment, and make a contact with a co-factor, but at the same time still have a large accessible surface?'. I describe here a method to get answers to these kinds of questions in a very quick and easy manner. The method described is partly based on the principles used in the design of relational databases, and its mode of operation is similar to the query methods used in a relational database environment. Although designed for aiding in molecular modeling, its applicability is much more general. The method has been implemented as part of a large molecular modeling package which copes with the numerous problems in systematic handling of protein structures, e.g. residue numbering. This also implies that many normal tools such as graphical analyses, I/O facilities, etc. are available on-line.

Secondary structures of rat lipolytic enzymes: Circular dichroism studies and relation to hydrophobic moments

To explore the secondary structures of lingual and pancreatic lipases, circular dichroism measurements were performed. Maximum average ellipticities were used to calculate the percentage of α-helices, β-sheets, and random coils. Lingual lipase had an ellipticity of −20235±140 deg cm 2/dmol(mean±SE) at 220nm suggesting 60% α-helix, 20% β-sheet and 20% random coil structure, but the mean ellipticity for pancreatic lipase was −14093±82 deg cm 2/dmol (mean ±SE) at 210nm suggesting a 34.8% α-helical, 25% β-sheet and 40% random coil secondary structure. An α-helical stretch of residues with a large hydrophobic moment (“globular” α-helix by hydrophobic moment plot) from amino acids 382 through 389 at the COOH-terminal end of lingual lipase was noted. This sequence, absent in pancreatic lipase, may account for the avid binding of lingual lipase to fat emulsion particles.

Statistical and functional analyses of viral and cellular proteins with N-terminal amphipathic alpha-helices with large hydrophobic moments: importance to macromolecular recognition and organelle targeting.

A total of 1,911 proteins with N-terminal methionyl residues were computer screened for potential N-terminal alpha-helices with strong amphipathic character. By the criteria of D. Eisenberg (Annu. Rev. Biochem. 53:595-623, 1984), only 3.5% of nonplastid, nonviral proteins exhibited potential N-terminal alpha-helices, 18 residues in length, with hydrophobic moment values per amino acyl residue ([muH]) in excess of 0.4. By contrast, 10% of viral proteins exhibited corresponding [muH] values in excess of 0.4. Of these viral proteins with known functions, 55% were found to interact functionally with nucleic acids, 30% were membrane-interacting proteins or their precursors, and 15% were structural proteins, primarily concerned with host cell interactions. These observations suggest that N-terminal amphipathic alpha-helices of viral proteins may (i) function in nucleic acid binding, (ii) facilitate membrane insertion, and (iii) promote host cell interactions. Analyses of potential amphipathic N-terminal alpha-helices of cellular proteins are also reported, and their significance to organellar or envelope targeting is discussed.