Arrangement Of Amino Acids Research Articles

Life sentences: Ontology recapitulates philology.

Life sentences: Ontology recapitulates philology.

A Periodic Table for Genetic Codes

This paper presents a new version of a periodic table for genetic codes using a ‘Leibnitz Number’ as a codon number or anticodon number, which is a natural binary code number and hence outwardly similar to the Gray code binary number. In the obtained periodic table or in the reformed table (a cube-shaped periodic table), the proteinaceous amino acids not only have periodicity, but also occupy mirror-symmetrical positions with respect to the xy-plane. Moreover, the cube-shaped periodic table allows a partial explanation of non-standard genetic codes and some predictions about providing potential candidates for non-standard genetic codons to be discovered in the future. By making a new format of a two-dimensional periodic table for anticodons as the primary reference point, all of the anticodon pairing with multiple codons can be intimately related to a mirror-symmetrical arrangement of amino acids with relation to the yz-plane in the two-dimensional periodic table.

Achieving stability and conformational specificity in designed proteins via binary patterning

We have developed a method to determine the optimal binary pattern (arrangement of hydrophobic and polar amino acids) of a target protein fold prior to amino acid sequence selection in protein design studies. A solvent accessible surface is generated for a target fold using its backbone coordinates and “generic” side-chains, which are constructs whose size and shape are similar to an average amino acid. Each position is classified as hydrophobic or polar according to the solvent exposure of its generic side-chain. The method was tested by analyzing a set of proteins in the Protein Data Bank and by experimentally constructing and analyzing a set of engrailed homeodomain variants whose binary patterns were systematically varied. Selection of the optimal binary pattern results in a designed protein that is monomeric, well-folded, and hyperthermophilic. Homeodomain variants with fewer hydrophobic residues are destabilized, while additional hydrophobic residues induce aggregation. Binary patterning, in conjunction with a force field that models folded state energies, appears sufficient to satisfy two basic goals of protein design: stability and conformational specificity.

A novel approach for the identification of unique tumor vasculature binding peptides using an E. coli peptide display library.

Tumor neovascularization is necessary for continued tumor growth and metastasis. During the process of endothelial cell (EC) recruitment and tumor infiltration, specific molecular markers unique for this interaction are expressed on the EC surface. Targeting these molecular markers would, in effect, allow for specific tumor targeting. Tripeptide sequence motifs have previously been reported that will bind to angiogenic tumor ECs. These sequences were identified from in vivo phage peptide display libraries. The purpose of this study was to use a more simplified bacterial peptide display library in an in vitro system to seek out peptide motifs with unique binding to tumor microvasculature. FliTrx is a bacterial peptide display library containing the entire repertoire of possible random dodecapeptides expressed on the flagella tip of E. coli. Two EC populations were used for the screening process, Matrigel invading cells (MAGIC) and tumor-derived endothelial cells (TDEC). MAGIC are obtained from ECs that infiltrate a subcutaneous fibroblast growth factor-containing Matrigel deposit, and TDEC are ECs selectively obtained from tumor vasculature. FliTrx cells were incubated with MAGIC at 4 degrees C to remove any potential clones displaying peptides that will bind to nonspecific EC surface targets. The non-binding cells were then incubated with TDEC, allowing for clones displaying potential binding peptides to bind tumor specific targets on TDECs. The bacterial population was then expanded and this "panning" process was carried out a total of five times. Peptide insert sequences from 100 bacterial colonies were analyzed for potential repetitive peptide motifs. Recurring peptide sequences were detected that were 3-mers (13 sequences) and 4-mers (4 sequences). Of the 3-mers, four repeated 3 times, whereas none of the 4-mers repeated more than twice. All of the repeated sequences were basic in charge, and arginine was the most commonly seen amino acid. A tripeptide basic-basic-nonpolar amino acid arrangement was the most prevalent charge sequence in all repetitive motifs (17 repeat sequences). Two test peptides showed TDEC binding specificity, and both conformed to the basic-basic-nonpolar motif. We report peptide sequences derived from panning an in vitro system designed to detect tumor-EC specific markers. These putative motifs may serve as molecular determinants for a novel therapeutic modality aimed at specifically targeting tumors through tumor angiogenic vessels.

Exocytosis requires asymmetry in the central layer of the SNARE complex.

Assembly of SNAREs (soluble N:-ethylmaleimide- sensitive factor attachment protein receptors) mediates membrane fusions in all eukaryotic cells. The synaptic SNARE complex is represented by a twisted bundle of four alpha-helices. Leucine zipper-like layers extend through the length of the complex except for an asymmetric and ionic middle layer formed by three glutamines (Q) and one arginine (R). We have examined the functional consequences of Q-R exchanges in the conserved middle layer using the exocytotic SNAREs of yeast as a model. Exchanging Q for R in Sso2p drastically reduces cell growth and protein secretion. When a 3Q/1R ratio is restored by a mirror R-->Q substitution in the R-SNARE Snc2p, wild-type functionality is observed. Secretion is near normal when all four helices contain Q, but defects become apparent when additional mutations are present in other layers. Using molecular dynamics free energy perturbation simulations, these findings are rationalized in structural and energetic terms. We conclude that the asymmetric arrangement of the polar amino acids in the central layer is essential for normal function of SNAREs in membrane fusion.

Binding of the GABA(A) receptor-associated protein (GABARAP) to microtubules and microfilaments suggests involvement of the cytoskeleton in GABARAPGABA(A) receptor interaction.

GABA(A) receptor-associated protein (GABARAP) was isolated on the basis of its interaction with the gamma2 subunit of GABA(A) receptors. It has sequence similarity to light chain 3 (LC3) of microtubule-associated proteins 1A and 1B. This suggests that GABARAP may link GABA(A) receptors to the cytoskeleton. GABARAP associates with tubulin in vitro. However, little is known about the mechanism for the interaction, and it is not clear whether the interaction occurs in vivo. Here, we report that GABARAP interacts directly with both tubulin and microtubules in a salt-sensitive manner, indicating the association is mediated by ionic interactions. GABARAP coimmunoprecipitates with tubulin and associates with both microtubules and microfilaments in intact cells. The cellular distribution is altered by treatment with taxol, nocodazole, and cytochalasin D. The tubulin binding domain was located at the N terminus of GABARAP by using synthetic peptides and deletion constructs and is marked by a specific arrangement of basic amino acids. The interaction between GABARAP and actin might be mediated by other proteins. These results demonstrate the GABARAP interacts with the cytoskeleton both in vitro and in cells and suggest a role of GABARAP in the interaction between GABA(A) receptors and the cytoskeleton. Such interactions are presumably needed for receptor trafficking, anchoring, and/or synaptic clustering. The structural arrangement of the basic amino acids present in the tubulin binding domain of GABARAP may aid in recognition of the potential of tubulin binding activity in other known proteins.

An integrated approach to the analysis and modeling of protein sequences and structures. III. A comparative study of sequence conservation in protein structural families using multiple structural alignments

The information required to generate a protein structure is contained in its amino acid sequence, but how three-dimensional information is mapped onto a linear sequence is still incompletely understood. Multiple structure alignments of similar protein structures have been used to investigate conserved sequence features but contradictory results have been obtained, due, in large part, to the absence of subjective criteria to be used in the construction of sequence profiles and in the quantitative comparison of alignment results. Here, we report a new procedure for multiple structure alignment and use it to construct structure-based sequence profiles for similar proteins. The definition of “similar” is based on the structural alignment procedure and on the protein structural distance (PSD) described in paper I of this series, which offers an objective measure for protein structure relationships. Our approach is tested in two well-studied groups of proteins; serine proteases and Ig-like proteins. It is demonstrated that the quality of a sequence profile generated by a multiple structure alignment is quite sensitive to the PSD used as a threshold for the inclusion of proteins in the alignment. Specifically, if the proteins included in the aligned set are too distant in structure from one another, there will be a dilution of information and patterns that are relevant to a subset of the proteins are likely to be lost.In order to understand better how the same three-dimensional information can be encoded in seemingly unrelated sequences, structure-based sequence profiles are constructed for subsets of proteins belonging to nine superfolds. We identify patterns of relatively conserved residues in each subset of proteins. It is demonstrated that the most conserved residues are generally located in the regions where tertiary interactions occur and that are relatively conserved in structure. Nevertheless, the conservation patterns are relatively weak in all cases studied, indicating that structure-determining factors that do not require a particular sequential arrangement of amino acids, such as secondary structure propensities and hydrophobic interactions, are important in encoding protein fold information. In general, we find that similar structures can fold without having a set of highly conserved residue clusters or a well-conserved sequence profile; indeed, in some cases there is no apparent conservation pattern common to structures with the same fold. Thus, when a group of proteins exhibits a common and well-defined sequence pattern, it is more likely that these sequences have a close evolutionary relationship rather than the similarities having arisen from the structural requirements of a given fold.

Endoplasmic Reticulum Retention Determinants in the Transmembrane and Linker Domains of Cytochrome P450 2C1

The cytochrome P450 2C1 N-terminal signal anchor sequence mediates direct retention of the protein in the endoplasmic reticulum and consists of a hydrophobic transmembrane domain, residues 3-20, followed by a hydrophilic linker, residues 21-28. Fusions of the N-terminal 21 or 28 amino acids of P450 2C1 to green fluorescent protein resulted in endoplasmic reticulum localization of the chimera in transfected cells. Disruption of microtubules by nocodazole treatment resulted in redistribution into a punctate pattern for the 1-21, but not for the 1-28, chimera indicating that the linker was preventing transport from the endoplasmic reticulum but was not required for retrieval to the endoplasmic reticulum from the pre-Golgi compartment. In the 1-28 chimera, mutations of residues 21-23 (KQS) in the linker resulted in redistribution of the chimera after nocodazole treatment. Mutations in the transmembrane domain affected both direct retention in the endoplasmic reticulum and retrieval from the pre-Golgi compartment, and although structural requirements for each process are distinct, in both cases the arrangement of amino acids and distribution of hydrophobicity are critical. In contrast, the linker region exhibits a sequence-specific requirement for direct retention in the endoplasmic reticulum.

A cysteine-rich motif in poliovirus protein 2C(ATPase) is involved in RNA replication and binds zinc in vitro.

Protein 2C(ATPase) of picornaviruses is involved in the rearrangement of host cell organelles, viral RNA replication, and encapsidation. However, the biochemical and molecular mechanisms by which 2C(ATPase) engages in these processes are not known. To characterize functional domains of 2C(ATPase), we have focused on a cysteine-rich motif near the carboxy terminus of poliovirus 2C(ATPase). This region, which is well conserved among enteroviruses and rhinoviruses displaying an amino acid arrangement resembling zinc finger motifs, was studied by genetic and biochemical analyses. A mutation that replaced the first cysteine residue of the motif with a serine was lethal. A mutant virus which lacked the second of four potential coordination sites for zinc was temperature sensitive. At the restrictive temperature, RNA replication was inhibited whereas translation and polyprotein processing, assayed in vitro and in vivo, appeared to be normal. An intragenomic second-site revertant which reinserted the missing coordination site for zinc and recovered RNA replication at the restrictive temperature was isolated. The cysteine-rich motif was sufficient to bind zinc in vitro, as assessed in the presence of 4-(2-pyridylazo)resorcinol by a colorimetric assay. Zinc binding, however, was not required for hydrolysis of ATP. 2C(ATPase) as well as its precursors 2BC and P2 were found to exist in a reduced form in poliovirus-infected cells.

Activity of the Human Centrosomal Kinase, Nek2, Depends on an Unusual Leucine Zipper Dimerization Motif

Nek2 is a human cell cycle-regulated kinase that is structurally related to the mitotic regulator, NIMA, of Aspergillus nidulans. Localization studies have shown that Nek2 is a core component of the centrosome, the microtubule organizing center of the cell, and functional approaches suggest a possible role for Nek2 in centrosome separation at the G2/M transition. Here, we have investigated the importance of an unusual leucine zipper coiled-coil motif present in the C-terminal noncatalytic domain of the Nek2 kinase. Glycerol gradient centrifugation indicated that endogenous Nek2 is present in HeLa cells as a salt-resistant 6 S complex, the predicted size of a Nek2 homodimer. Recombinant Nek2 overexpressed in insect cells also formed a 6 S complex, whereas a Nek2 mutant specifically lacking the leucine zipper motif was monomeric. Using yeast two-hybrid interaction analyses and coprecipitation assays, we found that Nek2 can indeed form homodimers both in vivo and in vitro and that this dimerization specifically required the leucine zipper motif. Moreover, deletion of the leucine zipper prevented a trans-autophosphorylation reaction on the C-terminal domain of Nek2 and strongly reduced Nek2 kinase activity on exogenous substrates. Finally, we emphasize that the Nek2 leucine zipper described here differs from classical leucine zippers in that it exhibits a radically different arrangement of hydrophobic and charged amino acids. Thus, this study reveals not only an important mechanism for the regulation of the Nek2 kinase but, furthermore, highlights an unusual organization of a leucine zipper dimerization motif.

A novel E2F binding protein with Myc-type HLH motif stimulates E2F-dependent transcription by forming a heterodimer.

The human embryonal carcinoma cells NEC14 can be induced to differentiate morphologically by the addition of 10(-2) M N, N'-hexamethylene-bis-acetamide and cease to grow in several days. Transcription factors of the E2F/DP family have been shown to be closely related to the regulation of cell proliferation. To analyse cellular proteins which interact with E2F in NEC14 cells, cDNA clones encoding E2F binding proteins were isolated from a lambdaZAP II NEC14 cell library with the 32P-labeled GST (Glutathione S-transferase)-E2F-1 fusion protein as a probe. One of the clones encodes E2FBP1 which has the helix-loop-helix (HLH) motif, but lacks the basic domain and the zipper structure usually found at N- and C-terminal sides to the HLH motif, respectively. The arrangement of amino acids in the helix 1 and helix 2 regions is quite similar to those of Mxi and Mad, but different from those of E2F-1 and DP-1. Western blot analysis of the immunoprecipitates prepared with anti-E2FBP1 antibody showed that E2FBP1 associates with both E2F-1 and DP-1 in vivo. E2FBP1 alone has no DNA binding activity, but bind to the E2F site through heterodimerization with E2F-1 but not with DP-1. Although E2FBP1 lacks the transactivation domain, it stimulates E2F site-dependent transcription in cooperation with E2F-1.

Determination of lumenal orientation of microsomal 50-kDa esterase/N-deacetylase.

The amino acid arrangements responsible for the insertion and specific lumenal orientation of proteins having an uncleaved signal-peptide-like anchor are poorly understood. A 50-kDa protein having a hydrophobic N-terminus similar to the lumenal glycoprotein 11beta-hydroxysteroid dehydrogenase [Ozols, J. (1995) J. Biol. Chem. 270, 2305-2312] was identified in detergent-solubilized microsomes. The posttranslational modifications and the membrane orientation of the 50-kDa protein were investigated using the approaches of protein structure analysis. Sequence analysis of the entire 50-kDa protein showed a lack of structural relatedness to the steroid dehydrogenase beyond the membrane binding segment. Structure analysis of peptides revealed that carbohydrate is attached at Asn-77 and Asn-281, implying that these sites of the 50-kDa protein are oriented toward the lumenal side of the endoplasmic membrane (ER). Specific enzymatic deglycosylation on the intact protein identified the two glycans as high mannose carbohydrate rather than of the complex type, suggesting that the protein had not undergone further trafficking steps beyond the lumen of ER. Chemical modification of cysteinyl residues showed a lack of free thiols in the intact protein. Peptide mapping identified one disulfide bond between Cys-115 and -340 further restricting the bulk of the protein to the lumenal compartment. Proteolysis of intact and solubilized microsomes showed that the 50-kDa protein is resistant to fragmentation at the conditions which led to the removal of the membranous segments from cytochrome b5 and the NADH-cytochrome b5 reductase. The proposed model of the 50-kDa protein predicts one transmembrane segment at the N-terminus, flanked by positively charged residues on the cytosolic surface and negatively charged residues on the lumenal side of the hydrophobic domain, with most of the polypeptide projecting into the lumen of the ER. The stated similarities in the topology between 11beta-steroid dehydrogenase and 50-kDa protein envision their transmembrane segment consisting of a basic residue(s) followed by an array of some 17 hydrophobic residues containing the Ala-Tyr-Tyr-X-Tyr cluster, where X represents a hydrophobic amino acid, which terminates with acidic residues. It is proposed here that such a motif may constitute a lumenal targeting signal for a set of single-membrane-spanning proteins that are otherwise structurally and functionally unrelated.

Interspecies sequence differences in the Mip protein from the genus Legionella: implications for function and evolutionary relatedness.

The nucleotide sequence of the mip genes and their inferred amino acid sequences were determined from 35 Legionella species and compared with the published sequences for L. pneumophila, L. micdadei and L. longbeachae. The sequences were 69-97% conserved at the nucleotide level and 82-99% at the amino acid level, with total conservation of amino acids determined to be associated with sites known to be involved in peptidyl prolyl cis-trans isomerase activity. No apparent difference could be determined in the arrangement of amino acids that would predict a functional difference in Mip from species associated with disease and Mip from species isolated only from the environment. Additionally, a phylogenetic comparison of the sequences with published 16S RNA sequences, using both genetic distance and maximum parsimony methods, was performed. Few relationships were apparent that were well supported by both data sets, the most robust being a clade comprising ([(cincinnatiensis, longbeachae, sainthelensi, santicrucis) gratiana] (moravica, quateirensis, shakespearei, worsleiensis) anisa, bozemanii, cherrii, dumoffii, gormanii, jordanis, parisiensis, pneumophila, steigerwaltii, tucsonensis, and wadsworthii).

FAP52, a Novel, SH3 Domain-containing Focal Adhesion Protein

Src-homology 3 (SH3) domain is a 60-70-amino acid motif present in a large variety of signal transduction and cytoskeletal proteins. We used reverse transcriptase-polymerase chain reaction with degenerate and specific primers and chicken brain mRNA to clone a cDNA that codes for a novel SH3 domain-containing protein. The sequence predicts a 448-amino acid polypeptide with a molecular mass of 51, 971 daltons. In the amino terminus, it shows a very high propensity for alpha-helicity, suggesting coiled-coil and possibly a higher order oligomeric arrangement. In the carboxyl terminus, there is a unique SH3 sequence. In Northern blotting, a major 3.7-kilobase and a minor 7.2-kilobase transcript was detected in most chicken tissues. In immunofluorescence microscopy and immunoelectron microscopy on cultured chicken fibroblasts, the protein was localized to focal adhesions in which it showed a distinct codistribution with the focal adhesion proteins vinculin, talin, and paxillin. Phosphoamino acid analysis showed that in cultured chicken heart fibroblasts, the protein contains phosphoserine, but no phosphothreonine or phosphotyrosine, and that the phosphorylation is not dependent on fibronectin. We propose this protein the name FAP52, for Focal Adhesion Protein of 52 kDa, and suggest that it forms part of the multimolecular complex constituting focal adhesion sites.

Transient Changes of the Conformation of Hemagglutinin of Influenza Virus at Low pH Detected by Time-resolved Circular Dichroism Spectroscopy

Membrane fusion of influenza virus is mediated by a conformational change of the viral membrane protein hemagglutinin (HA) triggered by low pH. By near UV CD spectroscopy, which is sensitive to the arrangement and mobility of aromatic amino acids in proteins, we have monitored continuously with a time resolution of 5 s the kinetics of structural alterations of the ectodomain of HA isolated from different influenza virus strains (H1 (A/PR 8/34), H2 (A/Japan), and H3 (X31)). To establish a functional correlation to structural alterations of the HA ectodomain reflected by the CD, we have measured the kinetics of the virus-erythrocyte fusion and of the inactivation of fusion by low pH preincubation of viruses. At acidic pH we found a multiphasic behavior of the CD signal recorded at 283 nm. Upon lowering the pH we detected first an increase of the CD amplitude, which is associated with the formation of a fusion-competent state of HA. The initial increase was followed by a continuous decline of CD amplitude, which can be ascribed to a transformation into a fusion-inactivated conformation that is in its early phase reversible as found for A/Japan. The half-time of the different phases of the CD signal depended on the virus strain, the temperature, and the acidic pH. The results support recent hypotheses that the fusion-competent conformation is an intermediate of the fusion-inactivated structure of HA.

Combinatorial manipulation of three key active site residues in glycinamide ribonucleotide transformylase.

The enzyme glycinamide ribonucleotide transformylase (EC 2.1.2.2) has previously been shown to have three key polar active site residues important for catalysis: N106, H108 and D144. Mutations of any of these three residues lead to substantially decreased catalytic activity, although none of them are completely irreplaceable. In order to determine whether any alternative arrangement of amino acids at these three positions could lead to an active protein, all three of these residues were simultaneously subjected to saturation site-directed mutagenesis. The resulting combinatorial library of mutant genes was screened for those encoding active proteins using functional complementation. Glycinamide ribonucleotide transformylase was found to be capable of tolerating no more than one mutation amongst these key residues, since the only proteins found to be sufficiently active to allow growth of auxotrophic cells on selective media were the wild-type and enzymes containing a single mutation to one of these residues. It seems likely that no enzymes containing two or more mutations of these three residues possess significant catalytic activity. The combinatorial approach used could prove to be quite useful in protein engineering and protein evolution experiments.

Amino Acid Oligomer Microspheres as Drug Delivery Systems. II. Oligomerization of NCAs Initiated with L-Tyrosine Methyl Ester

Oligomerizations of various amino acid N-carboxyanhydrides (NCAs) were prepared in the presence of L-tyrosine methyl ester (Tyr.Me). Tyr.Me was found to be an efficient initiator to form oligopeptides consisting of L-aspartic acid, L-glutamic acid, L-phenylalanine, and L-tyrosine with random arrangements of amino acids. In acidic solution, the oligopeptides formed microspheres which were capable of encapsulating macromolecular drugs such as insulin and heparin. These microspheres were considered to be potential oral drug delivery systems.

Characterization of a Novel Strain of Bacillus thuringiensis

Bacillus thuringiensis is a well-known species of entomopathogenic bacteria that is widely used as a biopesticide against many insect pests. Insecticidal proteins, coded for by genes located in plasmids, form typical parasporal, crystalline inclusions during sporulation. In this report, an unusual strain of B. thuringiensis subserovar oyamensis (LBIT-113), isolated from living larvae of Anopheles pseudopunctipennis in Mexico, was characterized by its ultrastructure, the protein composition of its parasporal crystal, plasmid pattern, and toxicological properties against several insect and noninsect targets. The parasporal crystal is enclosed within the spore's outermost envelope (exosporium), as determined by transmission electron microscopy, and exhibits a square, flat shape. Its main components are two proteins with sizes of 88 and 54 kDa. Despite some crystal morphology resemblance, both proteins are immunologically unrelated to the Cry IIIA protein, as shown by immunoblot analysis, when probed with antisera raised against the 88-kDa protein and the Cry IIIA protein. Partial N-terminal sequence of the 88-kDa protein revealed a unique amino acid arrangement among the Cry proteins. Solubilization of the crystal proteins was achieved at 3.3 M NaBr, and its digestion with trypsin showed only one ca. 60-kDa peptide, as observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The patterns of three plasmids of strain LBIT-113 were considerably different from those of B. thuringiensis subspp. kurstaki, tenebrionis, and israelensis. Parasporal crystals showed no toxicity to larvae of four species of caterpillar, three species of mosquito, two species of beetle, one species of cricket, one species of ant, one species of aphid, one species of nematode, one species of ostracod, one species of ameba, and one species of rotifer.

1] Apolipoproteins: Pathophysiology and clinical implications

The term apolipoprotein was initially coined by John Oncley to refer to the protein constituents of plasma lipoproteins. Plasma apolipoproteins are of interest principally because of their central role in plasma lipid transport and, hence their role in atherogenesis. Over the past several decades, a great deal has been learned about apolipoprotein structure and function. The regulation of apolipoprotein gene expression and the molecular mechanisms of apolipoprotein function are beginning to be revealed. Given the rate of progress in this research, therapeutic applications that target the expression or metabolism of specific apolipoproteins can be expected in the near future. Central to the functions of all apolipoproteins (apo) is the ability to bind phospholipids that reside in specialized regions termed “amphiphathic helices.” The characteristic feature of the amphipathic helix is the spatial arrangement of hydrophobic and hydrophilic amino acids. The hydrophobic face of the helix is intercalated between the fatty acyl chains of phospholipids, whereas the hydrophilic face is located close to the polar head groups of phospholipids. Such an orientation allows the interaction of protein domains with lipoprotein-modifying enzymes and cellular receptors that control the catabolism of lipoproteins (Lp) and their removal from the circulation. The apoB gene differs from other apolipoprotein genes in genomic organization, in that it contains 29 exons, 2 of which are extremely long. In addition to the amphipathic helix motifs, located mainly in the carboxyterminal half, apoB contains unique proline-rich sequences predicted to form amphipathic β sheets. The evolutionary relationship between apoB and the other apolipoproteins is, therefore, not clear. ApoD is not a member of the apolipoprotein gene family, as it differs from all other apolipoproteins in genomic organization, primary structure, and tissue sites of expression. Similar arguments can be used to qualify apo[a] and minor apolipoproteins, such as aport and apoJ, as atypical apolipoproteins.

Revealing the Architecture of a K + Channel Pore Through Mutant Cycles with a Peptide Inhibitor

Thermodynamic mutant cycles provide a formalism for studying energetic coupling between amino acids on the interaction surface in a protein-protein complex. This approach was applied to the Shaker potassium channel and to a high-affinity peptide inhibitor (scorpion toxin) that binds to its pore entryway. The assignment of pairwise interactions defined the spatial arrangement of channel amino acids with respect to the known inhibitor structure. A strong constraint was placed on the Shaker channel pore-forming region by requiring its amino-terminal border to be 12 to 15 angstroms from the central axis. This method is directly applicable to sodium, calcium, and other ion channels where inhibitor or modulatory proteins bind with high affinity.