Добірка наукової літератури з теми "Folded Peptides"

Half a century has passed since the hydrogen-bonded secondary structures of polypeptides and proteins were first recognized. An extraordinary wealth of conformational information is now available on peptides and proteins, which are formed of α-amino acid residues. More recently, the discovery of well-folded structures in oligopeptides containing β-amino acids has focused a great deal of current interest on the conformational properties of peptides constructed from higher homologues (ω) of α-amino acids. This review examines the nature of intramolecularly hydrogen-bonded conformations of hybrid peptides formed by amino acid residues, with a varying number of backbone atoms. The β-turn, a ubiquitous structural feature formed by two residue (αα) segments in proteins and peptides, is stabilized by a 10-atom (C 10 ) intramolecular 4→1 hydrogen bond. Hybrid turns may be classified by comparison with their αα counterparts. The available crystallographic information on hydrogen-bonded hybrid turns is surveyed in this review. Several recent examples demonstrate that individual ω-amino acid residues and hybrid dipeptide segments may be incorporated into the regular structures of α-peptides. Examples of both peptide helices and hairpins are presented. The present review explores the relationships between folded conformations in hybrid sequences and their counterparts in all α-residue sequences. The use of stereochemically constrained ω-residues promises to expand the range of peptide design strategies to include ω-amino acids. This approach is exemplified by well-folded structures like the C 12 (αγ) and C 14 (γγ) helices formed in short peptides containing multiply substituted γ-residues. The achiral γ-residue gabapentin is a readily accessible building block in the design of peptides containing γ-amino acids. The construction of globular polypeptide structures using diverse hybrid sequences appears to be a realistic possibility.

In this work, we report the self-assembly of chimeric peptides in which two distinctly folded domains can be organized into a finite peptide hexamer with precise control over multivalent ligand presentation and enhanced tumor cell targeting.

Abstract MHC (Major Histocompatibility Complex) class I molecules play a central role in the mammalian antiviral immune response by presenting the viral proteome at the cell surface for recognition by cytotoxic T lymphocytes. The folding of recombinant MHC class I molecules from denatured bacterial inclusion bodies and their assembly with the light chain beta-2 microglobulin largely depend on specific high-affinity peptides of usually eight or nine amino acids. To find the minimum peptide requirement for folding and stabilization of class I molecules, we have investigated the influence of dipeptides on the folding and peptide binding of recombinant HLA-A*02:01 (A2) and H-2Kb (Kb). In the presence of the dipeptide Gly-Leu (GL), both A2 and Kb folded well, even without a high-affinity peptide present, as measured by a tryptophan fluorescence assay. When folded in the presence of GL, they also bound much faster to their respective specific high-affinity peptides as when folded without GL. Kinetic analysis by fluorescence anisotropy revealed that the presence of GL enhances the on-rate of peptide binding to empty A2 and Kb by six to eight fold. Molecular dynamics simulation indicates that the dipeptide GL interacts with the C terminus of the peptide binding groove. In conclusion, our data demonstrate that the dipeptide GL improves the folding of A2 and Kb and induces a peptide-receptive conformation for enhanced peptide binding.

In the last few decades, development of novel experimental techniques, such as new types of disulfide (SS)-forming reagents and genetic and chemical technologies for synthesizing designed artificial proteins, is opening a new realm of the oxidative folding study where peptides and proteins can be folded under physiologically more relevant conditions. In this review, after a brief overview of the historical and physicochemical background of oxidative protein folding study, recently revealed folding pathways of several representative peptides and proteins are summarized, including those having two, three, or four SS bonds in the native state, as well as those with odd Cys residues or consisting of two peptide chains. Comparison of the updated pathways with those reported in the early years has revealed the flexible nature of the protein folding pathways. The significantly different pathways characterized for hen-egg white lysozyme and bovine milk α-lactalbumin, which belong to the same protein superfamily, suggest that the information of protein folding pathways, not only the native folded structure, is encoded in the amino acid sequence. The application of the flexible pathways of peptides and proteins to the engineering of folded three-dimensional structures is an interesting and important issue in the new realm of the current oxidative protein folding study.

Native disulfide formation is crucial to the process of disulfide-rich protein folding in vitro. As such, analysis of the disulfide bonds can be used to track the process of the folding reaction; however, the diverse structural isomers interfere with characterization due to the non-native disulfide linkages. Previously, a mass spectrometry (MS) based platform coupled with peptide demethylation and an automatic disulfide bond searching engine demonstrated the potential to screen disulfide-linked peptides for the unambiguous assignment of paired cysteine residues of toxin components in cobra venom. The developed MS-based platform was evaluated to analyze the disulfide bonds of structural isomers during the folding reaction of synthetic cardiotoxin A3 polypeptide (syn-CTX A3), an important medical component in cobra venom. Through application of this work flow, a total of 13 disulfide-linked peptides were repeatedly identified across the folding reaction, and two of them were found to contain cysteine pairings, like those found in native CTX A3. Quantitative analysis of these disulfide-linked peptides showed the occurrence of a progressive disulfide rearrangement that generates a native disulfide bond pattern on syn-CTX A3 folded protein. The formation of these syn-CTX A3 folded protein reaches a steady level in the late stage of the folding reaction. Biophysical and cell-based assays showed that the collected syn-CTX A3 folded protein have a β-sheet secondary structure and cytotoxic activity similar to that of native CTX A3. In addition, the immunization of the syn-CTX A3 folded proteins could induce neutralization antibodies against the cytotoxic activity of native CTX A3. In contrast, these structure activities were poorly observed in the other folded isomers with non-native disulfide bonds. The study highlights the ability of the developed MS platform to assay isomers with heterogeneous disulfide bonds, providing insight into the folding mechanism of the bioactive protein generation.

This study demonstrates the ability of travelling wave ion mobility-mass spectrometry to measure collision cross-sections of ions in the negative mode, using a calibration based approach. Here, negative mode ion mobility-mass spectrometry was utilised to understand structural transitions of calmodulin upon Ca2+ binding and complexation with model peptides melittin and the plasma membrane Ca2+ pump C20W peptide. Coexisting calmodulin conformers were distinguished on the basis of their mass and cross-section, and identified as relatively folded and unfolded populations, with good agreement in collision cross-section to known calmodulin geometries. Titration of calcium tartrate to physiologically relevant Ca2+ levels provided evidence for intermediately metalated species during the transition from apo- to holo-calmodulin, with collision cross-section measurements indicating that higher Ca2+ occupancy is correlated with more compact structures. The binding of two representative peptides which exemplify canonical compact (melittin) and extended (C20W) peptide-calmodulin binding models has also been interrogated by ion mobility mass spectrometry. Peptide binding to calmodulin involves intermediates with metalation states from 1–4 Ca2+, which demonstrate relatively collapsed structures, suggesting neither the existence of holo-calmodulin or a pre-folded calmodulin conformation is a prerequisite for binding target peptides or proteins. The biological importance of the different metal unsaturated calmodulin complexes, if any, is yet to be understood.

Twin-arginine-containing signal sequences mediate the transmembrane transport of folded proteins. The cognate twin-arginine translocation (Tat) machinery of Escherichia coli consists of the membrane proteins TatA, TatB, and TatC. Whereas Tat signal peptides are recognized by TatB and TatC, little is known about molecular contacts of the mature, folded part of Tat precursor proteins. We have placed a photo-cross-linker into Tat substrates at sites predicted to be either surface-exposed or hidden in the core of the folded proteins. On targeting of these variants to the Tat machinery of membrane vesicles, all surface-exposed sites were found in close proximity to TatB. Correspondingly, incorporation of the cross-linker into TatB revealed multiple precursor-binding sites in the predicted transmembrane and amphipathic helices of TatB. Large adducts indicative of TatB oligomers contacting one precursor molecule were also obtained. Cross-linking of Tat substrates to TatB required an intact twin-arginine signal peptide and disappeared upon transmembrane translocation. Our collective data are consistent with TatB forming an oligomeric binding site that transiently accommodates folded Tat precursors.

The major histocompatibility complex class Ib protein, Qa-1b, serves as a ligand for murine CD94/NKG2A natural killer (NK) cell inhibitory receptors. The Qa-1b peptide-binding site is predominantly occupied by a single nonameric peptide, Qa-1 determinant modifier (Qdm), derived from the leader sequence of H-2D and L molecules. Five anchor residues were identified in this study by measuring the peptide-binding affinities of substituted Qdm peptides in experiments with purified recombinant Qa-1b. A candidate peptide-binding motif was determined by sequence analysis of peptides eluted from Qa-1 that had been folded in the presence of random peptide libraries or pools of Qdm derivatives randomized at specific anchor positions. The results indicate that Qa-1b can bind a diverse repertoire of peptides but that Qdm has an optimal primary structure for binding Qa-1b. Flow cytometry experiments with Qa-1b tetramers and NK target cell lysis assays demonstrated that CD94/NKG2A discriminates between Qa-1b complexes containing peptides with substitutions at nonanchor positions P4, P5, or P8. Our findings suggest that it may be difficult for viruses to generate decoy peptides that mimic Qdm and raise the possibility that competitive replacement of Qdm with other peptides may provide a novel mechanism for activation of NK cells.

Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2009.
Title from electronic title page (viewed Oct. 5, 2009). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Chemistry. " Discipline: Chemistry; Department/School: Chemistry.

Since ancient times, substances derived from amphibian skins have been recognised to possess various medicinal properties, In recent years, the peptides from amphibian skin have attracted extensive attention for their profound significance in providing clues directed toward novel drug development, for better understanding of miscellaneous physiological and pathological processes, for elucidation of phylogenetic relationships and for improving taxonomy. Previously, the chemical complexity of the defensive skin secretion of the broad-folded frog, Hylarana latouchii, has not been studied in detail. In this thesis, parallel transcriptomic and peptidomic analyses of the electrically-stimulated skin secretion have enabled the identification and characterisation of seven, biologically-active peptides. Five of these peptides are antimicrobial peptides displaying differential growth-inhibitory activity toward test microorganisms and human cancer cell lines; two of which belong to the previously-identified brevinin-l and temp orin families, while the others show little structural similarity with other antimicrobial peptides and represent the prototypes of a novel peptide family - the hylaranins. Hylaranin-Ll and hylaranin-L2 (two 18-mer structurally-related but distinct peptides) are described in Chapter 3 and hylaranin-L3 (a unique l3-residue peptide) is described in Chapter 5. In Chapter 4, a novel Bowman-Birk-type trypsin inhibitory peptide, pLR-HL, belonging to the pLRlranacyclin family, was identified. By substitution of its Lys8 residue with Phe, the Phe8-pLR-HL analogue was found to be transformed into a chymotrypsin inhibitor. The II -residue canonical reactive loops within each peptide could exert corresponding protease inhibitory activity independently, In Chapter 6, a peptide structurally-related to bombesin was isolated and found to possess contractile activity on rat urinary bladder and uterus smooth muscles. These data illustrate that amphibian skin continues to provide numerous novel peptides for furthering research in the fields of pharmaceutical science, biological chemistry, medical science, and systematics. It is expected that many more novel amphibian skin peptides are awaiting discovery.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2001.
Includes bibliographical references.
This thesis describes the development of iterative and combinatorial methods for identifying small peptide scaffolds able to support catalytic function. The incorporation of thiamine coenzyme functionality into small peptide scaffolds is achieved through the use of a coenzyme amino acid chimera (Taz). This thiazole amino acid can be alkylated to generate the active thiazolium species. A series of designed peptides containing the Taz coenzyme chimera revealed that an increasingly structured and hydrophobic peptidyl environment can dramatically enhance the acidity of the thiazolium C2 methine, the initial step in the catalytic pathway. A combinatorial approach is developed to permit a more rapid screening of large libraries of ppa peptide sequences for unique functional properties. The method used a fluorescent 13-diketone probe to identify a family of similar sequences that incorporate a functional primary amine at the center of the hydrophobic core of the PPa peptide structure. The function of the amine is enhanced by the provision of the hydrophobic peptidyl environment and is able to modestly enhance aldol condensation and retro-aldol reactions. Finally, a new trimeric Apa oligomer structure is evaluated as a functional peptide scaffold. Initially, the structural requirements of the motif are explored through a series of sequence modifications. These studies lead to the incorporation of a primary amine at a unique site in the scaffold that greatly enhances the reactivity of the amine. It is further demonstrated that this increased functional ability is directly related to the folded stability of the trimer.
by Kevin A. McDonnell.
Ph.D.

Die Pektat-Lyasen gehören zu einer Proteinfamilie, die meistens von pflanzenpathogenen Mikroorganismen sekretiert werden. Die Enzyme katalysieren den Abbau von Polygalakturonsäure, einem Hauptbestandteil in
pflanzlichen Mittellamellen und Primärzellwänden. Der Abbau der alpha-1,4-verbrückten Galakturonsäurereste erfogt durch eine beta-Eliminierungsreaktion, dabei entsteht ein Produkt mit einer ungesättigten C4-C5 Bindung am nicht reduzierenden Ende, das durch spektroskopische Messungen beobachtet werden kann. Für die enzymatische Reaktion der Pektat-Lyasen ist Calcium nötig und das pH-Optimum der Reaktion liegt bei pH 8.5. Alle bis jetzt bekannten Strukturen der Pektat- und Pektin-Lyasen haben das gleiche Strukturmotiv - eine rechtsgängige parallele beta-Helix. Die Struktur der Pektat-Lyase aus Bacillus subtilis (BsPel) ist im Komplex mit Calcium gelöst worden. BsPel ist ein monomeres Protein mit einer ungefähren Molekularmasse von 43 kDa, das keine Disulfidbrücken enthält. Dies erlaubte sowohl eine effiziente rekombinante Expression des Wildtypproteins, als auch von destabilisierten Mutanten im Cytoplasma von E. coli. Parallele beta-Helices sind relativ große, jedoch verhältnismäßig einfach aufgebaute Proteine. Um detailliertere Informationen über die kritischen Schritte bei der in vitro-Faltung von parallelen beta-Helices zu erhalten, sollte in der vorliegenden Arbeit versucht werden, den Faltungsmechanismus dieses Proteins näher zu charakterisieren. Dabei sollte vor allem die Frage geklärt werden, welche Wechselwirkungen für die Stabilität dieses Proteins einerseits und für die Stabilität von essentiellen Faltungsintermediaten andererseits besonders wichtig sind.

Rückfaltung von BsPel, ausgehend vom guanidiniumchlorid-denaturierten Zustand, war bei kleinen Proteinkonzentrationen und niedrigen Temperaturen vollständig möglich. GdmCl-induzierte Faltungsübergänge waren aber nicht reversibel und zeigten eine apparente Hysterese. Kinetische Messungen des Fluoreszenz- und CD-Signals im fernen UV ergaben eine extreme Denaturierungsmittelabhängigkeit der Rückfaltungsrate im Bereich des Übergangmittelpunktes. Der extreme Abfall der Rückfaltungsraten mit steigender Denaturierungsmittelkonzentration kann als kooperative
Entfaltung eines essentiellen Faltungsintermediats verstanden werden. Dieses Faltungsintermediat ist temperaturlabil und kann durch den Zusatz Glycerin im Renaturierungspuffer stabilisiert werden, wobei sich die Hysterese verringert, jedoch nicht vollständig aufgehoben wird. Durch reverse Doppelsprungexperimente konnten zwei transiente Faltungsintermediate nachgewiesen werden, die auf zwei parallelen Faltungswegen liegen und beide zum nativen Zustand weiterreagieren können. Fluoreszenzemissionsspektren der beiden Intermediate zeigten, daß beide schon nativähnliche Struktur aufweisen. Kinetische Daten von Prolin-Doppelsprungexperimenten zeigten, daß Prolinisomerisierung den geschwindigkeitsbestimmenden Schritt in der Reaktivierung des denaturierten Enzyms darstellt. Desweiteren konnte durch Prolin-Doppelsprungexperimenten an Mutanten mit Substitutionen im Prolinrest 281 gezeigt werden, daß die langsame Renaturierung von BsPel nicht durch die Isomerisierung der einzigen cis-Peptidbindung an Prolin 281 verursacht wird, sondern durch die Isomerisierung mehrerer trans-Proline. Die beiden beobachteten transienten Faltungsintermediate sind somit wahrscheinlich zwei Populationen von Faltungsintermediaten mit nicht-nativen X-Pro-Peptidbindungen, wobei sich die Populationen durch mindestens eine nicht-native X-Pro-Peptidbindung unterscheiden.

Der Austausch des Prolinrestes 281 gegen verschiedene Aminosäuren (Ala, Ile, Leu, Phe, Gly) führte zu einer starken Destabilisierung des nativen Proteins und daneben auch zu einer Reduktion in der Aktivität, da die Mutationsstelle in der Nähe der putativen Substratbindetasche liegt. Die Rückfaltungskinetiken der Prolinmutanten war bei 10°C annähernd gleich zum Wildtyp und die geschwindigkeitsbestimmenden Schritte der Faltung waren durch die Mutation nicht verändert. Die durch die Mutation verursachte drastische Destabilisierung des nativen Zustands führte zu einem reversiblen Entfaltungsgleichgewicht bei pH 7 und 10°C. GdmCl-induzierte Faltungsübergänge der Mutante P281A zeigten bei Messungen der Tryptophanfluoreszenzemission und der Aktivität einen kooperativen Phasenübergang mit einem Übergangsmittelpunkt bei 1.1 M GdmCl. Durch die Übereinstimmung der Faltungsübergänge bei beiden Messparametern konnten die Faltungsübergänge nach dem Zwei-Zustandsmodell ausgewertet werden. Dabei wurde eine freie Sabilisierungsenthalpie der Faltung für die Mutante von - 64.2 ± 0.4 kJ/mol und eine Kooperativität des Übergangs
von - 58.2 ± 0.3 kJ/(mol·M) bestimmt.


BsPel enthält, wie die meisten monomeren rechtsgängigen parallelen beta-Helix-Proteine, einen internen Stapel wasserstoffverbrückter Asparagin-Seitenketten. Die Mehrheit der erzeugten Mutanten mit Substitutionen im Zentrum der Asn-Leiter (N271X) waren als enzymatisch aktives Protein zugänglich. Die Auswirkung der Mutation auf die Stabilität und Rückfaltung wurde an den Proteinen BsPel-N271T und BsPel-N271A näher analysiert. Dabei führte die Unterbrechung des Asparaginstapels im Inneren der beta-Helix zu keiner drastischen Destabilisierung des nativen Proteins. Allerdings führten diese Mutationen zu einem temperatur-sensitiven Faltungsphänotyp und die Hysterese im Denaturierungsübergang wurde verstärkt. Offenbar wird durch die Unterbrechung des Asparaginstapel ein essentielles, thermolabiles Faltungsintermediat destabilisiert. Der Asparaginstapel wird somit bei der Faltung sehr früh ausgebildet und ist wahrscheinlich schon im Übergangszustand vorhanden.
Pectate lyases belong to a family of proteins secreted by plant pathogenic microbes. The enzymes cleave alpha-1,4 linked galacturonic acid by a beta-elimination that results in an unsaturated product, which can be quantified spectrophotometrically. Calcium is essential for the activity and the pH-optimum is near 8.5. All known structures of pectate and pectin lyases have the same structural motif - a right handed parallel beta-helix. The structure of pectate lyase from Bacillus subtilis (BsPel) has been solved in complex with calcium. It is a monomeric protein, with a molecular mass of about 43 kDa and without disulfide bonds. This allows its high-yield recombinant expression in the cytoplasm of Escherichia coli. Parallel beta-helices are relative large proteins, however with a simple folding topology. The objective of this work was to characterize the folding mechanism of BsPel. In particular we investigated the role of the interactions of certain residues in the parallel beta-helix for the stability of the native protein and the stability of essential folding intermediates.

Refolding of BsPel was possible at low protein concentrations and low temperature. However, denaturation of BsPel was not freely reversible. De- and renaturation curves showed a large apparent hysteresis. Furthermore, the folding rate constant deduced from fluorescence and circulardichroism measurements showed a very strong dependence on denaturant concentrations near the midpoint of the renaturation transition. This can be explained with a cooperative unfolding of an essential folding intermediate. Upon stabilisation of the temperature-sensitive intermediate by addition of glycerol in the renaturation buffer, the hysteresis is reduced, but does not disappear. Reverse double mixing kinetic experiments have shown that two transient folding intermediates are on the folding pathway. These intermediates are on parallel pathways and both can fold to the native state. Fluorescence emission spectra have shown the native-like structure of both intermediates. Furthermore, data from proline double mixing kinetic experiments revealed that isomerization of peptidyl-prolyl bonds was responsible for the slow kinetics in the reactivation of the enzyme. However, the isomerization of the single cis-peptidyl-prolyl bond at Pro281 was not responsible for the slowest folding phase observed, but rather the isomerization of other trans-peptidyl-prolyl bonds. Thus, both transient folding intermediates observed probably represent two populations of folding intermediates with non-native X-Pro-peptide bonds. The difference of the two populations is at least one non-native X-Pro-peptide bond.

Mutations of the proline 281 against various residues (Ala, Ile, Leu, Phe, Gly) resulted in a strong destabilization of the native protein. Also, the activity of the mutant proteins was strong reduced due to the position of the mutation site near the putative active center of the protein. At 10°C the kinetic folding behavior of the proline mutants was not significant changed. However, the strong destabilization of the native state in the proline mutants resulted in a reversible folding equilibrium at pH 7 and 10°C. The unfolding of the P281A mutant was reversible as determined by fluorescence emission and enzyme activity measurements. The coincidence of these detected transitions is consistent with a two-state equilibrium transition. At pH 7 and 10°C the delta G°(H₂O) for folding of P281A was - 64.2 ± 0.4 kJ/mol, with a midpoint of the transition at 1.1 M GdmCl and a cooperativity of - 58.2 ± 0.3 kJ/(mol·M).

BsPel has an asparagine ladder in turn 2 of the parallel beta-helix with extensive network of side-chain hydrogen bonds between the Asn residues. Such an Asn-ladder is a conserved feature of many monomeric beta-helices crystallized so far. The middle Asn residue (271) was selected and exchanged for various residues. Most of the mutants were expressed at 25°C as soluble and active proteins but with a significant reduction in yield. Mutants N271T and N271A were selected to study the stability and refolding of these proteins in comparison with the wild-type protein. The substitution in the Asn-ladder did not drastically destabilize the native protein, but caused a temperature-sensitive-folding (tsf) phenotype with an increased hysteresis in the de- and renaturation transition curves. In addition, the disruption of the Asn-ladder resulted in destabilization of an essential, thermosensitive folding intermediate. Thus, the Asn-ladder is formed very early during the folding, probably well before the transition state of folding.

In silico investigation on protein domains structure and linear/structural motifs can strongly boost functional analyses and technological design. Protein surface features study is crucial to understanding Protein-Protein Interactions (PPI); in particular, surface and pockets conservation and variation, in terms of hydrophobicity, steric hindrance and electrostatics can act as driving forces in protein evolution and functional specialization. Therefore, molecular modeling and structure comparison techniques play an important role in shedding light on “protein behavior” and this PhD work took advantage from integrating computational approaches based on some known molecular modeling methods, such as e.g. Homology Modeling, Fold Recognition, Ab initio Modeling, PBE (Poisson-Boltzmann Electrostatics), Protein-peptide Docking and Hydropathy Analysis with structure and sequence comparison and scanning tools and, of course, with feedback from wet lab analyses performed by co-workers. Such an integrative approach was followed along investigations on a number of different biological systems: • Surface determinants in H5N1 type A Influenza viruses: Here, an analysis of surface determinants from H5N1 haemagglutinin, involved in host-viral interaction, was completed and then published. Genomic variation is very high in influenza A viruses. However, viral evolution and spreading are strongly influenced by immunogenic features and capacity to bind host cells, depending in turn on the two major capsidic proteins (haemagglutinin and neuraminidase). Current analyses of viral evolution are based on serological and primary sequence comparison; however, comparative structural analysis of capsidic proteins can provide functional insights on surface regions possibly crucial to antigenicity and cell binding. We performed molecular modeling and extensive structural comparison of influenza virus haemagglutinin and of their domains and sub-regions to investigate type- and/or domain specific variation. We found that structural closeness and primary sequence similarity are not always tightly related; moreover, type-specific features could be inferred when comparing surface properties of haemagglutinin subregions, monomers and trimers, in terms of electrostatics and hydropathy. Focusing on H5N1, we found that the variation at the receptor binding domain (RBD) surface intriguingly relates to branching of still circulating clades from those ones that are no longer circulating. Recent evidence on the association between electrostatic fingerprints at the haemagglutinin receptor binding surface and the evolutionary success and spreading of H5N1 avian influenza clades prompted us to perform further integrated phylogenetic and structural bioinformatic analysis in H9N2 viruses. In fact, influenza A virus is a zoonotic agent with a significant impact both on public health and poultry industry and switch to human host has been reported for both H5N1 and H9N2 viruses. We performed the evolutionary analysis of a large and non-redundant viral strain dataset, leading to clustering of H9N2 viruses in five groups. Then and according to recent evidence on H5N1, congruence resulted among phylogenetic data and surface electrostatic fingerprints from structural comparison. In particular, surface feature fingerprints could be inferred that relate group specific variation in electrostatic charges and isocontour to well-known hemagglutinin sites involved in modulation of immune escape and host specificity. Results from this second work strengthen suggestion that when integrating up-to-date phylogenetic analyses with sequence-based and structural investigation of surface features may represent a front-end strategy for inferring trends and relevant mechanisms in influenza virus evolution. • Domain architecture variation in mammalian protein trafficking: Human VAMP7b is the most interesting variant among those produced by alternative splicing of the encoding gene SYBL1. Production of VAMP7b variants is determined by skipping of exon 6 which in turn results in coding sequence frameshift. We found that this event is conserved in other mammalian species. VAMP7b shares with the main isoform the N-terminal, inhibitory longin domain and the first half of the SNARE motif. In mammals, VAMP7b is a truncated protein in which the C-terminal half of the SNARE motif and the transmembrane region are replaced by short and variable peptides. Intriguingly instead, only in human and apes sequence frameshift determined by exon 6 skipping results in the creation of a novel unique domain of unknow function, hence human VAMP7b is not truncated but even 40 residues longer than the main isoform. Since existence of such “long” isoform and of its unique domain at protein level were confirmed by specific antibodies, we embarked on in silico dissection of the novel domain by position specific matrix sequence analysis and by ab initio structural modeling. Moreover, since the N-terminal region of the SNARE motif is conserved and it is known to mediate intramolecular binding to the Longin domain, we investigated both in vivo (by two-hybrid in yeast analysis) and in vitro (by NMR analysis) on conservation of the closed conformation. Furthermore, SCL of both VAMP7b and Ykt6b was investigated using GFP and RFP chimeras. Last but not least, b isoforms of the longin genes were analyzed by qPCR and found to be developmentally regulated. • Binding motif regulating neurite outgrowth and guidance: Fine tuning of PPIs by variation in domain architecture or by changing local motifs by surface features modulation can regulate both extracellular and intracellular signaling pathways. Extracellular PPIs can play a central role in heterologous recognition (e.g. host-pathogen) as well as in homologous signaling among cells from the same organism. Proteins exposed at the plasma membrane (PM) can interact each other and with the extracellular matrix (ECM) to provide positional information and guidance cues. Cell adhesion molecules (CAMs) are PM proteins mediating either attractive or repulsive signals by homo- and heterophilic interactions of their extracellular domains (Eds). CAM EDs are most often composed by Ig-like or Fibronectin type III fold repeats. Current evidence suggests that the four N-terminal Ig 1-4 domains of CAM EDs play a major role in such homo- or heterophilic interactions and in particular an important interaction motif is contributed by repeat Ig2. In our lab, biomimetic peptides have been developed by reproducing the known or predicted interaction motifs from the Ig2 domain of human L1CAM and the single Ig domain of human LINGO1, i.e. two proteins that play a crucial role in neurite outgrowth and guidance and in neuronal differentiation. Based on the somehow surprising structural and sequence conservation of the motif region (even when proteins show very different ED architectures), we started investigating on variation and conservation of the putative motif region by means of homology search, regular expression and finally by structural modeling and comparison. Preliminary results highlighted strong conservation of the central Arg residue in the interaction motif, while in other positions of the motif residue properties rather than specific residues are conserved. Such evidence is in agreement with finding that mutation of such residue in L1CAM is responsible for a severe neurological disorder, while mutations at other residues of the motif, results in less severe phenotype. This suggests the motif is an epitope positionally conserved around the central Arg allowing limited, but significant structural variability in surrounding sequence. In order to check such a hypothesis, a structural superposition of the Ig domains containing the interaction motif was performed, confirming that the peptide motif itself is positionally conserved but the highest positional and structural conservation concerns the central Arg residue. Experiments with peptides mutated in the central Arg showed biological activity of these peptides in terms of neuritogenesis signalling. These works carry out a bioinformatic protocol for the characterization of interaction determinants and their functional modulation, easily transportable to other proteins.
Gli studi in silico aventi per oggetto la struttura di domini proteici e di motif sia strutturali che lineari, sono in grado di fornire un importante apporto in termini di comprensione di funzione e nelle biotecnologie. Lo studio delle caratteristiche a carico della superficie proteica si rivelano essenziali nella comprensione delle Interazioni Proteina-Proteina (PPI); in particolare, la conservazione e variazione della superficie proteica e delle relative cavità in termini di idrofobicità, ingombro sterico e caratteristiche elettrostatiche, possono essere considerate come la forza in grado di guidare l’evoluzione e la specializzazione funzionale delle proteine stesse. Alla luce di quanto sopra esposto, tecniche come la Modellistica Molecolare ed il confronto tra strutture giocano un ruolo importante nel chiarire il modus operandi delle proteine e questo progetto di Dottorato ha proprio sfruttato l’approccio integrato di alcune ben note tecniche di biologia computazionale basate sulla Modellistica Molecolare come, ad esempio, Homology Modeling, Fold Recognition, Ab initio Modeling, PBE (Poisson-Boltzmann Electrostatics), Protein-peptide Docking e Hydropathy Analysis con confronto di sequenze e strutture. Elemento indispensabile e prezioso, ovviamente, il feedback ottenuto dagli esperimenti al banco effettuati dai nostri collaboratori. Questo approccio integrato è stato dunque applicato a differenti sistemi biologici: • Individuazione di determinanti di superficie in virus influenzali di tipo A H5N1: è stata effettuata un’analisi dei determinanti di superficie a carico dell’emoagglutinina proveniente dal virus influenzale H5N1, coinvolta nell’interazione virus-ospite. Questo lavoro ha già condotto ad una pubblicazione. La variazione genomica è elevata nei virus influenzali di tipo A. L’evoluzione e la diffusione dei virus sono molto influenzate dalle caratteristiche immunogeniche e dalla capacità del virus, di interagire con le cellule dell’ospite tramite le due più importanti proteine presenti sul capside virale: l’emoagglutinina e la neuraminidasi. Le analisi oggi a disposizione sono basate sul confronto dell’attività sierologica e di sequenze primarie; alla luce di ciò, l’analisi strutturale di queste proteine capsidiche può essere in grado di svelare delle conoscenze a riguardo di certe regioni presenti sulla superficie proteica che possono essere cruciali per l’antigenicità e per il legame alle cellule dell’ospite. L’emoagglutinina, sezionata nei suoi domini e subdomini, è stata da noi studiata con metodi di Modellistica Molecolare e sottoposta a confronti strutturali fini, per individuare quelle variazioni che potessero risultare tipo/dominio specifiche. Abbiamo evidenziato che la vicinanza strutturale e la similarità di sequenza primaria non sempre sono correlate; in più, caratteristiche tipo-specifiche di sottoregioni dell’emoagglutinina, monomeri e trimeri, possono essere rivelate grazie al confronto delle loro proprietà di superficie, (in termini di elettrostatica ed idrofobicità) appartenenti a sottoregioni dell’emoagglutinina, monomeri e trimeri. In questo lavoro ci siamo focalizzati sul virus H5N1 e abbiamo scoperto che il dominio di legame recettoriale dell’emoagglutinina (RBD) presenta delle variazioni tra clade circolanti e non più circolanti. Le recenti scoperte riguardanti l’associazione tra la disposizione delle cariche al RBD ed il successo in termini evolutivi e di diffusione del virus H5N1 ci hanno spinto ad eseguire analisi integrate di filogenesi e biologia strutturale a carico dei virus H9N2. Infatti, l’influenza A è un agente zoonotico in grado di produrre un grosso impatto sia sulla salute pubblica che sull’industria del pollame, avendo la capacità di effettuare il salto d’ospite, come riportato proprio per H5N1 ed H9N2. Abbiamo effettuato un’analisi evoluzionistica su un grande dataset non ridondante di ceppi virali e questo ci ha consentito di individuare cinque gruppi di virus H9N2. In accordo con le precedenti analisi effettuate per H5N1, abbiamo ottenuto accordo tra i dati filogenetici con quelli ottenuti dalle analisi di confronto strutturale. In particolare, emerge che la variazione della disposizione delle cariche coincide con quella di siti noti dell’emoagglutinina coinvolti nell’evasione al sistema immunitario e nella specificità d’ospite. I risultati ottenuti da questo secondo lavoro pongono l’accento sull’importanza dell’integrazione tra analisi di tipo filogenetiche e di biologia strutturale nella scoperta di nuovi meccanismi evolutivi dei virus dell’influenza. • Variazione dell’architettura di domini in proteine di mammifero coinvolte nel traffico vescicolare: la proteina umana VAMP7b è la più interessante tra quelle prodotte per splicing alternativo del gene SYBL1. La produzione di VAMP7b è causata dal salto dell’esone 6 che si traduce in uno slittamento della sequenza codificante. Abbiamo scoperto che questo evento è conservato in altre specie di mammiferi. VAMP7b condivide con l’isoforma principale il dominio inibitorio longin N-terminale e la prima metà dello SNARE motif. Nei mammiferi, VAMP7b è una proteina tronca in cui al C-terminale metà dello SNARE motif e la regione transmembrana sono sostituite da peptidi corti e variabili. È molto interessante notare come negli uomini e nelle scimmie antropomorfe lo slittamento della regione codificante determinato dal salto dell’esone 6 abbia prodotto un nuovo dominio di funzione sconosciuta: proprio per questo VAMP7b umana non è tronca, ma addirittura 40 residui più lunga rispetto all’isoforma principale. Dal momento che l’esistenza di questa isoforma “lunga” ed il suo nuovo dominio sono stati confermati a livello proteico grazie all’ausilio di specifici anticorpi, abbiamo effettuato una dissezione in silico del nuovo dominio adoperando un’analisi di sequenza di tipo matrice posizione-specifica (PSI-BLAST), seguita da da Modellistica Strutturale di tipo ab initio. In più, dal momento che la regione N-terminale dello SNARE motif è conservata ed è nota nel mediare il legame intramolecolare al dominio Longin, abbiamo appurato la conservazione della conformazione chiusa sia in vivo (saggio del doppio ibrido in lievito) che in vitro (analisi NMR). Inoltre, la localizzazione subcellulare (SCL) di VAMP7b e Ykt6b è stata studiata adoperando chimere contenenti GFP e RFP. Non ultimo, le isoforme b dei geni longin sono stati analizzati tramite qPCR e si è scoperto essere regolate durante lo sviluppo. • Motif di legame con azione regolatoria sulla crescita e l’indirizzamento neuronale: La regolazione fine delle interazioni proteina-proteina che avviene grazie alle variazioni nell’architettura dei domini o dal cambiamento di motif locali indotto dalla modulazione di caratteristiche di superficie, è in grado di regolare i percorsi di segnalazione sia a livello intra- che extracellulare. Le interazioni proteina-proteina extracellulari possono giocare un ruolo fondamentale nel riconoscimento eterologo (es. ospite-patogeno) come nella segnalazione omologa tra cellule appartenenti allo stesso organismo. Le proteine esposte in membrana plasmatica (PM) possono interagire le une con le altre e con la matrice extracellulare (ECM) per consentire informazioni posizionali e segnali di indirizzamento. Le molecole di adesione cellulare (CAMs) sono proteine della membrana plasmatica in grado di mediare segnali sia di natura attrattiva che repulsiva grazie ad interazioni omo- ed eterofiliche a carico dei loro domini extracellulari (EDs). Questi ultimi sono composti per la magior parte da domini ripetuti aventi fold di tipo Ig o Fibronectina di tipo III. Le attuali conoscenze suggeriscono che i 4 domini extracellulari N-terminali di tipo Ig siano importanti nelle interazioni omo- o eterofiliche ed in modo particolare il dominio Ig2 è provvisto di un importante motif di interazione. Nel nostro laboratorio abbiamo sviluppato dei peptidi biomimetici che riproducono i motif di interazione conosciuti o predetti appartenenti al dominio Ig2 di L1CAM umana e al singolo dominio Ig di LINGO1 umana, proteine, queste, che giocano un ruolo fondamentale nella crescita, nell’indirizzamento e nel differenziamento neuronale. Sulla base della conservazione strutturale della regione del motif (anche tra proteine con architetture molto diverse dei loro EDs), abbiamo iniziato a studiarne la variazione di sequenza mediante analisi per omologia e per espressioni regolari, per infine tornare al livello strutturale mediante Modellistica Molecolare. I risultati preliminari indicano una forte conservazione dell’Arginina centrale presente nel motif d’interazione, mentre nelle altre posizioni del motif si osserva la conservazione di proprietà dei residui piuttosto che la presenza di specifici residui. Questa evidenza è in accordo con il dato di fatto che la mutazione dell’Arginina in L1CAM è responsabile di un serio disordine neurologico, mentre mutazioni a carico di altri residui del motif causano un fenotipo meno grave. Questo suggerisce che il motif è un epitopo posizionalmente conservato attorno all’Arginina centrale in grado di consentire una variabilità limitata ma significativa nella sequenza circostante. Per verificare quest’ipotesi è stata effettuata una superimposizione strutturale dei domini Ig contenenti il motif d’interazione: il risultato ha confermato che il peptide contenente il motif è di per sé conservato posizionalmente e che la conservazione maggiore sia a livello posizionale che struturale è a carico del residuo centrale di Arginina. Esperimenti con peptidi mutati nell’Arginina centrale hanno dimostrato un’attività in termini di segnalazione nella neuritogenesi. Questi lavori hanno consentito di sviluppare un protocollo bioinformatico per la caratterizzazione di determinanti d’interazione e della loro modulazione funzionale, facilmente trasportabile su altre proteine.

Decreased ACE activity is a common finding in patients with adult respiratory distress syndrome (ARDS) and in animal models of lung injury. The nature of this effect is unknown. Fibrin, also a common finding in lung injury, is degraded to small peptides by proteolytic enzymes. Peptide 6A, corresponding to amino acid residues 43-47 of the BB-chain of fibrin (cgen), is produced by plasmin degradation of fibrin and has been shown to inhibit ACE in vitro. The purpose of the present investigation was to study whether peptide 6A inhibits pulmonary ACE in vivo and, if so, determine its effect of hemodynamic changes induced by bradykinin (BK) in the rabbit. We investigated the effect of peptide 6A an the hydrolysis of a synthetic ACE substrate, benzoyl-phe-ala-pro (BPAP) in anesthetized rabbits and in isolated lungs. Peptide 6A caused a reversible, dose-dependent inhibition of BPAP hydrolysis. The ID 50 for peptide 6A inhibition of ACE hydrolysis of BPAP was approximately 1 micrcmole.In both isolated rabbit lupgp and in the intact animal, BK injection elicited a dose dependent increase in pulmonary arterial pressure. In intact animals, this was accompanied by a dose dependent decrease in systemic arterial pressure.In both preparations, responses to BK were potentiated by addition of 1 micromole of peptide 6A. In isolated lungs, co-injection of peptide 6A significantly increased the pulmonary artery pressure response to every dose of BK except the highest (1 mg). In the anesthetized rabbit, 1 micromole of peptide 6A significantly (p<0.05) increased the pulmonary hypertensive response to 100 and 200 nanograms of BK and significantly (p<0.05) decreased the systemic hypotensive response following 100 , 200 , 300 and 400 nanograms of BK. The amount of BK needed to increase pulmonary arterial pressure was 1000-fold greater in the isolated lungs than in the intact animals. Peptides of this type might contribute to decreased ACE activity in patients with ARDS and may potentiate BK induced pulmonary hypertension and systemic hypotension in these patients.

Leukocyte elastase (EL) cleaves peptides from the N-terminal fibrinogen (fbg) Aα and B6 chains. The Aα peptide (Aα 1-21) and the as yet unidentified BIB peptide contain thrombin (TB)cleavage sites. As we were interested in fbg proteolysis in sepsis and leukemia, we have tried to measure these peptides (generated by incubating a crude granulocyte extract with fbg) using available fibrinopeptide A (FPA) - und Bß 15-42-antigen-RIA techniques. As the indirect measurement of Aα 1-21 in terms of FPA releasable by TB treatment in vitro (TB-inducible FPA, TIFPA) requires the use of a highly specific anti-FPA-antiserum to avoid cross reaction of Aα 1-21 we tested the specificity of several antisera. Only R2, provided by Dr. J. Owen, proved suitable whereas two commercial antisera measured 6-15% of Aα 1-21 as apparent FPA. Simultaneous recovery of FPA and Aα 1-21 from plasma using R2 was between 80-100% after precipitation of cross-reacting fbg by ethanol, whereas bentonite adsorbed Aα 1-21 and the EL induced BIB peptide. TIFPA was fully recovered after incubation with plasma (37°C, 2 hr) whereas cross-reactivity of Aα 1-21 in the FPA-assay did not increase. EL-induced proteolysis in plasma in terms of TIFPA generation did not occur unless the normal granulocyte-plasma ratio was increased about 300-400 fold. The Bß 15-42 antigen RIA allowed quantitative measurement of the as yet undefined EL-induced Bß peptide, since the "Bß 15-42" concentration measured was equal to the amount of FPB (measured as Bß 1-13) releasable from the EL-induced BIB-peptide by TB. The immunoreactivity of this peptide is stable in plasma and completely recovered after ethanol precipitation. This finding suggests that the EL-induced BIB peptide is longer than Bß 1-4 2 and not susceptible to C-terminal degradation of the Bß41/42 region which is crucial for recognition of the peptide by the antiserum (supported by Deutsche Forschungs-gemeinschaft).

Surgical site infections and chronic wounds, especially those caused by antibiotic-resistant microorganisms, result in hospitalization and fatalities each year. Methods to prevent these infections, such as cleaning and preparing medical tools, have had minimal success in preventing infections. Further, antibiotic treatments have become less successful in treating infections and wounds as a result of antibiotic-resistant bacteria. Antimicrobial peptides (AMP) are a possible treatment solution. AMPs are oligopeptides that occur in nature or can be synthesized in vitro which possess a broad spectrum of antimicrobial activity against bacteria and other harmful microorganisms. AMPs operate by disrupting the packing arrangements of biomembranes in prokaryotes through their insertion into negatively charged phospholipid bilayers. However, many AMP products have failed clinical trials because of their difficulty to be delivered at high concentrations in an active form. This project proposes the use of bicontinuous microemulsions (BMEs) to encapsulate and deliver AMPs. BMEs are thermodynamically stable monophasic solutions consisting of surfactant, oil, aqueous media, and sometimes a cosurfactant. They are optically clear and consist of surfactant monolayers that separate oil and water nanodomains. Several different BME systems composed of biocompatible oils such as isopropyl myristate and limonene were evaluated for their ability to encapsulate melittin, a model AMP, and to test the system’s antimicrobial activity. AMPs are typically cationic, and the following hypothesis is being tested, that BMEs created with anionic surfactants would induce a more highly folded, hence more biologically active, conformation for melittin. We are currently measuring the antimicrobial activity of BME-encapsulated melittin against several prominent bacteria that are present in chronic wounds and surgical site infections through multiple antimicrobial assays. We will also assess the structure and microenvironment of melittin using circular dichroism and fluorescence spectroscopy, respectively, and the impact of melittin on the structure of BMEs through small-angle X-ray scattering.

The molecular basis of platelet-fibrin interactions has been investigated by using synthetic peptides as potential inhibitors of binding fibrin protofibrils and fibrinogen to ADP-stimulated platelets, adhesion of fibrin fibers to the platelet surface, and platelet-mediated clot retraction. Synthetic peptides RGDS and HHLGGAKQAGDV, corresponding to regions of the fibrinogen α and γ chains previously identified as platelet recognition sites, inhibited the binding of radiolabelled soluble fibrin oligomers to ADP-stimulated platelets with IC50 values of 12 and 40 μM, respectively. The IC50 values obtained with fibrinogen as the ligand were 3-fold higher. Synthetic GPRP and GHRP, corresponding to the N-terminal sequences of the fibrin α and β chains, were minimally effective in blocking soluble fibrin oligomer binding to ADP-stimulated platelets. The extent of fibrin:platelet adhesion was determined with a microfluorimetric technique which measures the quantity of fluorescein-labelled fibrin attached to the surface of platelets. The signal obtained from the brightly fluorescent platelet:fibrin adducts was time- and concentration-dependent, and was fully inhibited by a monoclonal antibody directed against the glycoprotein II:IIIa complex (HP1-1D, kindly provided by Dr. W. Nichols). Inhibition of fibrin:platelet adhesion by RGDS, HHGGAKQAGDV, and GHRP all exhibited a similar, linear dependence on the peptide concentration, reaching 1/2 maximum at about 200 μM, suggesting nonspecific effects. GPRP inhibited fibrin assembly but did not appear to have specific effects on fibrin:platelet adhesion. The time course of clot retraction was followed by right angle light scattering intensity measurements. Only RGDS affected clot retraction, causing a 4-fold decrease in rate at 230 μM. These results indicate that fibrinogen and fibrin protofibrils, which are obligatory intermediates in the fibrin assembly pathway, share a set of common platelet recognition sites located at specific regions of the α and γ chains of the multinodular fibrin(ogen) molecules. The RGDS site is also involved in mediating interactions between the three dimensional fibrin network and ADP-stimulated platelets.

Previous studies from our laboratory (J. Biol. Chem. 260:10714,1985; J. Clin. Invest. 78:1631,1986) provide evidence that a monoclonal antibody (3C1) directed against the heavy chain region of factor XIa (FXIa) recognizes an epitope near a substrate binding site for FIX and a binding site for high molecular weight kininogen (HMWK). The present studies were carried out to determine whether these two sites are identical or different. Another heavy-chain-specific murine monoclonal antibody (5F7) was found to recognize an epitope distinct from that recognized by 3C1 since 3C1 did not compete with 5F7 for binding to FXI in a solid-phase radioimmunoassay. Antibody 3C1 was a competitive inhibitor of F-XIa-catalyzed F-IX activation, assayed by the release of a 3H-labeled activation peptide from FIX, whereas 5F7 had no effect on F—IX activation by FXIa. In contrast, 5F7 (which also inhibited F-XIIa-catalyzed F-XI activation in the presence of HMWK and kaolin) completely blocked FXI binding to immobilized HMWK at concentrations 1,000-fold lower than 3C1. Finally, HMWK had no effect on F-IX activation by FXIa. We therefore conclude that two separate and distinct domains are present in the heavy-chain region of FXI, one of which is a substrate binding site for FIX and the other a binding site for HMWK. A 15,000 Mr peptide containing the HMWK binding site was isolated using cyanogen bromide digests of factor XI which were bound to and eluted from a ,5F7 antibody affinity column and further purified using high performance liquid chromatography. Gas phase sequencing studies are in progress to characterize this peptide and place its sequence within the known structure of the heavy chain of FXIa. In conclusion, our antibodies have defined two domains within the heavy chain region of FXI: one defined by 5F7 is near the HMWK binding site, whereas the other, recognized by 3C1, is a substrate binding site for FIX. Finally, a peptide domain in the heavy chain of FXI that compriaes the HMWK binding site has been identified and isolated.

Human neutrophil elastase (HNE) cleaves the Aα21-22 bond of fibrinogen thus releasing the fibrinopeptide A (FPA)-containing fragment Aαl-21. Plasma Aal-21 levels reflect in vivo HNE activity and peptide levels are increased in cigarette smokers and patients with chronic lung disease. To further explore the HNE-fibrinogen interaction, we set out to develop an animal model. The digestion of purified baboon and marmoset fibrinogen by human thrombin, HNE and extracts of baboon and marmoset neutrophils was monitored with a specific radioimmunoassay for human FPA. Thrcmbin produced quantitative release (2 mol/mol fibrinogen) of FPA. In contrast, HNE and the neutrophil extracts did not release FPA, but rather, produced quantitative release of a larger, FPA-containing fragment. Immunochemically, this fragment was clearly distinguishable from FPA in that in vitro thrombin treatment increased its immunoreactivity 1,000-fold (thrombin increasable FPA or TIFPA). TIFPA release by the neutrophil extracts was blocked by α1-proteinase inhibitor, a specific HNE inhibitor (MeO-Suc-Ala2-Pro-ValCH2Cl) and an anti-HNE IgG, indicating that elastase was the responsible proteinase and that there was homology between the human and primate enzymes. The products of HNE and neutrophil extract proteolysis of the primate fibrinogens were then separated by high performance liquid chromatography and the TIFPA-containing fractions were subjected to amino acid sequence analysis. The FPA-containing fragments each consisted of 21 amino acids, had minor substitutions when compared with human A α] -21 [Baboon: Aα(3) Ser - Thr; Marmoset Aα(l) Ala - Thr, Aα(3) Ser - Thr, Aα(ll) Glu - Ala], and exhibited complete crossreactivity with the human peptide. Using the TIFPA assay, there was good recovery of primate or human Aαl-21 added to primate blood and the mean peptide level in 8 healthy marmosets was similar to that in man (0.5 nM and 0.4 nM, respectively). In conclusion, (1) the Aα;21 -22 bond of baboon and marmoset fibrinogen is a cleavage site for human and primate elastase, (2) baboon and marmoset Aal-21 can be measured with the assay for the human peptide, and (3) the primate serves as a useful model for the study of elastase-fibrinogen interactions.

Trigramin (Mr weight 10 kDa), an acidic, cysteine rich peptide purified into homogeneity from Trimeresurus gramineus snake venom contained a single protein chain with EAGE at the NH2 terminal end. It inhibited platelet aggregation induced by various agents without affecting release reaction. It blocked competitively the binding of 125I-fibrincgen to AEP stimulated and chymotrypsin treated platelets (Ki= 2 × 10−8). 125-I trigramin bound to intact and to AEP stimulated platelets in a saturable manner (approx. 16,000 sites per platelet). However, AEP increased 5 fold, the binding affinity of trigramin to platelets (to Kd = 4 × 10−8M) suggesting that AEP is changing the conformation of receptors associated with GPIIb/GPIIIa complex. The binding of trigramin to thran-basthenic platelets was markedly reduced. The binding to normal platelets was significantly inhibited by EDTA and by monoclonal antibodies directed against GPIIb/GPIIIa complex but not by the antibodies directed against GPIIb or GPIIIa molecules. The binding of 125I-trigramin to AEP-stimulated platelets was inhibited by RGES (IC50 = 125 μM) and by YHHLGGAKOAGDV (C-terminal fragment of fibrinogen gamma chain, IC50 = 250 μM) suggesting that these or similar peptide sequences are required for interactions of various ligands with GPIIb/GPIIIa complex

Active site-directed inactivation of a serine protease with a thioester derivative of a peptide-chloromethyl ketone followed by reaction of the unique thiol group generatedin the presence of hydroxylamine with a fluorophore-iodoacetamide hasbeen investigated as a new method for covalent incorporation of extrinsic fluorescence probes into the active sites of blood coagulation proteases. The specificity of labeling by this method was evaluated by quantitation of the reactions between human thrombin, acetylthioacetyl-D-Phe-Pro-ArgCH2Cl (ATA-FPRCK) and 5-iodoacetamidofluorescein(IAF).ATA-FPRCK was synthesized by reaction of FPRCK with succinimidylacetylthioacetate and purified by chromatography on SP-Sephadex and Sephadex G10. Titrations of the loss of thrombin chromogenic substrate activity with ATA-FPRCK were linear, with end points of 1.1-1.2 mol ATA-FPRCK added/mol active sites, consistent with a reaction stoichiometry of 1 and the ∽90% purity of the compound estimated by reverse-phase HPLC.Inactivation of thrombin wasquantitatively correlated with incorporation of the thioester, with a maximum of 1.04 mol/mol active sites.IAF labeling of ATA-FPR-thrombin inthe presence of 0.1M NH20H yielded a maximu of 0.96 mol IAF incorporated/mol active sites in a reaction accompanied by loss of the thiol group. Incorporation of ATA-FPRCK wasdependent on thefunctional thrombin active site, asdemonstrated by less than 4%thioester or IAF incorporation for the enzyme previously inactivated with FPRCK. I conclude that active site-selective fluorescence labelingcan be achieved by the method described here with the advantage of a wide choice in the properties of theprobe incorporated. In addition, a 2.3-fold difference in fluorescenceintensity was observed for 2,6-ANS derivatives of ATA-FPR-thrombin andATA-D-Phe-Phe-Arg-thrombin, indicating that the spectral properties ofenvironmentally sensitive fluorescence probes are influenced by the structure of the peptide inhibitor.Supported in part by a grant from the American National Red Cross.

This paper presents an efficient and novel computational protein prediction methodology called Kineto-Static Compliance Method. Successive Kineto-Static Fold Compliance is a methodology for predicting a protein molecule’s motion under the effect of an inter-atomic force field without the need for molecular dynamic simulation. Instead, the chain complies under the Kineto-Static effect of the force field in such a manner that each rotatable joint changes by an amount proportional to the effective torque on that joint. This process successively iterates until all of the joint torques have converged to zero. This configuration is equivalent to a stable, globally optimized potential energy state of the system or, in other words, the final conformation of the protein. This methodology is implemented in a computer software package named ProtoFold. In this paper, we have used Protofold to predict the final conformation of a small peptide chain segment, an alpha helix, and the Triponin protein chains from a denatured configuration. The results show that torques in each joint are minimized to values very close to zero, which demonstrates the method’s effectiveness for protein conformation prediction.

Cationic polypeptide proteins found in the seeds of the tropical plant Moringa oleifera have coagulation efficiencies similar to aluminum and ferric sulfates without their recalcitrant nature. Although these proteins possess great potential to augment or replace traditional coagulants in water treatment, harvesting active protein from seeds is laborious and not cost-effective. Here, we describe an alternative method to express and secrete active M. oleifera coagulant protein (MO) in Bacillus subtilis. A plasmid library containing the MO gene and 173 different types of secretory signal peptides was created and cloned into B. subtilis strain RIK1285. Fourteen of 440 clones screened were capable of secreting MO with yields ranging from 55 to 122 mg/L of growth medium. The coagulant activity of the highest MO secreting clone was evaluated when grown on Luria broth, and cell-free medium from the culture was shown to reduce turbidity in a buffered kaolin suspension by approximately 90% compared with controls without the MO gene. The clone was also capable of secreting active MO when grown on a defined synthetic wastewater supplemented with 0.5% tryptone. Cell-free medium from the strain harboring the MO gene demonstrated more than a 2-fold reduction in turbidity compared with controls. Additionally, no significant amount of MO was observed without the addition of the synthetic wastewater, suggesting that it served as a source of nutrients for the effective expression and translocation of MO into the medium.

Original Objectives: 1. Purify and biochemically characterize RB60 orthologs in higher plant chloroplasts; 2. Clone the gene(s) encoding plant RB60 orthologs and determine their structure and expression; 3. Manipulate the expression of RB60; 4. Assay the effects of altered RB60 expression on thylakoid biogenesis and photosynthetic function in plants exposed to different light conditions. In addition, we also examined the gene structure and expression of RB60 orthologs in the non-vascular plant, Physcomitrella patens and cloned the poly(A)-binding protein orthologue (43 kDa RB47-like protein). This protein is believed to a partner that interacts with RB60 to bind to the psbA5' UTR. Thus, to obtain a comprehensive view of RB60 function requires analysis of its biochemical partners such as RB43. Background & Achievements: High levels of sunlight reduce photosynthesis in plants by damaging the photo system II reaction center (PSII) subunits, such as D1 (encoded by the chloroplast tpsbAgene). When the rate of D1 synthesis is less than the rate of photo damage, photo inhibition occurs and plant growth is decreased. Plants use light-activated translation and enhanced psbAmRNA stability to maintain D1 synthesis and replace the photo damaged 01. Despite the importance to photosynthetic capacity, these mechanisms are poorly understood in plants. One intriguing model derived from the algal chloroplast system, Chlamydomonas, implicates the role of three proteins (RB60, RB47, RB38) that bind to the psbAmRNA 5' untranslated leader (5' UTR) in the light to activate translation or enhance mRNA stability. RB60 is the key enzyme, protein D1sulfide isomerase (Pill), that regulates the psbA-RN :Binding proteins (RB's) by way of light-mediated redox potentials generated by the photosystems. However, proteins with these functions have not been described from higher plants. We provided compelling evidence for the existence of RB60, RB47 and RB38 orthologs in the vascular plant, Arabidopsis. Using gel mobility shift, Rnase protection and UV-crosslinking assays, we have shown that a dithiol redox mechanism which resembles a Pill (RB60) activity regulates the interaction of 43- and 30-kDa proteins with a thermolabile stem-loop in the 5' UTR of the psbAmRNA from Arabidopsis. We discovered, in Arabidopsis, the PD1 gene family consists of II members that differ in polypeptide length from 361 to 566 amino acids, presence of signal peptides, KDEL motifs, and the number and positions of thioredoxin domains. PD1's catalyze the reversible formation an disomerization of disulfide bonds necessary for the proper folding, assembly, activity, and secretion of numerous enzymes and structural proteins. PD1's have also evolved novel cellular redox functions, as single enzymes and as subunits of protein complexes in organelles. We provide evidence that at least one Pill is localized to the chloroplast. We have used PDI-specific polyclonal and monoclonal antisera to characterize the PD1 (55 kDa) in the chloroplast that is unevenly distributed between the stroma and pellet (containing membranes, DNA, polysomes, starch), being three-fold more abundant in the pellet phase. PD1-55 levels increase with light intensity and it assembles into a high molecular weight complex of ~230 kDa as determined on native blue gels. In vitro translation of all 11 different Pill's followed by microsomal membrane processing reactions were used to differentiate among PD1's localized in the endoplasmic reticulum or other organelles. These results will provide.1e insights into redox regulatory mechanisms involved in adaptation of the photosynthetic apparatus to light stress. Elucidating the genetic mechanisms and factors regulating chloroplast photosynthetic genes is important for developing strategies to improve photosynthetic efficiency, crop productivity and adaptation to high light environments.