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Membrane proteins reside in lipid bilayers and are typically extracted from this environment for study, which often compromises their integrity. In this work, we ejected intact assemblies from membranes, without chemical disruption, and used mass spectrometry to define their composition. From Escherichia coli outer membranes, we identified a chaperone-porin association and lipid interactions in the b-barrel assembly machinery. We observed efflux pumps bridging inner and outer membranes, and from inner membranes we identified a pentameric pore of TonB, as well as the protein-conducting channel SecYEG in association with F1FO adenosine triphosphate (ATP) synthase. Intact mitochondrial membranes from Bos taurus yielded respiratory complexes and fatty acid–bound dimers of the ADP (adenosine diphosphate)/ATP translocase (ANT-1). These results highlight the importance of native membrane environments for retaining small-molecule binding, subunit interactions, and associated chaperones of the membrane proteome.

Three different tetraphenylalanine (FFFF) based peptides that differ at the N- and C-termini have been synthesized by using standard procedures to study their ability to form different nanoassemblies under a variety of conditions. The FFFF peptide assembles into nanotubes that show more structural imperfections at the surface than those formed by the diphenylalanine (FF) peptide under the same conditions. Periodic DFT calculations (M06L functional) were used to propose a model that consists of three FFFF molecules defining a ring through head-to-tail NH3 +¿-OOC interactions, which in turn stack to produce deformed channels with internal diameters between 12 and 16 Å. Depending on the experimental conditions used for the peptide incubation, N-fluorenylmethoxycarbonyl (Fmoc) protected FFFF self-assembles into a variety of polymorphs: ultra-thin nanoplates, fibrils, and star-like submicrometric aggregates. DFT calculations indicate that Fmoc-FFFF prefers a parallel rather than an antiparallel ß-sheet assembly. Finally, coexisting multiple assemblies (up to three) were observed for Fmoc-FFFF-OBzl (OBzl = benzyl ester), which incorporates aromatic protecting groups at the two peptide terminals. This unusual and noticeable feature is attributed to the fact that the assemblies obtained by combining the Fmoc and OBzl groups contained in the peptide are isoenergetic. Variety show! Three different tetraphenylalanine-based peptides that differ at the N- and C-termini have been synthesized by using standard procedures to study their ability to form different nanoassemblies (e.g., nanotubes, see figure) under a variety of conditions., Peer Reviewed, Postprint (author's final draft)

This short-term innovative research proposal was focused on the structural characterization of peptide-based, self-assembled nanostructures of specific dimensions that are controlled by the geometry and physicochemical properties of the assembly interface. Our objectives were to (i) explore solution conditions that allow the production of nanostructures of select structural properties, specifically via the use of gradient hydrophobic interfaces, and (ii) characterize the conformation, fibril formation, and network properties of these peptide-based materials via a suite of circular dichroic spectroscopy (CD), oscillatory rheology, microscopy (AFM, SEM, TEM), and scattering (SANS) methods. We have successfully achieved the aims of this STIR project, and have clearly demonstrated that (i) beta-hairpin structures can be formed by peptides with gradient hydrophobic faces comprising non-natural amino acids, (ii) these peptides are competent for fibril formation, and (iii) fibril formation and branching (as indicated by resulting network mechanical properties) can be modulated by modifications of the gradient of the hydrophobic interface. Loose packing of the designed fibrils is indicated, on the basis of initial SANS analysis, suggesting opportunities to modify the fibril interface with a range of chemically and electronically diverse hydrophobic amino acids, which will provide new opportunities to make smart materials for in-situ sensing and device fabrication., The original document contains color images.

The phosphatidylethanolamine binding proteins (pebps) are an evolutionarily conserved family of proteins recently implicated in mitogen-activated protein (MAP) kinase pathway regulation, where they are called raf kinase inhibitory proteins. Here, we describe the cloning, cellular localization, and partial characterization of a new member, pebp-2, with potential roles in male fertility. Expression data show that pebp-2 is a testis-specific 21-kDa protein found within late meiotic and haploid germ cells in a stage-specific pattern that is temporally distinct from that of pebp-1. Sequence analyses suggest that pebp-2 forms a distinct subset of the pebp family within mammals. Database analyses revealed the existence of a third subset. Analysis suggests that the specificity/regulation of the distinct pebps subsets is likely to be determined by the amino terminal 40 amino acids or the 3' untranslated region, where the majority of sequence differences occur. Protein homology modeling suggests that pebp-2 protein is, however, topologically similar to other pebps and composed of Greek key fold motifs, a dominant beta sheet formed from five anti-parallel beta strands forming a shallow groove associated with a putative phosphatidylethanolamine binding site. The pebp-2 gene is intronless and data suggest that it is a retrogene derived from pebp-1. Further, pebp-2 colocalizes with members of the MAP kinase pathway in late spermatocytes and spermatids and on the midpiece of epididymal sperm. These data raise the possibility that pebp-2 is a novel participant in the MAP kinase signaling pathway, with a role in spermatogenesis or posttesticular sperm maturation.

Activins are involved in many physiological and pathological processes and, like other members of the transforming growth factor-beta superfamily, signal via type II and I receptor serine kinases. Ligand residues involved in type II receptor binding are located in the two anti-parallel beta strands of the TGF-beta proteins, also known as the fingers. Activin-A mutants able to bind ActRII but unable to bind the activin type I receptor ALK4 define ligand residues involved in ALK4 binding and could potentially act as antagonists. Therefore, a series of FLAG-tagged activin-A/C chimeras were constructed, in each of which eight residues in the wrist loop and helix region (A/C 46-53, 54-61, 62-69, and 70-78) were replaced. Additionally, a chimera was generated in which the entire wrist region (A/C 46-78) was changed from activin-A to activin-C. The chimeras were assessed for ActRII binding, activin bioactivity, as well as antagonistic properties. All five chimeras retained high affinity for mouse ActRII. Of these, only A/C 46-78 was devoid of significant activin bioactivity in an A3 Lux reporter assay in 293T cells at concentrations up to 40 nM. A/C 46-53, 54-61, 62-69, and 70-78 showed activity comparable with wild type activin-A. When tested for the ability to antagonize ligands that signal via activin type II receptors, such as activin-A and myostatin, only the A/C 46-78 chimera showed antagonism (IC50, 1-10 nM). Additionally, A/C 46-78 decreased follicle-stimulating hormone release from the LbetaT2 cell line and rat anterior pituitary cells in primary culture in a concentration-dependent manner. These data indicate that activin residues in the wrist are involved in ALK4-mediated signaling. The activin antagonist A/C 46-78 may be useful for the study and modulation of activin-dependent processes. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

The phosphatidylethanolamine binding proteins (pebps) are an evolutionarily conserved family of proteins recently implicated in mitogen-activated protein (MAP) kinase pathway regulation, where they are called raf kinase inhibitory proteins. Here, we describe the cloning, cellular localization, and partial characterization of a new member, pebp-2, with potential roles in male fertility. Expression data show that pebp-2 is a testis-specific 21-kDa protein found within late meiotic and haploid germ cells in a stage-specific pattern that is temporally distinct from that of pebp-1. Sequence analyses suggest that pebp-2 forms a distinct subset of the pebp family within mammals. Database analyses revealed the existence of a third subset. Analysis suggests that the specificity/regulation of the distinct pebps subsets is likely to be determined by the amino terminal 40 amino acids or the 3' untranslated region, where the majority of sequence differences occur. Protein homology modeling suggests that pebp-2 protein is, however, topologically similar to other pebps and composed of Greek key fold motifs, a dominant beta sheet formed from five anti-parallel beta strands forming a shallow groove associated with a putative phosphatidylethanolamine binding site. The pebp-2 gene is intronless and data suggest that it is a retrogene derived from pebp-1. Further, pebp-2 colocalizes with members of the MAP kinase pathway in late spermatocytes and spermatids and on the midpiece of epididymal sperm. These data raise the possibility that pebp-2 is a novel participant in the MAP kinase signaling pathway, with a role in spermatogenesis or posttesticular sperm maturation.

Activins are involved in many physiological and pathological processes and, like other members of the transforming growth factor-beta superfamily, signal via type II and I receptor serine kinases. Ligand residues involved in type II receptor binding are located in the two anti-parallel beta strands of the TGF-beta proteins, also known as the fingers. Activin-A mutants able to bind ActRII but unable to bind the activin type I receptor ALK4 define ligand residues involved in ALK4 binding and could potentially act as antagonists. Therefore, a series of FLAG-tagged activin-A/C chimeras were constructed, in each of which eight residues in the wrist loop and helix region (A/C 46-53, 54-61, 62-69, and 70-78) were replaced. Additionally, a chimera was generated in which the entire wrist region (A/C 46-78) was changed from activin-A to activin-C. The chimeras were assessed for ActRII binding, activin bioactivity, as well as antagonistic properties. All five chimeras retained high affinity for mouse ActRII. Of these, only A/C 46-78 was devoid of significant activin bioactivity in an A3 Lux reporter assay in 293T cells at concentrations up to 40 nM. A/C 46-53, 54-61, 62-69, and 70-78 showed activity comparable with wild type activin-A. When tested for the ability to antagonize ligands that signal via activin type II receptors, such as activin-A and myostatin, only the A/C 46-78 chimera showed antagonism (IC50, 1-10 nM). Additionally, A/C 46-78 decreased follicle-stimulating hormone release from the LbetaT2 cell line and rat anterior pituitary cells in primary culture in a concentration-dependent manner. These data indicate that activin residues in the wrist are involved in ALK4-mediated signaling. The activin antagonist A/C 46-78 may be useful for the study and modulation of activin-dependent processes. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

Cells respond profoundly to the mechanical rigidity and three-dimensional nanotopology of substrates, as well as the spatial and temporal arrangements of extracellular cues. We summarize the latest developments in probing and engineering biocompatible nanofibrous extracellular environments at the cell and molecular level for applications in tissue engineering and biological research. This will, in turn, guide further development of three-dimensional nanofibrous scaffolds in order to elicit specific cellular responses for relevant applications. © 2006 Elsevier Ltd. All rights reserved.

Basic molecular structural principles of biological materials. Molecular structures of various materials of biological origin, including collagen, silk, bone, protein adhesives, GFP, self-assembling peptides. Molecular design of new biological materials for nanotechnology, biocomputing and regenerative medicine. Graduate students are expected to complete additional coursework. Description from course home page: This course, intended for both graduate and upper level undergraduate students, will focus on understanding of the basic molecular structural principles of biological materials. It will address the molecular structures of various materials of biological origin, such as several types of collagen, silk, spider silk, wool, hair, bones, shells, protein adhesives, GFP, and self-assembling peptides. It will also address molecular design of new biological materials applying the molecular structural principles. The long-term goal of this course is to teach molecular design of new biological materials for a broad range of applications. A brief history of biological materials and its future perspective as well as its impact to the society will also be discussed. Several experts will be invited to give guest lectures.

Basic molecular structural principles of biological materials. Molecular structures of various materials of biological origin, including collagen, silk, bone, protein adhesives, GFP, self-assembling peptides. Molecular design of new biological materials for nanotechnology, biocomputing and regenerative medicine. Graduate students are expected to complete additional coursework. Description from course home page: This course, intended for both graduate and upper level undergraduate students, will focus on understanding of the basic molecular structural principles of biological materials. It will address the molecular structures of various materials of biological origin, such as several types of collagen, silk, spider silk, wool, hair, bones, shells, protein adhesives, GFP, and self-assembling peptides. It will also address molecular design of new biological materials applying the molecular structural principles. The long-term goal of this course is to teach molecular design of new biological materials for a broad range of applications. A brief history of biological materials and its future perspective as well as its impact to the society will also be discussed. Several experts will be invited to give guest lectures.

Epoxide hydrolases are essential for the processing of epoxide-containing compounds in detoxification or metabolism. The classic epoxide hydrolases have an α/β hydrolase fold and act via a two-step reaction mechanism including an enzyme-substrate intermediate. We report here the structure of the limonene-1,2-epoxide hydrolase from Rhodococcus erythropolis, solved using single-wavelength anomalous dispersion from a selenomethionine-substituted protein and refined at 1.2 Å resolution. This enzyme represents a completely different structure and a novel one-step mechanism. The fold features a highly curved six-stranded mixed β-sheet, with four α-helices packed onto it to create a deep pocket. Although most residues lining this pocket are hydrophobic, a cluster of polar groups, including an Asp-Arg-Asp triad, interact at its deepest point. Site-directed mutagenesis supports the conclusion that this is the active site. Further, a 1.7 Å resolution structure shows the inhibitor valpromide bound at this position, with its polar atoms interacting directly with the residues of the triad. We suggest that several bacterial proteins of currently unknown function will share this structure and, in some cases, catalytic properties.

Cílem této práce bylo seznámit se s prostorovým uspořádáním molekul proteinů, s jejich získáváním, způsoby zápisu a veličinami sloužícími k jejich popisu. Predikce proteinových struktur je velice důležitá pro zjištění funkce budoucích proteinů. Dalším cílem bylo vytvoření funkce v prostředí MATLAB, která bude graficky reprezentovat prostorové uspořádání páteřní struktury proteinu formou Ramachandranova diagramu. Společně s touto funkcí vytvořit další funkci k vykreslení postraních řetězců aminokyselin a funkci k výpočtu stereochemické kvality. Nakonec vytvořit program pro kompletní vyhodnocení prostorového uspořádání molekuly proteinu a výpočtu stereochemické kvality a otestování programu na proteinových strukturách z veřejné databáze RSCB PDB., Objective of this work was to study the spatial arrangement of protein molecules, with their extraction, notations and variables used in the course descriptions. Prediction of protein structures is very important for determining the future function of proteins. Another objective was to create a function in MATLAB that will graphically represent the spatial arrangement of the backbone structure of the protein by form Ramachandran plot. Along with this function create another function to render the side-chains of amino acids and function to calculate the stereochemical quality. Finally, create a program for a complete assessment of the spatial arrangement of the protein molecules and calculating the stereochemical quality of and test program for protein structures from public databases RSCB PDB.

Cílem této práce bylo seznámit se s prostorovým uspořádáním molekul proteinů, s jejich získáváním, způsoby zápisu a veličinami sloužícími k jejich popisu. Predikce proteinových struktur je velice důležitá pro zjištění funkce budoucích proteinů. Dalším cílem bylo vytvoření funkce v prostředí MATLAB, která bude graficky reprezentovat prostorové uspořádání páteřní struktury proteinu formou Ramachandranova diagramu. Společně s touto funkcí vytvořit další funkci k vykreslení postraních řetězců aminokyselin a funkci k výpočtu stereochemické kvality. Nakonec vytvořit program pro kompletní vyhodnocení prostorového uspořádání molekuly proteinu a výpočtu stereochemické kvality a otestování programu na proteinových strukturách z veřejné databáze RSCB PDB., Objective of this work was to study the spatial arrangement of protein molecules, with their extraction, notations and variables used in the course descriptions. Prediction of protein structures is very important for determining the future function of proteins. Another objective was to create a function in MATLAB that will graphically represent the spatial arrangement of the backbone structure of the protein by form Ramachandran plot. Along with this function create another function to render the side-chains of amino acids and function to calculate the stereochemical quality. Finally, create a program for a complete assessment of the spatial arrangement of the protein molecules and calculating the stereochemical quality of and test program for protein structures from public databases RSCB PDB.

Cílem této práce bylo seznámit se s prostorovým uspořádáním molekul proteinů, s jejich získáváním, způsoby zápisu a veličinami sloužícími k jejich popisu. Predikce proteinových struktur je velice důležitá pro zjištění funkce budoucích proteinů. Dalším cílem bylo vytvoření funkce v prostředí MATLAB, která bude graficky reprezentovat prostorové uspořádání páteřní struktury proteinu formou Ramachandranova diagramu. Společně s touto funkcí vytvořit další funkci k vykreslení postraních řetězců aminokyselin a funkci k výpočtu stereochemické kvality. Nakonec vytvořit program pro kompletní vyhodnocení prostorového uspořádání molekuly proteinu a výpočtu stereochemické kvality a otestování programu na proteinových strukturách z veřejné databáze RSCB PDB., Objective of this work was to study the spatial arrangement of protein molecules, with their extraction, notations and variables used in the course descriptions. Prediction of protein structures is very important for determining the future function of proteins. Another objective was to create a function in MATLAB that will graphically represent the spatial arrangement of the backbone structure of the protein by form Ramachandran plot. Along with this function create another function to render the side-chains of amino acids and function to calculate the stereochemical quality. Finally, create a program for a complete assessment of the spatial arrangement of the protein molecules and calculating the stereochemical quality of and test program for protein structures from public databases RSCB PDB.

Cílem této práce bylo seznámit se s prostorovým uspořádáním molekul proteinů, s jejich získáváním, způsoby zápisu a veličinami sloužícími k jejich popisu. Predikce proteinových struktur je velice důležitá pro zjištění funkce budoucích proteinů. Dalším cílem bylo vytvoření funkce v prostředí MATLAB, která bude graficky reprezentovat prostorové uspořádání páteřní struktury proteinu formou Ramachandranova diagramu. Společně s touto funkcí vytvořit další funkci k vykreslení postraních řetězců aminokyselin a funkci k výpočtu stereochemické kvality. Nakonec vytvořit program pro kompletní vyhodnocení prostorového uspořádání molekuly proteinu a výpočtu stereochemické kvality a otestování programu na proteinových strukturách z veřejné databáze RSCB PDB., Objective of this work was to study the spatial arrangement of protein molecules, with their extraction, notations and variables used in the course descriptions. Prediction of protein structures is very important for determining the future function of proteins. Another objective was to create a function in MATLAB that will graphically represent the spatial arrangement of the backbone structure of the protein by form Ramachandran plot. Along with this function create another function to render the side-chains of amino acids and function to calculate the stereochemical quality. Finally, create a program for a complete assessment of the spatial arrangement of the protein molecules and calculating the stereochemical quality of and test program for protein structures from public databases RSCB PDB.

Cílem této práce bylo seznámit se s prostorovým uspořádáním molekul proteinů, s jejich získáváním, způsoby zápisu a veličinami sloužícími k jejich popisu. Predikce proteinových struktur je velice důležitá pro zjištění funkce budoucích proteinů. Dalším cílem bylo vytvoření funkce v prostředí MATLAB, která bude graficky reprezentovat prostorové uspořádání páteřní struktury proteinu formou Ramachandranova diagramu. Společně s touto funkcí vytvořit další funkci k vykreslení postraních řetězců aminokyselin a funkci k výpočtu stereochemické kvality. Nakonec vytvořit program pro kompletní vyhodnocení prostorového uspořádání molekuly proteinu a výpočtu stereochemické kvality a otestování programu na proteinových strukturách z veřejné databáze RSCB PDB., Objective of this work was to study the spatial arrangement of protein molecules, with their extraction, notations and variables used in the course descriptions. Prediction of protein structures is very important for determining the future function of proteins. Another objective was to create a function in MATLAB that will graphically represent the spatial arrangement of the backbone structure of the protein by form Ramachandran plot. Along with this function create another function to render the side-chains of amino acids and function to calculate the stereochemical quality. Finally, create a program for a complete assessment of the spatial arrangement of the protein molecules and calculating the stereochemical quality of and test program for protein structures from public databases RSCB PDB.

Cílem této práce bylo seznámit se s prostorovým uspořádáním molekul proteinů, s jejich získáváním, způsoby zápisu a veličinami sloužícími k jejich popisu. Predikce proteinových struktur je velice důležitá pro zjištění funkce budoucích proteinů. Dalším cílem bylo vytvoření funkce v prostředí MATLAB, která bude graficky reprezentovat prostorové uspořádání páteřní struktury proteinu formou Ramachandranova diagramu. Společně s touto funkcí vytvořit další funkci k vykreslení postraních řetězců aminokyselin a funkci k výpočtu stereochemické kvality. Nakonec vytvořit program pro kompletní vyhodnocení prostorového uspořádání molekuly proteinu a výpočtu stereochemické kvality a otestování programu na proteinových strukturách z veřejné databáze RSCB PDB., Objective of this work was to study the spatial arrangement of protein molecules, with their extraction, notations and variables used in the course descriptions. Prediction of protein structures is very important for determining the future function of proteins. Another objective was to create a function in MATLAB that will graphically represent the spatial arrangement of the backbone structure of the protein by form Ramachandran plot. Along with this function create another function to render the side-chains of amino acids and function to calculate the stereochemical quality. Finally, create a program for a complete assessment of the spatial arrangement of the protein molecules and calculating the stereochemical quality of and test program for protein structures from public databases RSCB PDB.

Cílem této práce bylo seznámit se s prostorovým uspořádáním molekul proteinů, s jejich získáváním, způsoby zápisu a veličinami sloužícími k jejich popisu. Predikce proteinových struktur je velice důležitá pro zjištění funkce budoucích proteinů. Dalším cílem bylo vytvoření funkce v prostředí MATLAB, která bude graficky reprezentovat prostorové uspořádání páteřní struktury proteinu formou Ramachandranova diagramu. Společně s touto funkcí vytvořit další funkci k vykreslení postraních řetězců aminokyselin a funkci k výpočtu stereochemické kvality. Nakonec vytvořit program pro kompletní vyhodnocení prostorového uspořádání molekuly proteinu a výpočtu stereochemické kvality a otestování programu na proteinových strukturách z veřejné databáze RSCB PDB., Objective of this work was to study the spatial arrangement of protein molecules, with their extraction, notations and variables used in the course descriptions. Prediction of protein structures is very important for determining the future function of proteins. Another objective was to create a function in MATLAB that will graphically represent the spatial arrangement of the backbone structure of the protein by form Ramachandran plot. Along with this function create another function to render the side-chains of amino acids and function to calculate the stereochemical quality. Finally, create a program for a complete assessment of the spatial arrangement of the protein molecules and calculating the stereochemical quality of and test program for protein structures from public databases RSCB PDB.