Protein Part-4

Proteomics and Bioinformatics

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Proteomics and BioinformaticsThe total complement of proteins present at a time in a cell or cell type is known as its proteome, and the study of such large-scale data sets defines the field of proteomics, named by analogy to the related field of genomics. Key experimental techniques in proteomics include Mass Spectrometry, which allows rapid highthroughput identification of proteins and sequencing of peptides. protein microarrays, which allow the detection of the relative levels of a large number of proteins present in a cell, and two-hybrid screening, which allows the systematic exploration of protein-protein interactions. The total complement of biologically possible such interactions is known as the interactome. A systematic attempt to determine the structures of proteins representing every possible fold is known as structural genomics.The large amount of genomic and proteomic data available for a variety of organisms, including the human genome, allows researchers to efficiently identify homologous proteins in distantly related organisms by sequence alignment. Sequence profiling tools can perform more specific sequence manipulations such as restriction enzyme maps, open reading frame analyses for nucleotide sequences, and secondary structure prediction. From this data phylogenetic trees can be constructed and evolutionary hypotheses developed using special software like ClustalW regarding the ancestry of modern organisms and the genes they express. The field of bioinformatics seeks to assemble, annotate, and analyze genomic and proteomic data, applying computational techniques to biological problems such as gene finding and cladistics.

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Structure Prediction and Simulation

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Complementary to the field of structural genomics, protein structure prediction seeks to develop efficient ways to provide plausible models for proteins whose structures have not yet been determined experimentally. The most successful type of structure prediction, known as homology modeling, relies on the existence of a "template" structure with sequence similarity to the protein being modeled; structural genomics' goal is to provide sufficient representation in solved structures to model most of those that remain. Although producing accurate models remains a challenge when only distantly related template structures are available, it has been suggested that sequence alignment is the bottleneck in this process, as quite accurate models can be produced if a "perfect" sequence alignment is known. Many structure prediction methods have served to inform the emerging field of protein engineering, in which novel protein folds have already been designed. A more complex computational problem is the prediction of intermolecular interactions, such as in molecular docking and protein-protein interaction prediction.The processes of protein folding and binding can be simulated using techniques derived from molecular dynamics, which increasingly take advantage of distributed computing as in the Folding@Home project. The folding of small alpha-helical protein domains such as the villin headpiece and the HIV accessory protein have been successfully simulated in silico, and hybrid methods that combine standard molecular dynamics with quantum mechanics calculations have allowed exploration of the electronic states of rhodopsins.

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History

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Proteins were recognized as a distinct class of biological molecules in the eighteenth century by Antoine Fourcroy and others, distinguished by the molecules' ability to coagulate or flocculate under treatments with heat or acid. Noted examples at the time included albumen from egg whites, blood, serum albumin, fibrin, and wheat gluten. Dutch chemist Gerhardus Johannes Mulder carried out elemental analysis of common proteins and found that nearly all proteins had the same empirical formula. The term "protein" to describe these molecules was proposed in 1838 by Mulder's associate Jons Jakob Berzelius. Mulder went on to identify the products of protein degradation such as the amino acid leucine for which he found a (nearly correct) molecular weight of 131 Da.The difficulty in purifying proteins in large quantities made them very difficult for early protein biochemists to study. Hence, early studies focused on proteins that could be purified in large quantities, e.g., those of blood, egg white, various toxins, and digestive/metabolic enzymes obtained from slaughterhouses. In the late 1950s, the Armour Hot Dog Co. purified 1 kg (= one million milligrams) of pure bovine pancreatic ribonuclease A and made it freely available to scientists around the world.
Linus Pauling is credited with the successful prediction of regular protein secondary structures based on hydrogen bonding, an idea first put forth by William Astbury in 1933. Later work by Walter Kauzmann on denaturation, based partly on previous studies by Kaj Linderstorm-Lang, contributed an understanding of protein folding and structure mediated by hydrophobic interactions. In 1949 Fred Sanger correctly determined the amino acid sequence of insulin, thus conclusively demonstrating that proteins consisted of linear polymers of amino acids rather than branched chains, colloids, or cyclos. The first atomic-resolution structures of proteins were solved by X-ray crystallography in the 1960s and by NMR in the 1980s. As of 2006, the Protein Data Bank has nearly 40,000 atomic-resolution structures of proteins. In more recent times, cryo-electron microscopy of large macromolecular assemblies and computational protein structure prediction of small protein domains are two methods approaching atomic resolution.

Text Source: Wikipedia Liscence NGU

Protein- Part-3

Enzymes:

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The best-known role of proteins in the cell is their duty as enzymes, which catalyze chemical reactions. Enzymes are usually highly specific catalysts that accelerate only one or a few chemical reactions. Enzymes carry out most of the reactions involved in metabolism and catabolism, as well as DNA replication, DNA repair, and RNA synthesis. Some enzymes act on other proteins to add or remove chemical groups in a process known as post-translational modification. About 4,000 reactions are known to be catalyzed by enzymes. The rate acceleration conferred by enzymatic catalysis is often enormous - as much as 1017-fold increase in rate over the uncatalyzed reaction in the case of orotate decarboxylase (78 million years without the enzyme, 18 milliseconds with the enzyme)The molecules bound and acted upon by enzymes are known as substrates. Although enzymes can consist of hundreds of amino acids, it is usually only a small fraction of the residues that come in contact with the substrate, and an even smaller fraction - 3-4 residues on average - that are directly involved in catalysis. The region of the enzyme that binds the substrate and contains the catalytic residues is known as the active site.

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Cell signaling and ligand transport:

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A mouse antibody against cholera that binds a carbohydrate antigen.Many proteins are involved in the process of cell signaling and signal transduction. Some proteins, such as insulin, are extracellular proteins that transmit a signal from the cell in which they were synthesized to other cells in distant tissues. Others are membrane proteins that act as receptors whose main function is to bind a signaling molecule and induce a biochemical response in the cell. Many receptors have a binding site exposed on the cell surface and an effector domain within the cell, which may have enzymatic activity or may undergo a conformational change detected by other proteins within the cell.Antibodies are protein components of adaptive immune system whose main function is to bind antigens, or foreign substances in the body, and target them for destruction. Antibodies can be secreted into the extracellular environment or anchored in the membranes of specialized B cells known as plasma cells. Whereas enzymes are limited in their binding affinity for their substrates by the necessity of conducting their reaction, antibodies have no such constraints. An antibody's binding affinity to its target is extraordinarily high.

Many ligand transport proteins bind particular small biomolecules and transport them to other locations in the body of a multicellular organism. These proteins must have a high binding affinity when their ligand is present in high concentrations, but must also release the ligand when it is present at low concentrations in the target tissues. The canonical example of a ligand-binding protein is hemoglobin, which transports oxygen from the lungs to other organs and tissues in all vertebrates and has close homologos in every biological kingdom.Transmembrane proteins can also serve as ligand transport proteins that alter the permeability of the cell membrane to small molecules and ions. The membrane alone has a hydrophobic core through which polar or charged molecules cannot diffuse. Membrane proteins contain internal channels that allow such molecules to enter and exit the cell. Many ion channel proteins are specialized to select for only a particular ion; for example, potassium and sodium channels often discriminate for only one of the two ions.

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Structural proteins:

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Structural proteins confer stiffness and rigidity to otherwise-fluid biological components. Most structural proteins are fibrous proteins; for example, actin and tubulin are globular and soluble as monomers, but polymerise to form long, stiff fibers that comprise the cytoskeleton, which allows the cell to maintain its shape and size. Collagen and elastin are critical components of connective tissue such as cartilage, and keratin is found in hard or filamentous structures such as hair, nails, feathers, hooves, and some animal shells.Other proteins that serve structural functions are motor proteins such as myosin, kinesin, and dynein, which are capable of generating mechanical forces. These proteins are crucial for cellular motility of single celled organisms and the sperm of many sexually reproducing multicellular organisms. They also generate the forces exerted by contracting muscles.

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Methods of study:

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As some of the most commonly studied biological molecules, the activities and structures of proteins are examined both in vitro and in vivo. In vitro studies of purified proteins in controlled environments are useful for learning how a protein carries out its function: for example, enzyme kinetics studies explore the chemical mechanism of an enzyme's catalytic activity and its relative affinity for various possible substrate molecules. By contrast, in vivo experiments on proteins' activities within cells or even within whole organisms can provide complementary information about where a protein functions and how it is regulated.

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Protein purification:

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In order to perform in vitro analysis, a protein must be purified away from other cellular components. This process usually begins with cell lysis, in which a cell's membrane is disrupted and its internal contents released into a solution known as a crude lysate. The resulting mixture can be purified using ultracentrifugation, which fractionates the various cellular components into fractions containing soluble proteins; membrane lipids and proteins; cellular organelles, and nucleic acids. Precipitation by a method known as salting out can concentrate the proteins from this lysate. Various types of chromatography are then used to isolate the protein or proteins of interest based on properties such as molecular weight, net charge and binding affinity. The level of purification can be monitored using various types of gel electrophoresis if the desired protein's molecular weight and isolelectric point are known, by spectroscopy if the protein has distinguishable spectroscopic features, or by enzyme assays if the protein has enzymatic activity. Additionally, proteins can be isolated according their charge using electrofocusing.

For natural proteins, a series of purification steps may be necessary to obtain protein sufficiently pure for laboratory applications. To simplify this process, genetic engineering is often used to add chemical features to proteins that make them easier to purify without affecting their structure or activity. Here, a "tag" consisting of a specific amino acid sequence, often a series of histidine residues (a "His-tag"), is attached to one terminus of the protein. As a result, when the lysate is passed over a chromatography column containing nickel, the histidine residues ligate the nickel and attach to the column while the untagged components of the lysate pass unimpeded.Cellular localization. Proteins in different cellular compartments and structures tagged with green fluorescent protein The study of proteins in vivo is often concerned with the synthesis and localization of the protein within the cell. Although many intracellular proteins are synthesized in the cytoplasm and membrane-bound or secreted proteins in the endoplasmic reticulum, the specifics of how proteins are targeted to specific organelles or cellular structures is often unclear. A useful technique for assessing cellular localization uses genetic engineering to express in a cell a fusion protein or chimera consisting of the natural protein of interest linked to a "reporter" such as green fluorescent protein (GFP). The fused protein's position within the cell can be cleanly and efficiently visualized using microscopy, as shown in the figure opposite.Through another genetic engineering application known as site-directed mutagenesis, researchers can alter the protein sequence and hence its structure, cellular localization, and susceptibility to regulation, which can be followed in vivo by GFP tagging or in vitro by enzyme kinetics and binding studies.

Text Source: Wikipedia Liscence NGU

Protein- Part-2

Protein structure:

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Most proteins fold into unique 3-dimensional structures. The shape into which a protein naturally folds is known as its native state. Although many proteins can fold unassisted simply through the structural propensities of their component amino acids, others require the aid of molecular chaperones to efficiently fold to their native states. Biochemists often refer to four distinct aspects of a protein's structure:

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Primary structure: The amino acid sequence

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Secondary structure : regularly repeating local structures stabilized by hydrogen bonds. The most common examples are the alpha helix and beta sheet. Because secondary structures are local, many regions of different secondary structure can be present in the same protein molecule.

Tertiary structure: the overall shape of a single protein molecule; the spatial relationship of the secondary structures to one another. Tertiary structure is generally stabilized by nonlocal interactions, most commonly the formation of a hydrophobic core, but also through salt bridges, hydrogen bonds, disulfide bonds, and even post translational modifications. The term "tertiary structure" is often used as synonymous with the term fold.

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Quarternary structure: the shape or structure that results from the interaction of more than one protein molecule, usually called protein subunits in this context, which function as part of the larger assembly or protein complex.

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Proteins are not entirely rigid molecules. In addition to these levels of structure, proteins may shift between several related structures while they perform their biological function. In the context of these functional rearrangements, these tertiary or quaternary structures are usually referred to as "conformations," and transitions between them are called conformational changes. Such changes are often induced by the binding of a substrate molecule to an enzyme's active site, or the physical region of the protein that participates in chemical catalysis. In solution all proteins also undergo variation in structure through thermal vibration and the collision with other molecules, see the animation on the right.

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Proteins can be informally divided into three main classes, which correlate with typical tertiary structures: globular, fibrous, and membrane proteins. Almost all globular proteins are soluble and many are enzymes. Fibrous proteins are often structural; membrane proteins often serve as receptors or provide channels for polar or charged molecules to pass through the cell membrane.
A special case of intramolecular hydrogen bonds within proteins, poorly shielded from water attack and hence promoting their own dehydration are called dehydrons.

Structure determination:

Discovering the tertiary structure of a protein, or the quaternary structure of its complexes, can provide important clues about how the protein performs its function. Common experimental methods of structure determination include X- ray crystollography and NMR spectroscopy, both of which can produce information at atomic resolution. Cryoelectron microscopy is used to produce lower-resolution structural information about very large protein complexes, including assembled viruses; a variant known as electron crystallography can also produce high-resolution information in some cases, especially for two-dimensional crystals of membrane proteins. Solved structures are usually deposited in the Protein Data Bank (PDB), a freely available resource from which structural data about thousands of proteins can be obtained in the form of Cartesian coordinates for each atom in the protein.
Many more gene sequences are known than protein structures. Further, the set of solved structures is biased toward proteins that can be easily subjected to the conditions required in X- ray crystollography one of the major structure determination methods. In particular, globular proteins are comparatively easy to crystallize in preparation for X-ray crystallography. Membrane proteins, by contrast, are difficult to crystallize and are underrepresented in the PDB. Structural genomics initiatives have attempted to remedy these deficiencies by systematically solving representative structures of major fold classes.
Cellular functions

Proteins are the chief actors within the cell, said to be carrying out the duties specified by the information encoded in genes. With the exception of certain types of RNA, most other biological molecules are relatively inert elements upon which proteins act. Proteins make up half the dry weight of an E.coli cell, whereas other macromolecules such as DNA and RNA make up only 3% and 20%, respectively. The set of proteins expressed in a particular cell or cell type is known as its proteome.

Text Source:Wikipedia Liscence NGU

Protein- Part-1

Biochemistry

Proteins are linear polymers built from 20 different L-α-amino acids. All amino acids possess common structural features, including an a carbon to which an amino group, a carboxyl group, and a variable side chain are bonded. Only proline differs from this basic structure as it contains an unusual ring to the N-end amine group, which forces the CO–NH amide moiety into a fixed conformation. The side chains of the standard amino acids have different chemical properties that produce three-dimensional protein structure and are therefore critical to protein function. The amino acids in a polypeptide chain are linked by peptide bonds formed in a dehydratopn reaction. Once linked in the protein chain, an individual amino acid is called a residue, and the linked series of carbon, nitrogen, and oxygen atoms are known as the main chain or protein backbone. The peptide bond has two resonance forms that contribute some double-bond character and inhibit rotation around its axis, so that the alpha carbons are roughly coplanar. The other two dihedral angles in the peptide bond determine the local shape assumed by the protein backbone.

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Synthesis:

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Proteins are assembled from amino acids using information encoded in genes. Each protein has its own unique amino acid sequence that is specified by the nucleotide sequence of the gene encoding this protein. The genetic code is a set of three-nucleotide sets called codons and each three-nucleotide combination stands for an amino acid, for example AUG stands for methionine. Because DNA contains four nucleotides, the total number of possible codons is 64; hence, there is some redundancy in the genetic code, with some amino acids specified by more than one codon. Genes encoded in DNA are first transcribed into pre-messenger RNA (mRNA) by proteins such as RNA polymerase. Most organisms then process the pre-mRNA (also known as a primary transcript) using various forms of Post transcriptional modificational to form the mature mRNA, which is then used as a template for protein synthesis by the ribosome. In prokaryotes the mRNA may either be used as soon as it is produced, or be bound by a ribosome after having moved away from the nucleoid. In contrast, eukaryotes make mRNA in the cell nucleus and then translocate it across the nuclear membrane into the cytoplasm, where protein synthesis then takes place. The rate of protein synthesis is higher in prokaryotes than eukaryotes and can reach up to 20 amino acids per second.
The process of synthesizing a protein from an mRNA template is known as translation. The mRNA is loaded onto the ribosome and is read three nucleotides at a time by matching each codon to its base pairing anticodon located on a transfer RNA molecule, which carries the amino acid corresponding to the codon it recognizes. The enzyme aminoacyl tRNA synthetase "charges" the tRNA molecules with the correct amino acids. The growing polypeptide is often termed the nascent chain. Proteins are always biosynthesized from N-terminus to C-terminus

The size of a synthesized protein can be measured by the number of amino acids it contains and by its total molecular mass, which is normally reported in units of daltons or the derivative unit kilodalton (kDa). Yeast proteins are on average 466 amino acids long and 53 kDa in mass. The largest known proteins are the titins, a component of the muscle sarcomere, with a molecular mass of almost 3,000 kDa and a total length of almost 27,000 amino acids.

Chemical synthesis:

Short proteins can also be synthesized chemically by a family of methods known as peptide synthesis, which rely on organic synthesis techniques such as chemical ligation to produce peptides in high yield. Chemical synthesis allows for the introduction of non-natural amino acids into polypeptide chains, such as attachment of fluorescent probes to amino acid side chains. These methods are useful in laboratory biochemistry and cell biology, though generally not for commercial applications. Chemical synthesis is inefficient for polypeptides longer than about 300 amino acids, and the synthesized proteins may not readily assume their native tertiary structure. Most chemical synthesis methods proceed from C-terminus to N-terminus, opposite the biological reaction.

Text Source:Wikipedia Liscence NGU

What Is NCBI ?

The National Center for Biotechnology Information (NCBI) is part of the United States National Library of Medicine (NLM), a branch of the National Institue of Health. The NCBI is located in Bethesda, Maryland and was founded in 1988 through legislation sponsored by Senator Claude Pepper. The NCBI houses genome sequencing data in GenBank and an index of biomedical research articles in PubMed Central and PubMed, as well as other information relevant to biotechnology. All these databases are available online through the Entrez search engine.The NCBI is directed by David Lipman, one of the original authors of the Blast sequence alignment program and a widely respected figure in Bioinformatics. He also leads an intramural research program, including groups led by Stephen Altschul (another Blast co-author), David Landsman, and Eugene Koonin.

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The NCBI has had responsibility for making available the GenBank DNA sequence database since 1992. GenBank coordinates with individual laboratories and other sequence databases such as those of the European Molecular Biology Laboratory (EMBL) and the DNA Database of Japan (DDBJ).Since 1992, NCBI has grown to provide other databases in addition to GenBank. NCBI provides Online Mendelian Inheritance In Man, the Molecular Modeling Database (3D protein structures), the Unique Human Gene Sequence Collection, a Gene Map of the Human genome, a Taxonomy Browser, and coordinates with the National Cancer Institute to provide the Cancer Genome Anatomy Project. The NCBI assigns a unique identifier (Taxonomy ID number) to each species of organism.The NCBI has software tools that are available by www browsing or by FTP. For example,BLAST is a sequence similarity searching program. BLAST can do sequence comparisons against the GenBank DNA database in less than 15 seconds.

Text Source:Wikipedia Liscence NGU