Most structural proteins are fibrous proteins; for example, actinand tubulin are globular and soluble as monomers, but polymerize to form long, stiff fibers that make up the cytoskeleton, which allows the cell to maintain its shape and size. Collagen and elastin are critical components of connective tissue such as cartilage, Sony VAIO VPCS12L9E/B battery
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. Sony VAIO VPCF11S1E/B battery
These proteins are crucial for cellular motility of single celled organisms and the sperm of many multicellular organisms which reproduce sexually. They also generate the forces exerted by contracting muscles.
As some of the most commonly studied biological molecules, the activities and structures of proteins are examined both in vitro and in vivo. Sony VAIO VPCYB3V1E/R Battery
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 thechemical mechanism of an enzyme's catalytic activity and its relative affinity for various possible substrate molecules. Sony VAIO VPCF23P1E Battery
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.
In order to perform in vitro analysis, a protein must be purified away from other cellular components. Sony VAIO VPCF23N1E Battery
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 usingultracentrifugation, which fractionates the various cellular components into fractions containing soluble proteins; Sony VAIO VPCY21S1E/L Battery
membrane lipids and proteins; cellularorganelles, 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. Sony VAIO VPCY21S1E/G battery
The level of purification can be monitored using various types of gel electrophoresis if the desired protein's molecular weight and isoelectric point are known, byspectroscopy if the protein has distinguishable spectroscopic features, or by enzyme assays if the protein has enzymatic activity. Sony VAIO VPCF24M1E battery
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. Sony VGP-BPS21A/B Battery
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. Sony VGP-BPS21/S Battery
A number of different tags have been developed to help researchers purify specific proteins from complex mixtures.
The study of proteins in vivo is often concerned with the synthesis and localization of the protein within the cell. Sony VGP-BPS21B Battery
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.Sony VGP-BPS21 Battery
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).[40] The fused protein's position within the cell can be cleanly and efficiently visualized using microscopy,[41] as shown in the figure opposite. Sony Vaio PCG-5G2L Battery
Other methods for elucidating the cellular location of proteins requires the use of known compartmental markers for regions such as the ER, the Golgi, lysosomes/vacuoles, mitochondria, chloroplasts, plasma membrane, etc. With the use of fluorescently tagged versions of these markers or of antibodies to known markers, Sony Vaio PCG-5G3L Battery
it becomes much simpler to identify the localization of a protein of interest. For example, indirect immunofluorescence will allow for fluorescence colocalization and demonstration of location. Fluorescent dyes are used to label cellular compartments for a similar purpose.[42] Sony Vaio PCG-5J1L Battery
Other possibilities exist, as well. For example, immunohistochemistry usually utilizes an antibody to one or more proteins of interest that are conjugated to enzymes yielding either luminescent or chromogenic signals that can be compared between samples, allowing for localization information. Sony Vaio PCG-5J2L Battery
Another applicable technique is cofractionation in sucrose (or other material) gradients using isopycnic centrifugation.[43] While this technique does not prove colocalization of a compartment of known density and the protein of interest, it does increase the likelihood, and is more amenable to large-scale studies. Sony Vaio PCG-5K2L Battery
Finally, the gold-standard method of cellular localization is immunoelectron microscopy. This technique also uses an antibody to the protein of interest, along with classical electron microscopy techniques. The sample is prepared for normal electron microscopic examination, Sony Vaio PCG-5L1L Battery
and then treated with an antibody to the protein of interest that is conjugated to an extremely electro-dense material, usually gold. This allows for the localization of both ultrastructural details as well as the protein of interest.[44] Sony VAIO VGN-SR51MF/W Battery
Through another genetic engineering application known as site-directed mutagenesis, Sony Vaio PCG-6S2L Battery
researchers can alter the protein sequence and hence its structure, cellular localization, and susceptibility to regulation. This technique even allows the incorporation of unnatural amino acids into proteins, using modified tRNAs,[45]and may allow the rational design of new proteins with novel properties. Sony Vaio PCG-6S3L Battery
The 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 2D electrophoresis, Sony Vaio PCG-6V1L Battery
which allows the separation of a large number of proteins, mass spectrometry,[48] which allows rapid high-throughput identification of proteins and sequencing of peptides (most often after in-gel digestion), protein microarrays,[49] which allow the detection of the relative levels of a large number of proteins present in a cell, and two-hybrid screening, Sony Vaio PCG-6W1L Battery
which allows the systematic exploration of protein–protein interactions.[50] The total complement of biologically possible such interactions is known as the interactome.[51] A systematic attempt to determine the structures of proteins representing every possible fold is known asstructural genomics.[52] Sony Vaio PCG-7111L Battery
The large amount of genomic and proteomic data available for a variety of organisms, including the human genome, allows researchers to efficiently identifyhomologous proteins in distantly related organisms by sequence alignment. Sequence profiling tools can perform more specific sequence manipulations such asrestriction enzyme maps, Sony Vaio PCG-71511M Battery
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. Sony Vaio PCG-6W3L Battery
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.
Complementary to the field of structural genomics, protein structure prediction seeks to Sony Vaio PCG-7113L Battery
develop efficient ways to provide plausible models for proteins whose structures have not yet been determined experimentally.[53] 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; Sony Vaio PCG-7133L Battery
structural genomics' goal is to provide sufficient representation in solved structures to model most of those that remain.[54] 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, Sony Vaio PCG-7Z1L Battery
as quite accurate models can be produced if a "perfect" sequence alignment is known.[55] Many structure prediction methods have served to inform the emerging field of protein engineering, in which novel protein folds have already been designed.Sony Vaio PCG-7Z2L Battery
A more complex computational problem is the prediction of intermolecular interactions, such as in molecular docking and protein–protein interaction prediction.[57]
The processes of protein folding and binding can be simulated using such technique as molecular mechanics, in particular, molecular dynamics and Monte Carlo, Sony Vaio PCG-8Y1L Battery
which increasingly take advantage of parallel and distributed computing (Folding@home project; molecular modeling on GPU). The folding of small alpha-helical protein domains such as the villin headpiece and the HIV accessory proteinhave been successfully simulated in silico, Sony Vaio PCG-8Y2L Battery
and hybrid methods that combine standard molecular dynamics with quantum mechanics calculations have allowed exploration of the electronic states of rhodopsins.
Most microorganisms and plants can biosynthesize all 20 standard amino acids, while animals (including humans) must obtain some of the amino acids from thediet. Sony Vaio PCG-8Z2L Battery
The amino acids that an organism cannot synthesize on its own are referred to as essential amino acids. Key enzymes that synthesize certain amino acids are not present in animals — such as aspartokinase, which catalyzes the first step in the synthesis of lysine, methionine, and threonine from aspartate. Sony Vaio PCG-8Z1L Battery
If amino acids are present in the environment, microorganisms can conserve energy by taking up the amino acids from their surroundings and downregulating their biosynthetic pathways.
In animals, amino acids are obtained through the consumption of foods containing protein. Sony Vaio PCG-7112L Battery
Ingested proteins are then broken down into amino acids throughdigestion, which typically involves denaturation of the protein through exposure to acid and hydrolysis by enzymes called proteases. Some ingested amino acids are used for protein biosynthesis, while others are converted to glucose through gluconeogenesis, or fed into the citric acid cycle. Sony Vaio PCG-6W2L Battery
This use of protein as a fuel is particularly important under starvation conditions as it allows the body's own proteins to be used to support life, particularly those found inmuscle.[62] Amino acids are also an important dietary source of nitrogen.
Proteins were recognized as a distinct class of biological molecules in the eighteenth century by Antoine Fourcroy and others,Sony Vaio PCG-5K1L Battery
distinguished by the molecules' ability to coagulate or flocculate under treatments with heat or acid. Noted examples at the time included albumin from egg whites, blood serum albumin, fibrin, and wheat gluten.
Proteins were first described by the Dutch chemist Gerardus Johannes Mulder and named by the Swedish chemist Jöns Jacob Berzelius in 1838. Sony VGP-BPL8 Battery
Mulder carried outelemental analysis of common proteins and found that nearly all proteins had the same empirical formula, C400H620N100O120P1S1.[63] He came to the erroneous conclusion that they might be composed of a single type of (very large) molecule. The term "protein" to describe these molecules was proposed by Mulder's associate Berzelius; Sony VGP-BPL8A Battery
protein is derived from the Greek word πρωτεῖος (proteios), meaning "primary",[64] "in the lead", or "standing in front".[65] 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.[63] Sony VGP-BPL9 Battery
Early nutritional scientists such as the German Carl von Voit believed that protein was the most important nutrient for maintaining the structure of the body, because it was generally believed that "flesh makes flesh." The central role of proteins as enzymes in living organisms was not fully appreciated until 1926, Sony VGP-BPS10 Battery
when James B. Sumner showed that the enzyme urease was in fact a protein.
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, Sony VGP-BPS10 Battery
egg white, various toxins, and digestive/metabolic enzymes obtained fromslaughterhouses. In the 1950s, the Armour Hot Dog Co. purified 1 kg of pure bovine pancreatic ribonuclease A and made it freely available to scientists; this gesture helped ribonuclease A become a major target for biochemical study for the following decades. Sony VGP-BPS10/S Battery
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 Linderstrøm-Lang, Sony VGP-BPS10A Battery
contributed an understanding of protein folding and structure mediated byhydrophobic interactions.
The first protein to be sequenced was insulin, by Frederick Sanger, in 1949. Sanger correctly determined the amino acid sequence of insulin, Sony VGP-BPS10A/B Battery
thus conclusively demonstrating that proteins consisted of linear polymers of amino acids rather than branched chains, colloids, or cyclols.[72] He won the Nobel Prize for this achievement in 1958.
The first protein structures to be solved were hemoglobin and myoglobin, by Max Perutz and Sir John Cowdery Kendrew, respectively, in 1958. Sony VGP-BPS10B Battery
The first atomic-resolution structures of proteins were solved by X-ray diffraction analysis in the 1960s (Perutz and Kendrew shared the 1962 Nobel Prize in Chemistry for these discoveries) and by NMR in the 1980s. As of 2009, the Protein Data Bank has over 55,000 atomic-resolution structures of proteins. Sony VGP-BPS11 Battery
In more recent times, cryo-electron microscopy of large macromolecular assemblies[76] and computational protein structure prediction of small protein domains[77]are two methods approaching atomic resolution.
In molecular biology protein structure describes the various levels of organization of protein molecules. Sony VGP-BPS12 Battery
Proteins are an important class of biological macromoleculespresent in all organisms. Proteins are polymers of amino acids. Classified by their physical size, proteins are nanoparticles (definition: 1–100 nm). Each protein polymer – also known as a polypeptide – consists of a sequence formed from 20 possible L-α-amino acids, Sony VGP-BPS13 Battery
also referred to as residues. For chains under 40 residues the term peptideis frequently used instead of protein. To be able to perform their biological function, proteins fold into one or more specific spatial conformations, driven by a number of non-covalent interactions such as hydrogen bonding, ionic interactions, Van Der Waals forces, and hydrophobic packing. Sony VGP-BPS13/B Battery
To understand the functions of proteins at a molecular level, it is often necessary to determine their three-dimensional structure. This is the topic of the scientific field of structural biology, which employs techniques such as X-ray crystallography, NMR spectroscopy, and dual polarisation interferometry to determine the structure of proteins. Sony VGP-BPS13/S Battery
Protein structures range in size from tens to several thousand residues [1] Very large aggregates can be formed from protein subunits: for example, many thousand actinmolecules assemble into a microfilament.
A protein may undergo reversible structural changes in performing its biological function. Sony VGP-BPS13/S Battery
The alternative structures of the same protein are referred to as different conformations, and transitions between them are called conformational changes.
The primary structure refers to amino acid linear sequence of the polypeptide chain. The primary structure is held together by covalent or peptide bonds, which are made during the process of protein biosynthesis or translation. Sony VGP-BPS13A/B Battery
The two ends of the polypeptide chain are referred to as the carboxyl terminus (C-terminus) and the amino terminus (N-terminus) based on the nature of the free group on each extremity. Counting of residues always starts at the N-terminal end (NH2-group), which is the end where the amino group is not involved in a peptide bond. Sony VGP-BPS13A/S Battery
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